CN1704470A - High metal-salt-resistant micro penicillium purpurogerum - Google Patents

Abstract

The invention relates to a novel Penicillium janthinellum GXCR, which is a Penicillium fungus and capable of resisting copper salts, enriching and evoluting copper and resisting many kinds of other metal salts. The invention also relates to the use of the Penicillium janthinellum GXCR.

Description

The penicillium janthinellum of the anti-metal-salt of one plant height

Technical field

The present invention relates to a kind of new Penicillium (Penicillium) fungi.More specifically, the present invention relates to a kind of high anti-mantoquita, can be used for the penicillium janthinellum (Penicillium janthinellum) of absorbing copper when having high anti-multiple other metal-salt concurrently.

Background technology

One, the effect of cupric ion in organism

Aerobiont need resemble trace (trace amount) element of copper (copper) and so on, and this dvielement generally has the dual-use function that obtains and lose electronics.In the organism that lives, cupric ion is with compound form and the coexistence of high density electron rich molecule, as sulphur or xitix etc. ^[1], perhaps participate in the structure of the structure of RNA and DNA ^[2-3]Or participate in other biological metabolism activity and promote biology growing ^[4-5]Therefore, cupric ion can be used as the catalytic center of many proteolytic enzyme, these albumen relate to multiple critical enzyme catalysis process, as electronics transport protein Cyt oxydase, the releasing toxic albumen of oxyradical such as copper/zinc SOD albumen, Vitamin C oxidase, diamine oxidase and phenol oxidase etc. ^[6-8]Because " hobby " participates in redox reaction, excessive copper can cause producing pair cell and have extremely toxic nocuity hydroxyl, thereby causes the peroxidation of lipid and the fracture of nucleic acid ^[9]

Two, copper is at intracellular transporting mechanism and transport factor

Under laboratory condition, copper is an excellent catalyzer, and oxidation destroys those electron rich molecules.But in cell it to have an also active metal of oxygen the same with other, with inactive form be chelated and transporte to cells in, be transported to the privileged site and the reaction zone of cell afterwards by cellular component ^[1]Up to the present, still have many details unclear for the component of participating in transhipment copper with to the mechanism of film transhipment.Comparatively speaking, in yeast, study relatively deeply about the metabolism of copper ^[10]In yeast, copper is with monovalence copper (Cu ⁺) form carry out transmembrane transport by Ctr1 is protein mediated, and be transported to tenuigenin copper transhipment or the copper transhipment chaperone (chaperones) of little solubility ^[11]In yeast, have 5 kinds of intracellular copper transport factors at least: be arranged in the Ccc2 (a kind of P type ATP enzyme) of Secretory Pathway and many copper oxydase Fet3, the Terminal oxidase of plastosome, the copper transcription factor of nucleus, the copper/zinc SODi albumen of cytosol, the Atx1p albumen of kytoplasm ^[9-13]Identified 4 kinds of different copper transhipment mediation albumen, the copper transhipment route of 3 kinds of mediation property wherein is by on the Ctr1 albumen copper being transported to 3 different cell sites ^[1]:

1) Cox17 albumen guiding copper is transported to plastosome and inserts in terminal oxidase one cytochrome C oxidase of its respiratory chain.

2) Lys7 albumen instructs copper to be positioned tenuigenin colloidal sol to make it form a kind of basic antioxidant enzyme CuZnSOD.

3) Atx1 then is directed to copper golgi body (post-Golgi) chamber, back, and finally inserts the necessary many copper oxydase of the ionic absorption Fet3 with high-affinity by means of Ccc2 (a kind of P type ATP enzyme).

4) Atx1p albumen is that the cupric ion in the transport factor of cell surface is transported to Ccc2 and Fet3, but also do not know this albumen whether with copper to plastosome, the copper in nucleus or the cytosol/proteic transhipment of zinc SOD1 relevant [13].

In the organism of studying, the transport factor that the copper metabolism relates to all has homology.Metal chaperone (Coppermetallochaperone) and film transhipment that copper is arranged in the transport factor that relates in the metabolism of zymic copper ^[1-3,13-20], wherein some and some human albumen homologies: yeast Atx1/ people Hah1, yeast Lys7/ people Ccs1, the sick albumen of yeast Cox17/ people Cox17, yeast Ccc2/ people Wilson ^[21]These presentation of results, in many aspects, the copper between the yeast and the mankind is similar with the interior stabiliser of ionic body.

Copper transporting pathway in vivo is not changeless, can change with the concentration of cupric ion.In yeast, under lower concentration copper bar spare, a kind of transhipment route is adopted in the intracellular transhipment of cupric ion.When mantoquita concentration was higher in the substratum, other side approach was adopted in the cupric ion transhipment fully ^[1]

Three, the anti-copper mechanism of organism

As other heavy metal, copper is one of element that constitutes the earth's crust.Under the state of nature, copper is present in the various mineral more, and its content generally is lower than 0.1%, belongs to trace heavy metal element ^[15]From the biological utilisation angle, the characteristics of heavy metal be can not be degraded and from environment, thoroughly disappear, can only be another kind of form from a kind of form transformation, become lower concentration, part from high density and can be in toxicity one transform between the nontoxic or low toxicity state, can accumulate enrichment in vivo ^[16]The part heavy metal can a certain amount of (trace) level or some special growth metabolism activities that certain ionic valence condition is participated in organism ^[17]

Biological in evolutionary process, in its cell paste, developed the system of cover induction, transhipment and a chelated copper, with the primary demand of keeping copper and the fine equilibrium between its toxic level ^[4,18-20,22], thereby organism more or less have a copper resistant, and develop and the complicated anti-copper mechanism of a cover.

Prokaryotic organism mainly rely on the ATPase Send out pump of P-type to regulate intravital copper ion concentration and obtain the resistance of copper ^[13,23-25]Eukaryote mainly relies on absorbing path and two kinds of mechanism of synthetic metallothionein(MT) of closing cupric ion and obtains copper resistant ^[26]In fact copper resistant relates to a plurality of metabolic processes and complicated mechanism, and for example relevant with copper resistant copper inner equilibrium process is overlapped with the metabolic part process of the oxyradical that causes cell damage ^[4,24]

Four, the pollution of copper and harm

Industrialized development being extensive use of of copper has in addition made the surrounding enviroment that comprise nature water, various organic waste water, trade waste such as matchmaker's footpath between fields, sedimentary mud, metal processing district etc. the copper staining phenomenon all occur ^[15,27-37]

Just can produce pollution after copper is excessive, pollute and generally can cause dwarfing, poor growth, the biomass of plant plant to descend significantly ^[27,38-39]Also can cause the metabolism disorder of human body after copper is excessive, can cause multiple disease, representative disease such as Menkes syndromes and Wilson disease ^[21,40-41], nerve degeneration disease (Neurodegenerative Diseases) ^[42], lethality motor neuron (fatal motorneuron disease) ^[3]Etc. many other diseases ^[44-45]Wherein one of reason is to cause DNA and RNA damage on molecular level ^[42]The toxicity of copper is oxygen dependence, and its process is followed the Harber-Weiss reaction, produces reactive oxygen intermediate ^[45]Or combine with biomolecules in inappropriate mode ^[19]

Heavy metal ion has two aspects to the influence of microflora: the development to microflora during lower concentration has promoter action, and restraining effect is then arranged during high density, the susceptibility difference of different monoid microorganisms, normally actinomycetes＞bacterium＞fungi ^[17,46-47]Soil is subjected to heavy metal contamination heavier, and microbial population quantity is fewer; The heavy metal of anti-single kind the more relatively; Anti-heavy metal ion more than 2 kinds or 2 kinds few; Microorganism in the soil is than anti-(anti-) property height of air microbe.With the copper staining is example, has the research report to show that (Cu＜100mg/L) 35 kinds of fungies are arranged there are 25 kinds of fungies in the intermediate pollution soil (1000mg Cu/L) to control soil, and highly polluted soil (10000mg Cu/L) only has 13 kinds of fungies ^[47-48]Airborne many microorganisms to the patience of heavy metal only about 10mM, as reported from air separation to 82 kinds of fungal isolates, 52 kinds of (the chain spore belongs to (Alernaria), Aspergillus (Aspergillus), Cladosporium (Cladosporium), Penicillium (Penicillium), Rhodotorula (Rhodotorula), grape ear mould etc.) only anti-a kind of metal (As of isolate ²⁺, Cd ²⁺, Co ²⁺, Cr ²⁺, Hg ²⁺, Ni ²⁺, Pb ²⁺), it is the metal more than 2 kinds of 10mM that 15 kinds of moulds and the anti-concentration of 1 primary yeast are only arranged ^{[27,38-39,49]}

Five, the improvement present situation of the pollution of copper and researchdevelopment direction thereof

As other heavy metal, the industry of copper staining is administered technology and is mainly adopted physicochemical method, as absorption, ion-exchange, film and chemical precipitation and solvent extraction etc., though effect is obvious, but investment, running cost is high, complicated operation is difficult to promote, and can't put to good use especially for the pollution of large watershed lower concentration ^[50]

The same with other heavy metal, because copper can not be another kind of elemental substance by the organism metabolism, at present to the biological treating of its pollution that causes mainly to remove (removal) or to reclaim the biological restoration (bioremedation) of (recovery), main path has two kinds: 1) some is woody, economic plants utilizes its absorption to heavy metal, accumulation, patience and filterability to remove heavy metal by plantation in polluted agricultural land, soil.2) utilize biological chemistry, biological effectiveness and biological activity characteristic, heavy metal is converted into the product (complex state, take off alkyl, change valence state etc.) of low toxicity, perhaps utilizes the affinity of heavy metal and microorganism to adsorb and the toxicity and the transfer ability of reduction heavy metal are solidified, transformed to biologic activity ^{[16,47,51-53]}

The living species that is used for the heavy metal contamination reparation at present mainly contains plant, microorganism, algae three major types ^{[13,16-18,28-31,33-34,49,51-63]}, the branch of dead volume and living body biological recovery technique is arranged ^{[29,49-50,57]}The mechanism of biological restoration roughly has: processes such as the active absorption of active somatic cell, passive absorption, leaching, parsing and conversion ^[54,61-62]

Six, the biological restoration developing direction of copper staining

The big area soil pollution that comprises many heavy metals of copper at present is based on the plant biological reparation, also cloned simultaneously many preventing from heavy metal gene (referring to Http:// www.ncbi.nlm.nih.gov: GenBank), the most representative be in multiple organism, be cloned into can regulate protein gene etc. with various heavy bonded binding peptide or metallothionein(MT) (MTs), metallothionein(MT) sample albumen, plant chelating element (phtochelatin) and metal resistance, and with the genetically modified organism of this type of gene constructed repairing heavy metal pollution ^{[21,45,50,64]}But overwhelming majority clone's report is that single heavy metal resistance gene is (referring to http://www.ncbi.nlm.nih.gov; GenBank).Though, change the plant of MTs protein gene and report much and in partial heavy metal contamination reparation, obtained comparatively satisfied result, because the inherent technical problem still exists in the genetically engineered research, as the expression efficiency instability of promotor, expressive site is uncertain in the plant acceptor, or the inner complicated reason of factor receptor causes the allos MTs albumen inactivation of expressing etc. ^[57]Thereby, limited further and applied.

One of developing direction of heavy metal contamination biological restoration is microorganism display technique (microbialdisplay technology), this is that peptide or albumen with heavy metal height affinity are made the surface of its localization and expression in microorganism by genetic engineering technique, improve the adsorptive power of microorganism to heavy metal, thereby eliminate the pollution of heavy metal or reclaim rare noble metal, but this technology itself can not strengthen the anti-tolerance level of microorganism to heavy metal ^[45]In recent years, in fungi, attempted utilizing the multiple copied plasmid of cupric inductive promotor (this promotor is from the yeast metallothionein gene), clones coding zinc refers to the gene and the transformed yeast (S.cerevisiae) of (zinc-finger) transcription factor, make up cell surface engineering yeast, with its recovery and removal cupric ion ^[62]

Another developing direction is to exempt from (not) culturing micro-organisms technology (uncultured microbes), and this technical development is also very rapid, becomes the new microbial population of seeking preventing from heavy metal and the approach of cloning a novelty of new heavy metal resistance gene.But this technology mainly concentrates on the evaluation of thalline at present.In bacterium by clone, sequencing analysis bacterium 16srRNA gene identification many resistance monoids (referring to http://www.ncbi.nlm.nih.gov; GenBank), even the problem of existence is to have found to exempt from (not) culturing micro-organisms, can't obtain its culture because of limit by prior art.

Seven, the problem in research of microorganism copper resistant and the application

The exploitation that high anti-copper has high anti-microorganism strains multiple other heavy metallic salt, that have high absorption capacity simultaneously concurrently is repaired in using significant in the microorganism of the clone of the theoretical investigation of organism copper resistant such as anti-copper pathways metabolism, anti-copper and the new gene of osmophilic strain, copper staining undoubtedly.This class bacterial strain also can further be developed as the F-strain of the genetic engineering bacterium that makes up the organic pollutant in the degradable heavy metallic salt one organism Composite pollution environment.But there are the following problems for the problem in microorganism copper resistant research at present and the application:

1) up to now, that the resistance level of the monoclonal antibody copper of finding in fungi is the highest is a kind of Candida albicans (Candida albicans), and its anti-copper level is the highest to be 50mM CuSO4 only also ^[26]

2) research for the relation of bacterial strain (Penicillium spp.) growth of heavy metal that comprises copper (Cu) and Penicillium, metabolism etc. is very extensive ^[65-66]Though report thinks that Penicilliumjanthineleum is the accumulator of modal zinc (Zn) ^[67]Also being separated in this kind bacterium can high anti-100mM AlCl ₃Strain system ^[68]But do not appear in the newspapers as yet so far high anti-mantoquita is arranged, enriching Cu and can separate out salt copper, resisting high-concentration (NaCl), have strain isolated system or other fungi kind of the Penicillium janthineleum of high anti-multiple other toxic heavy metal salt simultaneously again concurrently.

3) if certain microorganism can constantly grow in high density mantoquita environment, and its thalline has higher cupric ion adsorptive power, at least can accomplish once to add the removal ability over a long time that thalline can need not to be added with and reach the prolongation thalline, thereby minimizing operation link and technology reduce cost.But there are not in this respect research report, application example and bacterial classification patent at present.

Summary of the invention

One aspect of the present invention relates to a kind of microorganism penicillium janthinellum strain (Penicilliumjanthinellum), and it belongs to Penicillium (Penicillium) fungi, can high anti-mantoquita, and enrichment also can be separated out high anti-multiple other metal-salt of having concurrently of copper.This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) (Beijing, Zhong Guan-cun, one in north) on October 31st, 2003, and preserving number is CGMCC 1027.

Another aspect of the present invention relates to the application of penicillium janthinellum strain GXCR aspect absorption and/or anti-mantoquita and/or zinc salt.

Another aspect of the present invention relates to the application of penicillium janthinellum strain GXCR aspect absorption and/or anti-mantoquita or zinc salt metal-salt in addition.

Another aspect of the present invention relates to penicillium janthinellum strain GXCR in the application of soaking aspect the ore deposit.

Another aspect of the present invention relates to the application aspect penicillium janthinellum strain GXCR is used to administer the heavy metal environmental pollution in preparation the biological adsorption agent.

Another aspect of the present invention relates to the application in the genetic engineering bacterium of the organic pollutant of penicillium janthinellum strain GXCR in making up degradable heavy metallic salt-organism Composite pollution environment.

Description of drawings

Fig. 1: the ITS sequence of penicillium janthinellum strain GXCR.

Fig. 2: be presented in the triangular flask of 500ml volume, add the liquid nutrient medium PDL of 200ml, and inoculate 10 ⁵Individual penicillium janthinellum strain GXCR conidium, under differing temps, the 150rpm shaking table is cultivated the chart (the vertical bar line among the figure is the mean value standard error of 3 independent experiments) of the thalli growth amount after 7 days.

Fig. 3: be presented in the triangular flask of 500ml volume, add the liquid nutrient medium PDL of the different pH of 200ml, and inoculate 10 ⁵Individual penicillium janthinellum strain GXCR conidium, 32 ℃, the 150rpm shaking table is cultivated the chart (the vertical bar line among the figure is the mean value standard error of 3 independent experiments) of the thalli growth amount after 7 days.

Fig. 4: be presented in the triangular flask of 500ml volume, the liquid nutrient medium PDL of the metal-containing salt of the different pH of adding 200ml, and inoculate 10 ⁵Individual penicillium janthinellum strain GXCR conidium, 32 ℃ are descended cultivation in the time of 5 days, the chart of the thalli growth amount of this bacterium (◆: Cu; ■: Cr ⁶⁺▲: Pb; *: Zn, the vertical bar line among the figure are the mean value standard error of 3 independent experiments).

Fig. 5: show that penicillium janthinellum strain GXCR is containing 32 ℃ of pictures of cultivating 7 days upgrowth situation, wherein A:PDA-Cu on the PDA substratum of Cu and Mn; The Mn of B:PDA-Cu-5mM; The Mn-of C:PDA-Cu-5mM adds organotrophy (1% Tryptones, 0.5% yeast extract) (concentration of the digitized representation Cu among the figure).

Fig. 6: show that penicillium janthinellum strain GXCR is containing 32 ℃ of pictures of cultivating 7 days upgrowth situation, wherein A:PDA-Cu on the PDA substratum of Cu, Zn/Al; The Zn of B:PDA-Cu-5mM; The Zn-of C:PDA-Cu-5mM adds organotrophy (1% Tryptones, 0.5% yeast extract); The Al of D:PDA-Cu-5mM; The Al-of E:PDA-Cu-5mM adds organotrophy (1% Tryptones, 0.5% yeast extract) (concentration of the digitized representation Cu among the figure).

Fig. 7: show that penicillium janthinellum strain GXCR is containing 32 ℃ of pictures of cultivating 7 days upgrowth situation, wherein A:PDA-Cu on the PDA substratum of Cu and Cd; The Cd of B:PDA-Cu-2mM; The Cd-of C:PDA-Cu-2mM adds organotrophy (1% Tryptones, 0.5% yeast extract) (concentration of the digitized representation Cu among the figure).

Fig. 8: show the picture of the upgrowth situation of penicillium janthinellum strain GXCR on the PDA substratum, wherein A: do not add the PDA of Cu, cultivated 7 days for 32 ℃; B: do not add the PDA of Cu, cultivated 20 days for 32 ℃; C: contain the PDA of the Cu of 40mM, cultivate 20 big (C1: front gas is given birth to bacterium colony) for 32 ℃; C2: gas is given birth to the back side of bacterium colony); D: contain the PDA of the Cu of 40mM, cultivate more than the 20d for 32 ℃.

Fig. 9: the picture that the scanning electron microscope (SEM) of the aerial mycelium of demonstration penicillium janthinellum strain GXCR is observed, wherein A1-2: the PDA that does not add Cu cultivates more than 20 days at 32 ℃; B1-4: contain the PDA of the Cu of 40mM, cultivate more than 20 days for 32 ℃.

Figure 10: be presented at and do not add or add on the PDA of 40mM Cu, cultivate more than 20 days for 32 ℃, penicillium janthinellum strain GXCR gas is given birth to the energy spectrum analysis (EDAX) of bacterium colony front mycelium composition, A1: the PDA substratum that does not add Cu; A2: the gas that does not add on the PDA of Cu is given birth to bacterium colony; B1: the front gas on the PDA of interpolation 40mM Cu is given birth to bacterium colony; B2: the front gas on the PDA of interpolation 40mM Cu is given birth to the PDA substratum of bacterium colony below.

Figure 11: be presented in the triangular flask of 500ml volume, add the liquid nutrient medium PDL that contains different concns Cu of 200ml, and inoculate 10 ⁵Individual penicillium janthinellum strain GXCR conidium, 28 ℃, the 150rpm shaking table was cultivated after 5 days, and growth mycelia surface is to the chart (: biological absorption of biology absorption and the Cu accumulation in the somatic cells of Cu; ◇: biological accumulation (semi-invariant is too little, and summation curve overlaps with X-axis); Vertical bar line among the figure is the mean value standard error of 3 independent experiments).

Figure 12: be presented in the triangular flask of 500ml volume, in the deionized water that contains different concns Cu of adding 200ml (natural pH), the penicillium janthinellum strain GXCR thalline of inoculation 1.5g, 28 ℃, the 150rpm shaking table was cultivated 30 minutes, the chart (thalline of the NaOH pre-treatment (immersion) of ◇: 0.5mM that the NaOH of 0.5mM anticipates (immersions) penicillium janthinellum strain GXCR thalline of 30 minutes and adsorbs without the biology of the Cu on the penicillium janthinellum strain GXCR thalline surface of 0.5mM NaOH pre-treatment (state of nature); ◆: without the pretreated thalline of 0.5mM NaOH; Dotted line is represented control strain Penicillium citrinum (Penicillium citrinum).The vertical bar line is represented the mean value standard error of 3 independent experiments).

Penicillium janthinellum strain GXCR of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) (Beijing, Zhong Guan-cun, one in north) on October 31st, 2003, and preserving number is CGMCC 1027.

Embodiment

Describe the present invention in detail below with reference to embodiment.Should be appreciated that following embodiment is in order to understand the present invention better, rather than be restriction purpose of the present invention.

It is as follows to cultivate the used substratum of penicillium janthinellum strain GXCR:

1) PDA (1000ml): 20% (w/v) potato, 1.5% glucose, 1.5% agar, pH7.0

2) PDL: the PDA of no agar

The used substratum of culturing bacterium is as follows:

LB substratum (1000ml): Tryptones 10.0g; Yeast extract 5.0g; NaCl 5.0g;

pH?7.0

The used metal-salt Al of the present invention (Al ₂(SO ₄) ₃); Cd (CdCl ₂2.5H ₂O); Cr ⁶⁺(K ₂Cr ₂O ₇); Cr ³⁺(CrCl ₃6H ₂O); Cu (CuSO ₄5H ₂O); Mn (MnCl ₂4H ₂O); Ni (NiCl ₂6H ₂O); Pb (PbAc ₂3H ₂O); Zn (ZnSO ₄7H ₂O)) all available from Guangxi science equipment import and export corporation.

Embodiment 1: the acquisition of penicillium janthinellum strain

The collection of earth sample and the screening of fungal bacterial strain

Swim three from the upper, middle and lower in washwater waterways, copper mine mining area and gather bed mud (the following 10cm in top layer), the about 500g in every place, the vial of packing into.Take by weighing the mud sample 10g that adopts, put into the 250ml triangular flask, add sterilized water 100ml, fully stir with glass stick, after leaving standstill 30 minutes, get supernatant liquor 1ml, with sterilized water supernatant liquor is coated on the PDA flat board that contains 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mMCu after by 10 times of gradient dilutions, cultivated 7 days for 28 ℃, the candidate's fungal colony that grows is transferred to makes candidate strain behind the purifying on the PDA culture medium flat plate of the new Cu that contains respective concentration and preserve.All candidate's fungal bacterial strains that obtain are inoculated to the PDA flat board of the Cu that contains 40mM, cultivated 7 days for 28 ℃, fungal colony that can continued growth goes on the PDA flat board of the new Cu that contains 40mM, preserves after 28 ℃ of cultivations.Obtained the fungal bacterial strain of strain normal growth on the PDA of the Cu that contains 40mM flat board with this method, and be GXCR this strain number.

Embodiment 2:GXCR biological property and characteristic thereof

1) the solid culture feature of GXCR

GXCR bacterial strain on PDA 28 ℃ when cultivating, its bacterium colony is rounded or near circular, bacterium colony is flat, the cultivation initial stage (48 hours) aerial hyphae be white in color.Along with the increase of incubation time, because conidial generation, bacterium colony can become khaki (Fig. 8 B) by greyish-green gradually.Opticmicroscope mirror mirror result shows that this bacterium mycelia and sporophore are smooth, and nascent sporophore is finger-like one-level branch, and 1～4 column stigma is given birth on the top, and stigma pushes up 10～20 conidiums of concatenating, and conidium is circular or oval, according to above-mentioned morphological specificity and document ^[69], this Pseudomonas is in Penicillium (Penicillium) fungi.

2) chromosomal internal transcribed spacer district (the internal transcribed spacer of GXCR; ITS) dna sequence dna

The sequence of Eukaryotic chromosomal DNA internal transcribed spacer district ITS is the nucleotide sequence of the conservative rDNA of chromosomal DNA the preceding paragraph, because this sequence has the specificity of planting, therefore, it is the important standard molecular biological of the evaluation eukaryote species that are widely adopted now.

(a) preparation of total DNA of GXCR

In the PDL of 100ml substratum, inoculation 10 ⁵The conidium of individual GXCR was cultivated 48 hours down for 28 ℃, filtered with 210 mesh sieve thin,tough silk and collected thalline, with thieving paper moisture was blotted.Move in the mortar of-20 ℃ of precoolings, add the rapid grind into powder of liquid nitrogen.Immediately powder is changed in the 50ml centrifuge tube of precooling, the DNA extraction liquid of adding 10ml (NaCl of 200mM, the EDTA-Na of 4mM, 0.2% SDS, the Tris-HCl of 0.1M, pH 8.0), mixing slowly put upside down; Add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), behind the mixing 4 ℃, centrifugal 15 minutes of 12000rpm is transferred to supernatant liquor in the new sterilization centrifuge tube.Use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting more once is transferred to new centrifuge tube with supernatant, adds the LiCl of the 8M of 1/10 volume, and mixing was put 1 hour on ice.Then, 4 ℃, centrifugal 10 minutes of 12000rpm.Get supernatant, add 2.5 times of volume dehydrated alcohols, placed 30 minutes for-80 ℃ behind the mixing.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant liquor, and the precipitation piece is dissolved in 1 * TE damping fluid (Tris-HCl of 10mM (pH 8.0), the EDTA of 10mM) standby with 75% washing with alcohol 2 times after the drying at room temperature.

(b) be used for the primer of the ITS sequence of pcr amplification GXCR

Primer is pressed the bibliographical information design ^[70,71], primer is given birth to worker company by Shanghai and is synthesized.

Primer 1:5 '-TCCGTAGGTGAACCTGCGG-3 ' (SEQ ID NO:1)

Primer 2: 5 '-TCCTCCGCTTATTGATATGC-3 ' (SEQ ID NO:2)

(c) pcr amplification ^[70]

Reaction system:

10 * damping fluid (Takara company), 10 μ l

DNTPs (Takara company) 4 μ l

The dna profiling 20 μ g/2 μ l of GXCR thalline

Primer 1 (25pM) 4 μ l

Primer 2 (25pM) 4 μ l

DMSO (methyl-sulphoxide) 6 μ l

Taq-enzyme (5U/ μ l) (Takara company) 1 μ l

Sterilization deionized water (ddH ₂O) 69 μ l

Cumulative volume 100 μ l

PCR reaction conditions: 95 ℃ of fs, 2 minutes; 96 ℃ of subordinate phase (35 circulations), 30 seconds, 55 ℃, 1 minute, 72 ℃, 1 minute: 72 ℃ of phase IIIs, 10 minutes.

The PCR product reclaims:

PCR product 0.8% sepharose, electrophoresis is 1 hour under the 5v/cm condition, behind the ethidium bromide staining, cuts the target dna band at 600bp place under the long wavelength ultraviolet lamp, with the glue recovery test kit recovery DNA of China wink company.Standby with reclaiming in 1 * TE damping fluid that product is dissolved in 30 μ l.

(d) clone of PCR product:

Reagent adopts Shanghai to give birth to the pUCM-T Vector test kit of worker company, and by specification uses.After glue reclaims the recovery of PCR product, connect and be cloned into the pUCM-T carrier.

The ligation system is as follows:

10 * connection damping fluid, 1 μ l

PUCm-T carrier 1 μ l

The PCR product 1 μ l (10 μ g) that glue reclaims

Sterilization ddH ₂O 6 μ l

T4DNA ligase enzyme 1 μ l (1U)

Cumulative volume 10 μ l

Connect temperature condition: ligation system liquid is placed on 16 ℃, 24 hours.

The purifying that connects product:

To connect liquid and place 5 minutes stopped reactions at 65 ℃.Potassium ethanoate (KAc) and 2 times of volume dehydrated alcohols of adding 1/10 volume 5M.Place more than 30 minutes (or 20 ℃ spend the night) for-80 ℃.Centrifugal, remove supernatant, precipitation is with 75% washing with alcohol 4～5 times, and 12000pm is centrifugal, the careful ethanol liquid of drawing remnants.After the drying at room temperature, be dissolved in the ddH of 5 μ l ₂Among the O.

Transform:

Recombinant vectors DNA (PUCm-ITS dna fragmentation) aqueous solution that 5 μ l are connected, behind Escherichia coli JM109 competent cell (Takara company) mixing of 40 μ l, move in the electricimpulse cup of precooling, 200 Ω, 25 μ D, 12.5KV/cm electric shock, the electric shock back adds the SOC substratum (Invitrogen of 1ml precooling at once in the electricimpulse cup ^TMCompany), add a cover and shake up back sucking-off bacterium liquid, 37 ℃ of shaking tables were cultivated 1 hour, bacterium is coated on contains penbritin Amp (100 μ g/ml) and the surface is used on the LA culture medium flat plate of the IPTG (isopropyl-) (Shanghai give birth to worker company) of the 100mM of 20 μ l and the 20mg/ml X-gal of 100 μ l (worker company is given birth in Shanghai) coating in advance, 37 ℃ of cultivations 16 hours.Picking white transformant bacterium colony is transferred in containing penbritin Amp (100 μ g/ml), the LA culture medium flat plate on, cultivate after 16 hours 4 ℃ of preservations for 37 ℃.The plasmid of transformant extracts:

The transformant bacterium colony of picking white is seeded in the LB substratum of the 10ml that contains Amp, and 37 ℃ of shaking tables were cultivated 16 hours.Use quick alkaline lysis method of extracting plasmid then ^[72], cut (Promega company) with the PstI enzyme, 0.7% agarose gel electrophoresis is preserved the transformant thalline clone who is connected with the dna fragmentation about 600bp standby.Afterwards, the plasmid with quick these white transformants of alkaline lysis method of extracting is used for order-checking.Dna sequencing is entrusted upward, and sea base health bio-engineering corporation carries out.Sequence is referring to Fig. 1.

So obtained the ITS sequence (SEQ ID NO:3) of GXCR, dna sequencing is the result show, the ITS sequence total length 586bp (Fig. 1) of GXCR.Utilize the capable analysis software BlastN of nucleotide homology in Genbank (http://www.ncbi.nlm.nih.gov) database that the ITS sequence of GXCR is carried out homology comparative analysis discovery, the ITS sequence of the Penicillium spp. that the ITS sequence of GXCR and 102 strains have been reported has 〉=97% consistence, wherein (the Genbank call number: AY373921) homology is the highest: homology gets score value (Score)=1134 with the ITS sequence of Penicillium janthinellum ATCC 4845, E value (Expect)=0.0, consistence (Identities)=586/588 (99%), (Gaps)=2/588 (0%) at interval.

Result and combining form are identified in view of the above, and GXCR is accredited as penicillium janthinellum strain (Penicilliumjanthinellum), are numbered GXCR, and full name is Penicillium janthinellum GXCR.

3) growth temperature of penicillium janthinellum strain GXCR and pH scope

In the triangular flask of 500ml, add the PDL substratum of 200ml, afterwards, inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR, under differing temps, cultivated 7 days, filter the collection thalline with 210 mesh sieve thin,tough silk, the thalline of collecting places 65 ℃ of bakings to weigh after 4 hours, according to the dry cell weight (g/L) of this bacterium, the growth temperature range of this bacterium is 22-42 ℃ as can be known, and optimum growth temp is 32 ℃ (Fig. 2);

Under 32 ℃ of conditions of optimum growth temp, in the same way, measure dry cell weight, according to the dry cell weight (g/L) of this bacterium, the pH scope of the growth of this bacterium is 2.0-9.0 as can be known, optimum growh pH is 5.0 (Fig. 3).

4) the anti-copper of penicillium janthinellum strain GXCR bacterial strain and other reference strain relatively

For the ability of the high anti-mantoquita of verifying the GXCR bacterial strain, we have made following simultaneous test.

Example 1: with penicillium janthinellum strain GXCR thalline, be inoculated in the PDA of the Cu that contains 40mM respectively and do not add the PDA substratum of Cu, place 32 ℃ to cultivate 5 days available from 2 strain yeast Pichia pastoris, the P.methanolica of Invitrogen company and Saccharomyces cereivisae.Found that, have only penicillium janthinellum strain GXCR both can be simultaneously at the PDA of the Cu that contains 40mM with do not add on the PDA substratum of Cu and grow simultaneously.Three primary yeast bacterium can not grow on the PDA of the Cu that contains 40mM substratum, can only not add growth (table 1) on the PDA substratum of Cu.

Table 1 penicillium janthinellum strain GXCR and other saccharomycetic growth fraction are

+: growth;-: do not grow

Example 2: with penicillium janthinellum strain GXCR thalline with available from 3 strain pseudomonasputidas (Pseudomonas putida) 1.761, pseudomonasputida (P.putida) 1.781 and the pseudomonasputida (P.putida) 1.924 of Chinese common micro-organisms culture presevation administrative center CGMCC (Institute of Microorganism, Academia Sinica); Be inoculated in the PDA of the Cu that contains 40mM respectively and do not add the PDA substratum of Cu available from intestinal bacteria (Escherichia coli) DH10B of Invitrogen company with available from the e. coli jm109 of TakaRa company, place 32 ℃ to cultivate 5 days.Found that, have only penicillium janthinellum strain GXCR both can be simultaneously at the PDA of the Cu that contains 40mM with do not add on the PDA substratum of Cu and grow simultaneously.3 strain pseudomonas and 2 strain intestinal bacteria all can not grow on the PDA of the Cu that contains 40mM substratum, can only not add growth (table 2) on the PDA substratum of Cu.

The growth fraction of table 2 penicillium janthinellum strain GXCR and its bacterial isolates

+: growth :-: do not grow

Example 3: with do not inoculate in advance the Cu of any thalline and PDA, the 40mM Cu that contains 40mM that has inoculated penicillium janthinellum strain GXCR thalline LA, do not add the PDA of Cu and the LA culture medium flat plate (cultivate and all be contained in culture dish) of the Cu that do not add, do not cover the ware lid then, place a wet cotton balls in media surface, then, place outdoor (temperature is about: night 28-daytime 37 ℃) to place 10 days (keep cotton balls moistening between incubation period, it is moist to keep substratum).The result has only penicillium janthinellum strain GXCR thalli growth on the LA of the Cu of the PDA of the Cu of 40mM, 40mM substratum, do not occur and there is other assorted bacterium.At the LA culture medium flat plate of PDA that does not add Cu and the Cu that do not add, except that the bacterium colony of the penicillium janthinellum strain GXCR of inoculation in advance, still there is the bacterium colony of a large amount of assorted bacterium (table 3) to occur.These results have illustrated that to a certain extent GXCR is the anti-Cu bacterial strain of a plant height.

Table 3 is exposed to the yeast culture result on airborne PDA and the LA substratum

+: there is bacterium colony to form;-: no bacterium colony forms

5) penicillium janthinellum strain GXCR is to the resistance feature of metal-salt

A) pH and penicillium janthinellum strain GXCR's to single metal-salt resistance

In the triangular flask of 500ml, the PDL substratum of the different pH of the metal-containing salt of adding 200ml, afterwards, inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR was cultivated 7 days down for 32 ℃, filtered with 210 mesh sieve thin,tough silk and collected thalline, and the thalline of collection places 65 ℃ of bakings to weigh the dry cell weight of this bacterium (g/L) after 4 hours.(Cu of 40mM, the Cr of 2mM under the test salt concentration conditions ⁶⁺, the Pb of 5mM and the Zn of 40mM), this bacterium is to Cu, Cr ⁶⁺Descending with the pH increase with the resistance of Pb, is 3 o'clock at pH, and its resistance is the strongest.But, be 6 o'clock at pH, the strongest to the resistance of Zn, the variation of pH is to the resistance influence little (Fig. 4) of the Zn of this bacterium generally.

B) Cmin of the metal-salt of restriction penicillium janthinellum strain GXCR growth

Never contain on the PDA culture medium flat plate of Cu 32 ℃ of penicillium janthinellum strain GXCR colony edges of cultivating 5 days with the punch tool (diameter 0.2cm) of sterilization and buy the bacterium cake, the bacterium cake is inoculated on the PDA substratum (natural pH) that contains the different concns metal-salt, cultivated 7 days for 32 ℃, simultaneously make parallel control, observe the upgrowth situation of bacterium colony with the PDA culture that does not add metal-salt; In the triangular flask of 500ml, add the PDL substratum that contains different metal salt (natural pH) of 200ml, afterwards, inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR, 32 ℃, the 150rpm shaking table was cultivated 7 days, made parallel control with the PDL culture that does not add metal-salt simultaneously.Whether there is mycelium to form the concentration of each metal-salt of judgement restriction penicillium janthinellum strain GXCR growth in tangible colony growth and the PDL substratum according to whether having on the PDA substratum.The results are shown in Table 4.

The Cmin of the metal-salt of table 4 restriction penicillium janthinellum strain GXCR growth

Metal-salt	In solid medium PDA (mM) *	Among the liquid medium within PDL (mM) *
			??Al	??＞500	?＞500
??Cd	??50	?10
			??Cr 3+	??＞60	?＞60
??Cr 6+	??＞3	?3
			??Cu	??200	?200
??Mn	??＞200	?＞200
			??Na	??＞3	?＞3
??Ni	??20	?10
			??Pb	??50	?10
??Zn	??＞1200	?＞1200

^*Repeat 3 times

C) penicillium janthinellum strain GXCR is to the resistance of three kinds of metal salt mixture

In the triangular flask of 500ml, what add 200ml contains different concns, the PDL substratum of metal-salt of the same race (natural pH) not, afterwards, and inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR, 32 ℃, the 150rpm shaking table was cultivated 5 days, made parallel control with the PDL culture that does not add metal-salt simultaneously.Filter the collection thalline with 210 mesh sieve thin,tough silk, the thalline of collection places 65 ℃ of bakings to weigh the dry cell weight of this bacterium (g/L) after 4 hours.According to orthogonal experiment (L ₉(4 ³)) result, under salt concn (Cu of 20mM and 40mM, the Cd of 1mM and 3mM, the Pb of 1mM and 3mM, the Zn of 100mM and the 200mM) condition of design (table 5), table 6 is the result show, the size order of the F value between each is handled is: A _F＞C _F＞B _F＞D _F, its A _F, C _FAnd B _FValue is all greater than F _{(0.01 2,10)}=7.56, reach utmost point significant difference, but handle the value D of D _FLess than F _{(0.05 2,10)}=4.10, do not reach salient pole work level of difference.Illustrate that the single metal-salt under the design's concentration is Cu＞Cd＞Zn＞Pb (table 6) to the order of the growth effect of this bacterium.

Dunca ' s LSR (table 7) and variance analysis (table 8) result show: this bacterium is the Zn of the Pb-200mM of the Cd-3mM that contains 3mM (handling 3), Zn (the handling 9) mixing salt of the Cd-200mM of the Zn (handling 8) of the Pb-100mM of the Cu-3mM of the Zn of the Pb-200mM of the Cu-1mM of 20mM (handling 6), 40mM or the Cu-1mM of 40mM can grow in the PDL substratum.The Pb of the Cd-3mM of the Cu-1mM that contains 20mM (handling 4) mixing salt in the PDL substratum growth phase to relatively poor.But can not Pb (the handling 7) mixing salt of the Cd-1mM of the Cu-3mM of the Zn of the Cd-100mM of the Cu-3mM that contains 20mM (handling 5) or 40mM grow in the PDL substratum.

Three kinds of metal-salt combinations of table 5. are to the orthogonal experiment (L of penicillium janthinellum strain GXCR growth effect ₉(4 ³))

Handle	??A(mM?Cu 2+)	??C(mM?Cd 2+)	??D(mM?Pb 2+)	??B(mM?Zn 2+)	??X(DM，g)
						??1	??1(0)	??1(0)	??1(0)	??1(0)	??X11＝0.142 ??X12＝0.136
??2	??1(0)	??2(1)	??2(1)	??2(100)	??X21＝0.102 ??X22＝0.0999
						??3	??1(0)	??3(3)	??3(3)	??3(200)	??X31＝0.095 ??X32＝0.082
??4	??2(20)	??2(1)	??3(3)	??1(0)	??X41＝0.011 ??X42＝0.013
						??5	??2(20)	??3(3)	??1(0)	??2(100)	??X51＝0 ??X52＝0
??6	??2(20)	??1(0)	??2(1)	??3(200)	??X61＝0.091 ??X62＝0.076
						??7	??3(40)	??3(3)	??2(1)	??1(0)	??X71＝0 ??X72＝0
??8	??3(40)	??1(0)	??3(3)	??2(100)	??X81＝0.084 ??X82＝0.072
						??9	??3(40)	??2(1)	??1(0)	??3(200)	??X91＝0.063 ??X92＝0.054

DM: dry mycelium

Variance analysis between table 6. factor (Cu, Cd, Pb and Zn)

The variation source	From mean square and SS	Degree of freedom df	All square MS	Variance F *
					??Repeat ??A(Cu) ??B(Zn) ??C(Cd) ??D(Pb) ??Error?1 ??Error?2 ??Total?error	??0.0003 ??0.02 ??0.002 ??0.015 ??0.0003 ??0.0002 ??0.0002 ??0.0004	??1 ??2 ??2 ??2 ??2 ??2 ??8	??0.01 ??0.001 ??0.0075 ??0.00015 ??0.0001	??100 ※※????10 ※※????75 ※※????1.5
	??10

^*F _0.05(2，10)＝4.10；F _0.01(2，10)＝7.56

^*Utmost point significant difference

LSR value in table 7.Dunca ' the s multiple range test

Rank is apart from (K)	5% minimum significantly extreme difference LSR 0.05	1% minimum significantly extreme difference LSR 0.01
			??2 ??3 ??4 ??5 ??6 ??7 ??8 ??9	??0.0227 ??0.0237 ??0.0242 ??0.0246 ??0.0249 ??0.0250 ??0.0250 ??0.0250	??0.0327 ??0.0345 ??0.0354 ??0.0361 ??0.0367 ??0.0373 ??0.0378 ??0.0381

The significance of the penicillium janthinellum strain GXCR dry cell weight between the different metal salt mixture of table 8. is handled relatively

Handle	Average DM (g)	Mean number Xi-0	??Xi-0	??Xi-0.011	??Xi-0.059	??Xi-0.078	??Xi-0.084	??Xi-0.089	??Xi-0.101
										??1 ??2 ??3 ??6 ??8 ??9 ??4 ??5 ??7	??0.142 ??0.101 ??0.089 ??0.084 ??0.078 ??0.059 ??0.011 ??0 ??0	??0142 ※※????0.101 ※※????0.089 ※※????0.084 ※※????0.078 ※※????0.059 ※※????0.011 ??0	??0.142 ※※??0.101 ※※????0.089 ※※????0.084 ※※????0.078 ※※????0.059 ※※????0.011	??0.131 ※※????0.09 ※※????0.078 ※※????0.073 ※※????0.067 ※※????0.048 ※※	??0.083 ※※????0.042 ※※????0.03 ※????0.025 ※????0.019	??0.064 ※※????0.023 ??0.011 ??0.006	??0.058 ※※????0.017 ??0.005	??0.053 ※※????0.012	??0.041 ※※

^{※ ※}Greater than LSR _0.01Value reaches utmost point significant difference

D) influence of the growth of the interaction partners penicillium janthinellum strain GXCR between the metal-salt

I) existence of Mn can significantly improve the resistance of GXCR to Cu

Never contain on the PDA culture medium flat plate of Cu 32 ℃ of penicillium janthinellum strain GXCR colony edges of cultivating 5 days with the punch tool (diameter 0.2cm) of sterilization and buy the bacterium cake, the bacterium cake is inoculated in the Mn that contains different concns (40mM, 200mM, 400mM and 800mM) Cu, (40mM, 200mM, 400mM and 800mM) Cu-5mM and the Mn-of (40mM, 200mM, 400mM and 800mM) Cu-5mM adds organotrophy (1% Tryptones; 0.5% yeast extract) on the PDA substratum (natural pH), cultivated 7 days for 32 ℃, observe the upgrowth situation of bacterium colony.The result shows, penicillium janthinellum strain GXCR can growth on the PDA solid medium of the Mn of the Cu-5mM that contains 800mM (Fig. 5, B); Add organotrophy (1% Tryptones at the Mn-that contains (40mM, 200mM, 400mM and 800mM) Cu-5mM; 0.5% yeast extract) bacterium colony on though the PDA substratum less but significantly fine and close (Fig. 5, C).Show that Mn can effectively improve the resistance of penicillium janthinellum strain GXCR to Cu.

When ii) Zn or Al and Cu existed simultaneously, the growth to penicillium janthinellum strain GXCR did not produce the nontoxicity synergistic effect

Never contain on the PDA culture medium flat plate of Cu 32 ℃ of penicillium janthinellum strain GXCR colony edges of cultivating 5 days with the punch tool (diameter 0.2cm) of sterilization and buy the bacterium cake, the bacterium cake is inoculated in the Zn/Al that contains different concns (40mM, 200mM, 400mM and 800mM) Cu, (40mM, 200mM, 400mM and 800mM) Cu-5mM and the Zn/Al-of (40mM, 200mM, 400mM and 800mM) Cu-5mM adds organotrophy (1% Tryptones; 0.5% yeast extract) on the PDA substratum (natural pH), cultivated 7 days for 32 ℃, observe the upgrowth situation of bacterium colony.Fig. 6 result shows, with (the Fig. 6 that compares at the last Penicillium janthinellum GXCR bacterium colony of the PDA that only contains (40mM, 200mM, 400mM and 800mM) Cu, A), bacterium colony size on the PDA substratum of the Zn that contains (40mM, 200mM, 400mM and 800mM) Cu-5mM does not have obvious variation (Fig. 6, B), show when Zn and Cu exist simultaneously, not to the growth toxigenicity synergistic effect of penicillium janthinellum strain GXCR.Compare with the bacterium colony on the PDA substratum of the Zn that contains (40mM, 200mM, 400mM and 800mM) Cu-5mM (Fig. 6, B), at the Zn-of (40mM, 200mM, 400mM and 800mM) Cu-5mM interpolation organotrophy (1% Tryptones; 0.5% yeast extract) bacterium colony on the PDA substratum does not have obvious variation, but obviously densification of bacterium colony (Fig. 6, C).

With (the Fig. 6 that compares at the last penicillium janthinellum strain GXCR bacterium colony of the PDA that only contains (40mM, 200mM, 400mM and 800mM) Cu, A), bacterium colony size on the PDA substratum of the Al that contains (40mM, 200mM, 400mM and 800mM) Cu-5mM does not have obvious variation (Fig. 6, D), show when Al and Cu exist simultaneously, not to the growth toxigenicity synergistic effect of penicillium janthinellum strain GXCR.Compare with the bacterium colony on the PDA substratum of the Al that contains (40mM, 200mM, 400mM and 800mM) Cu-5mM (Fig. 6, D), at the Al-of (40mM, 200mM, 400mM and 800mM) Cu-5mM interpolation organotrophy (1% Tryptones; The significantly densification of bacterium colony on the PDA substratum 0.5% yeast extract) (Fig. 6, E).

When iii) Cd and Cu exist simultaneously, the growth of penicillium janthinellum strain GXCR is produced significant toxicity synergistic effect

Never contain on the PDA culture medium flat plate of Cu 32 ℃ of penicillium janthinellum strain GXCR colony edges of cultivating 5 days with the punch tool (diameter 0.2cm) of sterilization and buy the bacterium cake, the bacterium cake is inoculated in the Cd that contains different concns (40mM, 200mM, 400mM and 800mM) Cu, (40mM, 200mM, 400mM and 800mM) Cu-2mM and the Cd-of (40mM, 200mM, 400mM and 800mM) Cu-2mM adds organotrophy (1% Tryptones; 0.5% yeast extract) on the PDA substratum (natural pH), cultivated 7 days for 32 ℃, observe the upgrowth situation of bacterium colony.With (the Fig. 7 that compares at the last penicillium janthinellum strain GXCR bacterium colony of the PDA that only contains (40mM, 200mM, 400mM and 800mM) Cu, A), bacterium colony on the PDA substratum of the Cd that contains (40mM, 200mM, 400mM and 800mM) Cu-2mM obviously diminishes, (the Fig. 7 that almost can not grow of thalline, B), show when Cd and Cu exist simultaneously, to the growth generation intensive toxicity synergistic effect of penicillium janthinellum strain GXCR.Compare with the bacterium colony on the PDA substratum of the Cd of the Cu-2mM that contains 200mM that (Fig. 7 B), adds organotrophy (1% Tryptones at the Cd-of the Cu-2mM of 200mM; 0.5% yeast extract) bacterium colony on the PDA substratum obviously big and fine and close (Fig. 7, C).

The above results is also explanation simultaneously, and organotrophy can effectively improve the resistance of penicillium janthinellum strain GXCR to metal-salt.

6) penicillium janthinellum strain GXCR is to enrichment and the Cu of Cu ²⁺Separating out of compound

Never contain on the PDA culture medium flat plate of Cu 32 ℃ of penicillium janthinellum strain GXCR colony edges of cultivating 5 days with the punch tool (diameter 0.2cm) of sterilization and buy the bacterium cake, the bacterium cake is inoculated on the PDA substratum of the Cu that contains 40mM, 32 ℃, when cultivating 20 days, the bacterium colony surface of penicillium janthinellum strain GXCR appears at crystallisate (Fig. 8 C1 of the blueness that presents metalluster under the natural light, C2, D), and do not have on the contrast bacterium colony surface of not adding the penicillium janthinellum strain GXCR on the PDA substratum of Cu this phenomenon (Fig. 8 A, B).Cut at the PDA of the Cu that contains 40mM and do not contain on the PDA substratum of Cu, 32 ℃, when cultivating 20d, (size is 0.1 * 0.1 * 0.1cm) bacterium piece (together with the PDA substratum under the bacterium colony) to 3 of the penicillium janthinellum strain GXCR that cultivates, behind the metal spraying of surface, with FDAC S-570 type scanning electron microscope (SEM) scanning.Electron-microscope scanning is the result show: the penicillium janthinellum strain GXCR mycelium surface on the PDA of the Cu that contains 40mM has and has a large amount of xln and filling space (Fig. 9 B1-4) between the mycelium, but has (Fig. 9 A1-2) not adding the no xln in penicillium janthinellum strain GXCR thalline surface of growing on the PDA of Cu; The result who analyzes with Japanese Philips PV9900 type energy depressive spectroscopy (EDAX) shows: do not add the PDA of Cu and do not have Cu at the living bacterium colony of the gas that does not add the penicillium janthinellum strain GXCR on the PDA of Cu ²⁺The peak (Figure 10 A1, A2), the Cu of the front of the bacterium piece of the penicillium janthinellum strain GXCR on the PDA of the Cu that contains 40mM (face that the bacterium piece of aerial hyphae is promptly arranged) ²⁺Can spectrum peak very high (Figure 10 B1), on the contrary, detect Cu corresponding to the reverse side (being the face of the no mycelia of bacterium piece) of same bacterium piece ²⁺Can spectrum peak very low (Figure 10 B2).This result shows, the Cu that adds in the substratum by the enrichment of penicillium janthinellum strain GXCR mycelium, absorb justacrine (separating out) to the somatic cells surface.Thereby, caused adding that Cu content reduces in the PDA substratum of Cu.

7) penicillium janthinellum strain GXCR is to absorption of the biology of Cu and the accumulation of Cu in its cell

Annotate: wherein the mensuration of Cu ionic concn adopts atomic absorption method ^[73]

I) absorption of the biology under the long-time culture condition of successive and accumulation

In the triangular flask of 500ml, add the PDL substratum that contains different concns Cu (natural pH) of 200ml, afterwards, inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR, 28 ℃, the 150rpm shaking table was cultivated 5 days, filtered the collection thalline with 210 mesh sieve thin,tough silk and encased mycelium with filter paper more afterwards, was squeezed to no raffinate repeatedly.Thalline is changed in the triangular flask, the Tris-HCl of the 50mM of adding 100ml and EDTA (pH 7.0) mixed solution of 20mM, magnetic stirring apparatus is fully broken up thalline, again triangular flask is placed on 28 ℃ of shaking tables, 200rpm jolts 10 minutes, filter the collection thalline and collect residual elutriant with 210 mesh sieve thin,tough silk, encase mycelium with masking foil again, push repeatedly and collect after the remaining elutriant, centrifugal 5 minutes of 5000rpm, collect thalline respectively and merge all elutriants, repeat the wash-out thalline in the same way 4 times, merge the elutriant of collecting at last and be settled to 100ml with the elutriant that merges.The thalline of collecting behind the wash-out places 65 ℃ of bakings 4 hours.The Cu (biological intravital accumulation) that measures respectively in Cu in the filtrate (biological absorption) and the somatic cells with atomic absorption method measures.Measurement result shows that the thalline surface is a concentration dependent form to the biology absorption of Cu and the accumulation of intracellular Cu.Under the Cu of 20-40mM concentration, the mycelia surface is respectively 26.0mg Cu/g dry mycelium and 26.19mg Cu/g dry mycelium to the biological adsorptive capacity of Cu.But when the Cu of 60mM concentration, it is maximum that the accumulation of intracellular Cu reaches, but only be 0.176mg Cu/g dry mycelium.Therefore, compare the accumulation of intracellular Cu almost can ignore (Figure 11) with the biological adsorptive capacity of maximum Cu.

Ii) in containing the deionized water of Cu, alkali pre-soaking thalline that handle and not pre-treatment (state of nature) is to the quick bio absorption of Cu

In the triangular flask of 500ml, add the PDL substratum of 200ml, afterwards, inoculation 10 ⁵The conidium of individual penicillium janthinellum strain GXCR, 28 ℃, the 150rpm shaking table was cultivated 5 days, filtered the collection thalline with 210 mesh sieve thin,tough silk and encased mycelium with filter paper more afterwards, was squeezed to no raffinate repeatedly, centrifugal 5 minutes of 5000rpm.Divide two parts with the thalline after centrifugal: portion changes in the NaOH solution of 0.2M of 200ml, magnetic stirring apparatus is fully broken up thalline, left standstill 30 minutes, filter the collection thalline with 210 mesh sieve thin,tough silk and encase mycelium with filter paper more afterwards, be squeezed to no remaining NaOH liquid repeatedly, get then in the deionized water of the Cu that contains different concns that wherein 1.5g thalline changes 200ml over to, magnetic stirring apparatus is fully broken up thalline, 28 ℃, the 150rpm shaking table was cultivated after 30 minutes, filter the collection thalline and encase mycelium with 210 mesh sieve thin,tough silk with masking foil, be squeezed to no residual solution repeatedly, then thalline is changed in the triangular flask, the Tris-HCl of the 50mM of adding 100ml and the EDTA (pH7.0) of 20mM, magnetic stirring apparatus is fully broken up thalline, 200rpm jolts 10 minutes, filter the collection thalline with 210 mesh sieve thin,tough silk and encase mycelium with filter paper more afterwards, be squeezed to no residual solution repeatedly, collect thalline respectively and merge all elutriants, repeat the wash-out thalline in the same way 4 times with the elutriant that merges, merge the elutriant of collecting at last and be settled to 100ml.Meanwhile, use the same method processing one strain as the Penicillium citrinum Penicillium citrinum 3.4039 on the reference citrus (Chinese common micro-organisms culture presevation administrative center CGMCC, Institute of Microorganism, Academia Sinica).Measure Cu content (representing biological adsorptive capacity) in the filtrate respectively with atomic absorption method.Measurement result shows, no matter be the 0.2MNaOH pre-soaking handle 30 minutes penicillium janthinellum strain GXCR and 0.2M NaOH pre-soaking handle 30 minutes with reference to the Penicillium citrinum thalline or the penicillium janthinellum strain GXCR of (state of nature) and the thalline of the penicillium janthinellum strain of the reference of pre-soaking processing (state of nature) are not handled in pre-soaking, in the Cu concentration range of the 0-20mM that tests, its biological adsorptive power all along with Cu concentration increase and increase, and the biology absorption of the Cu of the penicillium janthinellum strain GXCR thalline handled of the NaOH pre-soaking of 0.2mM approximately is the penicillium janthinellum strain GXCR thalline handled without the NaOH pre-soaking of 0.2mM 3 times (Figure 12).

Penicillium janthinellum strain GXCR thalline and when the Cu of 20mM concentration with reference to the thalline of Penicillium citrinum, biological adsorptive capacity maximum, 0.2mM the penicillium janthinellum strain GXCR thalline handled 30 minutes of NaOH pre-soaking can reach 76.9mg Cu/g dry mycelium to the biology absorption of Cu, the penicillium janthinellum strain GXCR thalline of handling with the NaOH pre-soaking of 0.2M then is not a 38.1mg Cu/g dry mycelium (Figure 12); 0.2M the NaOH pre-soaking biological adsorptive capacity of Penicillium citrinum thalline of handling 30 minutes reference be 8.98mg Cu/g dry mycelium, the biological adsorptive capacity of Penicillium citrinum thalline of the reference of handling with the NaOH pre-soaking of 0.2M only is a 7.67mg Cu/g dry mycelium.The biological adsorptive capacity of the thalline that the NaOH pre-soaking of the 0.2M of penicillium janthinellum strain GXCR is handled is 8.65 times of biological adsorptive capacity of the thalline of the reference strain Penicillium citrinum handled of the NaOH pre-soaking of 0.2M.The biological adsorptive capacity of the thalline of the penicillium janthinellum strain GXCR that handles without the NaOH pre-soaking of 0.2M is 4.9 times (Figure 12) of the biological adsorptive capacity of Penicillium citrinum thalline of the reference handled without the NaOH pre-soaking of 0.2M.

From the trend analysis of the graphic representation of Figure 12, along with the further raising of Cu concentration, the adsorptive capacity of Cu has the possibility of further raising.

Reference

1.Valentine?S?J?and?Gralla?E?B(1997)Enhanced：Delivering?Copper?InsideYeast?and?Human?Science.278：817-818.

2.Ennifar?E，Walter?P，Dumas?P(2003)A?crystallographic?study?of?the?bindingof?13?metal?ions?to?two?related?RNA?duplexes.Nucleic?Acids?Res.31(10)：2671-2682.

3.OHalloran?T?V?and?Culotta?V?C(2000)Metallochaperones，anIntracellular?Shuttle?Service?for?Metal?Ions.J?Biol?Chem.275(33)：25057-25060.

4.Culotta?V?C，Joh?H?D，Lin?S?J，et?al.(1995)A?physiological?role?forSaccharomyces?cerevisiae?copper/zinc?superoxide?dismutase?in?copperbuffering.J?Biol?Chem.270：29991-29997.

5.Fogarty?R?V，Tobin?J?M(1996)Fungal?melanins?and?their?interactions?withmetals.Enzyme?Microb?Technol.19(4)：311-317.

6.Radisky?D，and?Kaplan?J(1999)Regulation?of?transition?metal?transportacross?the?yeast?plasma?membrane..J?Biol?Chem.274：4481-4484.

7.Pe a?M?M?O，Lee?J，and?Thiele?D?J(1999)A?Delicate?Balance：Homeostatic?Control?of?Copper?Uptake?and?Distribution.J?Nutr.129：1251-1260.

8.Beaudoin?J?and?Labbé?S(2001)The?Fission?Yeast?Copper-sensingTranscription?Factor?Cufl?Regulates?the?Copper?Transporter?Gene?Expressionthrough?an?Acel/Amtl-like?Recognition?Sequenc J?Biol?Chem.276(18)：15472-15480.

9.Himelblau?E，Mira?H，Lin?S?J，et?al.(1998)，Identification?of?a?FunctionalHomolog?of?the?Yeast?Copper?Homeostasis?Gene?ATX1 from?Arabidopsis，Plant?Physiol.117(4)：1227-1234.

10.De?Freitas?J，Wintz?H，Kim?JH，et?al.(2003)Yeast，a?model?organism?for?ironand?copper?metabolism?studies.Biometals.16(1)：185-197.

11.Radisky?D?and?Kaplan?J(1999)Regulation?of?Transition?Metal?Transportacross?the?Yeast?Plasma?Membrane.J?Biol?Chem，8：4481-4484.

12.Glerum?D?M，Shtanko?A?and?Tzagoloff?A(1996)Characterization?ofCOX17，a?Yeast?Gene?Involved?in?Copper?Metabolism?and?Assembly?ofCytochrome?Oxidase.J?Biol?Chem?Jun.271(24)：14504-14509.

13.Culotta?V?C，Lin?S?J，Schmidt?P，et?al.(1999)Interacellular?pathways?ofcopper?trafficking?in?yeast?and?humans.Adv.Exp?Med?Biol.448：247-254.

14.Vulpe?C?D?and?Packman?S.(1995)Cellular?copper?transport.Annu?RevNutr.15：293-322.

15. Cong Yan state, the As-Is analysis in Wei Lihua (2002) edatope heavy metal contamination source, modern agriculture, 1:18-20.

16. the Xia Li river, magnificent Lopa Nationality, Li Xiangdong (1998) heavy metal contamination biological restoration mechanism and progress, nuclear agricultural science newspaper, 12 (1): 59-64.

17. Chen Su China, Sun Tie, Zhou Qixing, interaction and applied research thereof between Wu Guoping (2002) microorganism and the heavy metal, Chinese Journal of Applied Ecology, 13 (2): 239-24219.

18.Koch?K，Pena?M，Thiele?D?J(1997)Copper-binding?motifs?in?catalysisi，transport，detoxification?and?signaling.Chem?Biol.4：549-560.

19.Eide?D?J(1998)The?molecular?biology?of?metal?ion?transport?inSaccharomyces?cerevisiae.Annu?Rev?Nutr.Annu?Rev?Nutr.18：441-469.

20.LabbéS?and?Thiele?D?J(1999)Pipes?and?wiring：the?regulation?of?copperuptake?and?distribution?in?yeast.Trends?Microbiol.7：500-505.

21.Bull?P?C，and?Cox?D?W(1994)Wilson?disease?and?Menkes?disease：newhandles?on?heavy?metal?transport.Trends?Genet.Sci.10：248-252.

22.Tamai?K.T，Gralla?E?B，et?al.(1993)Yeast?and?mammalianmetallothioneins?functionally?substitute?for?yeast?copper-zinc?superoxidedismutase.Proc?Natl?Acad?Sci?USA.90：8013-8017.

23.Bayle?D，Wangler?S，Weitzenegger?T，Steinhilber?W，Volz?J，PrzybylskiM，Schafer?K?P，Sachs?G?&?Melchers?K(1998)Properties?of?the?P-typeATPases?encoded?by?the?copAP?operons?of?Helicobacter?pylori?andHelicobacter?felis.J?Bacteriol.180：317-329.

24.Odermatt?A，Suter?H，Krapf?R，Solioz?M(992)Pase?operon?involved?incopper?resistance?by?Enterococcus?hirae.Ann?N?Y?Acad?Sci.30；671：484-486.

25.Ge?Z.&?Taylor?D?E(1996)Helicobacter?pylori?genes?hpcopA?and?hpcopPconstitute?a?cop?operon?involved?in?copper?export.FEMS?Microbiol.Lett.145：81-188.

26.Weissman?Z，Berdicevsky?I，Cav?ari?B?Z，Kornitzer?D(2000)The?highcopper?tolerance?of?Candida?albicans?is?mediated?by?a?P-type?ATPase.Pro?NatlAcad.Sci?USA，97(7)：3520-3525.

27. Zhou Dongmei, Wang Yujun, Hao Xiuzhen etc. (2002) copper mine district heavy metal contamination differentiation rule preliminary study, agriculture environmental protection, 21 (23): 225-227.

28.Wang?L，Chua?H，Wong?PK，et?al.(2003)Ni2+removal?and?recovery?fromelectroplating?effluent?by?Pseudomonas?putida?5-x?cell?biomass.J?Environ?SciHealth?Part?A?Tox?Hazard?Subst?Environ?Eng.38(3)：521-531.

29.Cantafio?A?W，Hagen?K?D，Lewis?G?E，et?al.(1995)Pilot-scale?seleniumbioremediation?of?San?Joaquin?drainage?water?with?Thauera?selenatis，Appliedand?Environmental?Microbiology，62：3298-3303.

30.Woitke?P，Wellmitz?J，Helm?D，et?al.(2003)Analysis?and?assessment?ofheavy?metal?pollution?in?suspended?solids?and?sediments?of?the?riverDanube.Chemosphere.51(8)：633-642

31.Chiang?KY，Yoi?SD，Lin?HN，et?al.(2001)Stabilization?of?heavy?metalsin?sewage?sludge?composting?process.Water?Sci?Techno.44(10)：95-100.

32.Linnik R P and Zaporozhets O A. (2003) Solid-phase reagent for molecularspectroscopic deter minute ation of heavy metal speciation in natural water.AnalBioanal Chem.375 (8): 1083-1088.

33.Iqbal?M?and?Saeed?A(2003)Removal?of?heavy?metals?from?contaminatedwater?by?petiolar?felt-sheath?of?palm.Environ?Technol.23(10)：1091-1098.

34.Ibeanusi?V?M，Phinney?D，Thompson?M(2003)Removal?and?recovery?ofmetals?from?a?coal?pile?runoff.Environ?Monit?Assess.84(1-2)：35-44.

35.Cape?JN，Fowler?D，Davison?A(2003)Ecological?effects?of?sulfurdioxide，fluorides，and?minor?air?pollutants：recent?trends?and?researchneeds.Environ?Int?29(2-3)：201-211.

36.Samecka-Cymerman?A，Kempers?AJ(1996)Bioaccumulation?of?heavymetals?by?aquatic?macrophytes?around?Wroclaw，Poland.Ecotoxicol?EnvironSaf.35(3)：242-247.

37. Wang Qing people, Liu Xiumei, heavy metal contamination situation and the feature of Dong Yiting etc. (2002) typical industry district and dirty irrigated area plant, agriculture environmental protection, 21 (2): 115-118.

38.Sauge-Merle?S，Cuine?S，Carrier?P，et?al.(2003)Enhanced?toxic?metalaccumulation?in?engineered?bacterial?cells?expressing?Arabidopsis?thalianaphytochelatin?synthase，Appl?Environ?Microbiol，69(1)：490-494.

39.Cunningham?S?D，Berti?W?R，Hugang?J?W?W(1995)Phytoremediation?ofcontaminated?soils[J]Trends?Biotechnol，13：393-403.

40.Pufahl?R?A，Singer?C?P，Peariso?K?L，et?al.(1997)Metal?Ion?ChaperoneFunction?ofthe?Soluble?Cu(I)Receptor?Atxl.Science.278(5339)：853-556.

41.Price?D?L，Wong?P?C，Markowska?A?L，et?al.(2000)TheValue?of?Transgenic?Models?for?the?Study?of?Neurodegenerative?Diseases.Annals?NYAS?Online?920：179-191.

42.Halliwell，B.&?Gutteridge，J.M.C.(1984)Oxygen?toxicity，oxygenradicals，transition?metals?and?disease.Biochem?J.219：1-14.

43.Hamza?I?and?Gitlin?J?D(2002)Copper?chaperones?for?cytochrome?coxidase?and?human?disease.J?Bioenerg?Biomembr.34(5)：381-388.

44.Jezowska-Bojczuk?M，Szczepanik?W，Lesniak?W，et?al.(2002)DNA?andRNA?damage?by?Cu(II)-amikacin?complex.Eur?J?Biochem.269(22)：5547-56.

45. the bright spring of cogongrass, Xue Yanning, the progress of Wang Huixin (2001) microorganism display technique in the heavy metal contamination biological restoration, biotechnology progress, 21 (5): 48-51.

46.Wang?S-F，et?al.(1993)Action?in?tensity?of?nitrogen-fixing?and?denitrifyingbacteria?in?Chernozem?polluted?by?heavy?metals，Chin?J?ApplEcol，2(2)：174-177(in?Chinese).

47.Hiroki?M(1992)Effects?of?heavy?metal?contamination?on?soil?microbialpopulation，Soil?Sci?Plant，38：141-147.

48. Chen Su China, Sun Tie, Zhou Qixing, interaction and applied research thereof between Wu Guoping (2002) microorganism and the heavy metal, Chinese Journal of Applied Ecology 13 (2): 239-242.

49.Chaney?R?L，Malik?M，Li?Y?M，et?al.(1997)Phytoremediation?of?soilmetals，Current?Opinion?in?Biotechnology，8：279-284.

50. Song Fei, Guo Yuwen, the research of the plumbous combined pollution of Liu Xiaoyi etc. (1996) heavy metal in soil cadmium zinc, ACTA Scientiae Circumstantiae, 16 (4): 431-436.

51.Leung?W?C，Chua?H，Lo?W(2001)Biosorption?of?heavy?metals?bv?bacteriaisolated?from?activated?sludge.Appl?Biochem?Biotechnol.91-93：171-184.

52.Walter?I，Martinez?F，Alonso?L，et?al.(2002)Extractable?soil?heavy?metalsfollowing?the?cessation?of?biosolids?application?to?agricultural?soil.EnvironPollut.117(2)：315-321.

53.Baumann?T，Muller?S，Niessner?R(2002)Migration?of?dissolved?heavymetal?compounds?and?PCP?in?the?presence?of?colloids?through?a?heterogeneouscalcareous?gravel?and?a?homogeneous?quartz?sand-pilot?scaleexperiments.Water?Res.36(5)：1213-1223.

54.Kaewsam?P(2002)Biosorption?of?copper(II)from?aqueous?solutions?bypre-treated??biomass?of?marine?algae?Padina?sp.Chemosphere.47(10)：1081-1085.

55.Ritchie?SM，Bhattacharyya?D(2002)Membrane-based?hybrid?processes?forhigh?water?recovery?and?selective?inorganic?pollutant?separation.JHazard.92(1)：21-32.

56.Cotter-Howells J D, conta minute ated land of Caporn S (1996) Remediation of byformation of heavy metal phosphates, Applied Geochemistry, 11:335-342.

57, Jiao Fangchan, hair snow, Li Runzhi (2002) metal binding protein gene and the application in removing heavy metal contamination thereof, heredity, 24 (1): 82-86.

58. Li Haihua, Liu Jianwu, the Japanese plum people waits heavy metal contamination and crop enrichment progress in (2000) soil one botanical system, Agricultural University Of He'nan's journal, 34 (1): 30-34.

59.Nuhoglu?Y，Malkoc?E，Gurses?A，et?al.(2002)The?removal?of?Cu(II)fromaqueous?solutions?by?Ulothrix?zonata.Bioresour?Technol.85(3)：331-333.

60. Chen Yong gives birth to, Sun Qijun, Chen Jun etc. (1997) Biosorption of Heavy Metals technical study, environmental science progress, 5 (6): 34-43.

61.Rao?D?V，Shivannavar?C?T，Gaddad?S?M(2002)Bioleaching?of?copperfrom?chalcopyrite?ore?by?fungi.Indian?J?Exp?Biol.40(3)：319-324.

62.Kuroda?K，Ueda?M，Shibasaki?S，et?al.(2002)Cell?surface-engineered?yeastwith?ability?to?bind，and?self-aggregate?in?response?to，copper?ion.ApplMicrobiol?Biotechnol.59(2-3)：259-264.

63. Xu Ya is together, the historian, and Zhang Ming (2001) pollutes controlling microbial engineering, Chemical Industry Press, Beijing.

64.Hicks，J.B.，A.Hinen，and?G.R.Fink.1979.Propenies?of?yeasttransfomation.Cold?spring?harbor?Symp.Quant.Biol.43：1305-1313.

65.Kozlovskii?AG，Zhelifonova?VP，Vinokturova?NG，Ozerskaia?SM(2000)Effect?on?microelements?on?biosynthes?of?secondary?metabolites?in?the?fungusPenicillium?citrinum?Thom?VKM?F-1079.[Article?in?Russian]Mikrobiologiia.69(5)：642-646.

66.Adamcova?J，Proksa?B，Fuska?J(1992)Regulation?of?biosynthesis?ofvermiculin?and?vermistatin?in?Penicillium?vermiculatum.Folia?Microbiol(Praha).37(1)：50-52.

67.Zamani?B，Knezek?BD，Flegler?SL，Beneke?ES，Dazzo?FB(1985).Autoradiographic?method?to?screen?for?soil?microorganisms?which?accumulatezinc.Appl?Environ?Microbiol.49(1)：137-142.

68.Zhang?D，Duine?JA，Kawai?F(2002)The?extremely?high?Al?resistance?ofPenicillium?janthineleum?F-13?is?not?caused?by?internal?or?externalsequestration?of?Al.Biometals.15(2)：167-174.

69. the power of encouraging is flat, classification of fungi is learned, China Forest press, 1984.

70. military ripple, the screening of the Aspergillus strain of (2001) one strain produce flocculants such as Wei Dong, JOURNAL OF MICROBIOLOGY 21 (6): 3-5.

71.Narutaki?S，Takatori?K，et?al(2002)Identification?of?fungi?based?on?thenucleotide?sequence?homology?of?their?internal?transcribed?spacer?1(ITS1)region?J?Pharm?Sci?Technol?2002?Mar-Apr；56(2)：90-98.

72.Sambrook，J.，E.F.Fritsch，and?T.Maniatis(1989)Molecular?Cloning：ALaboratory?Manual，2nd?ed.，Cold?Spring?Harbor?Laboratory?Press，Cold?SpringHarbor，NY.

73. Li Yuzhen, Deng Hong skin of bamboo atomic absorption analysis application manual.Beijing science tech publishing house, 1990:151-165.

The IO40269-sequence table

SEQUENCE?LISTING

＜110〉Guangxi University

＜120〉penicillium janthinellum of the anti-metal-salt of a plant height

<130>IO40269

<160>3

<170>Patent?In?version?3.1

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<400>1

tccgtaggtg?aacctgcgg???????????????????????????????????????????????????19

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<400>2

tcctccgctt?attgatatgc??????????????????????????????????????????????????20

<210>3

<211>586

<212>DNA

＜213〉penicillium janthinellum (Penicillium janthinellum)

<400>3

tccgtaggtg?aacctgcgga?aggatcatta?ccgagtgagg?gccctctggt?ccaacctccc??????60

acccgtgttt?atcataccta?gttgcttcgg?cgggcccgcc?gtcatggccg?ccggggggca?????120

cccgcccccg?ggcccgcgcc?cgccgaagcc?cccctgaacg?ctgtctgaag?attgcagtct?????180

gagcgattag?ctaaatcagt?taaaactttc?aacaacggat?ctcttggttc?cggcatcgat?????240

gaagaacgca?gcgaaatgcg?ataagtaatg?tgaattgcag?aattcagtga?atcatcgagc?????300

tttgaacgca?cattgcgccc?cctggtattc?cggggggcat?gcctgtccga?gcgtcattgc?????360

The IO40269-sequence table

tgccctcaag?cacggcttgt?gtgttgggcc?cccgcccccc?ggctcccggg?gggcgggccc?????420

gaaaggcagc?ggcggcaccg?cgtccggtcc?tcgagcgtat?ggggcttcgt?cacccgctct?????480

gtaggcccgg?ccggcgcccg?ccggcgaccc?ccctcaatct?ttctcaggtt?gacctcggat?????540

caggtaggga?tacccgctga?acttaagcat?atcaataagc?ggagga????????????????????586

Claims (8)

1. a new penicillium janthinellum strain (Penicillium janthinellum) GXCR, preserving number is CGMCC 1027.

2. the penicillium janthinellum strain GXCR of claim 1, the dna sequence dna that it is characterized in that the internal transcribed spacer district is shown in SEQ ID NO:3.

3. the penicillium janthinellum strain GXCR of claim 1 is characterized in that high anti-mantoquita, and enrichment also can be separated out copper, simultaneously the metal-salt beyond the high anti-copper removal.

4. the application of the penicillium janthinellum strain GXCR of claim 1 aspect absorption and/or anti-mantoquita and/or zinc salt.

5. the application aspect the metal-salt of the penicillium janthinellum strain GXCR of claim 1 beyond absorption and/or anti-mantoquita or zinc salt.

6. the penicillium janthinellum strain GXCR of claim 1 is in the application of soaking aspect the ore deposit.

7. the penicillium janthinellum strain GXCR of claim 1 is used to administer the application aspect the biological adsorption agent of heavy metal environmental pollution in preparation.

8. the application in the genetic engineering bacterium of the organic pollutant of the penicillium janthinellum strain GXCR of claim 1 in making up degradable heavy metallic salt-organism Composite pollution environment.

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