CN1948459A - Cladosporium endogenic fungus capable of producing veralkol - Google Patents

Abstract

This invention has clone Stilbene synthase gene (sts) from Parthenocissi Tricuspidatae endogeny eumycete, the coding sequence similarity between Stilbene synthase gene and Parthenocissi Tricuspidatae is 95.25%,this eumycete belongs to Cladosporium sp by 18SrDNA series assessment. High performance liquid chromatogram and mass chromatographic analysis indicate that this eumycete can also composite resveratrol by culture in vitro. The analysis about non- coding part of endogeny eumycete sts gene indicates that: it is very different from regulatory element of sts gene which is in plants. Importing exogenous gene(such as VHb,ipt and iaaM and so on ) in endogeny eumycete can promote endogeny eumycete growing.

Description

Can produce the Cladosporium endogenetic fungus of trans-resveratrol

One, technical field:

The present invention relates to be separated to from the endogenetic fungus of parthenocissus a Cladosporium fungi-Cladosporium caulis, this fungi contains stilbene synthase gene (sts gene) crucial in the trans-resveratrol route of synthesis that the coding region is 1179bp.The present invention also utilizes genetic engineering technique that this bacterium has been carried out genetic improvement, makes metabolism and the speed of growth of this bacterium under the restricted condition of oxygen be significantly improved than non-transgenic bacterium.The invention belongs to technical field of bioengineering.

Two, background technology:

(Resveratrol Res) belongs to stilbene compound to trans-resveratrol, and chemistry is called 3,4 ', and (3,4 ', 5-trihydrolystilbene), molecular formula is C to 5-trihydroxy--stilbene ₁₄H ₁₂O ₃, relative molecular weight is 228.25, has suitable, anti-two kinds of structures, transconfiguration is more stable.The form that trans-resveratrol often combines with glycosides with glucose exists, extensively be present on a small quantity in the middle of natural phant such as grape, giant knotweed, black false hellebore, Semen Cassiae and peanut or the fruit, up to the present in 21 sections, 31 72 kind of plant that belong to, found trans-resveratrol at least with the form of free state.

Trans-resveratrol is the secondary metabolite of plant, and it all is transformed under the katalysis of different enzymes by precursors such as coumaric acids via the phenylalanine approach with polyphenols such as flavonoid compound, furocoumarin(e)s.Wherein, trans-resveratrol is that (Stilbene synthase, STS) catalytic substrate malonyl--CoA and benzene propionyl-CoA are synthetic by the stilbene synthase.In addition, research is discovery also, and catalytic reaction of stilbene synthase (STS) and chalcone synthetase (chalcone synthase, CHS) catalytic reaction is very similar, all condensation reactions of the malonyl coenzyme A of catalysis coumaric acyl coenzyme A and three molecules, only the former discharges four CO ₂Molecule, and the latter discharges three, thus formed different benzene ring structures, obtain different product (trans-resveratrol and naringenin phenyl styryl ketone).And the protein similarity of STS and CHS also reaches about 70% (Goodwin etc., 2000).

The plant of stilbene synthase gene is a lot of only to the aversion response (as physical abuse, uviolizing, environment-stress etc.) of poor environment the time, and just can excite this gene transcription and translate to the stilbene synthase, and then instantaneous synthesizing resveratrol.Simultaneously, trans-resveratrol exists only in (as Vitis, Arachis) in the edible food of minority, and content is very low, and it is very big influenced by kind, growth conditions etc.In addition, though most plant contains the substrate of stilbene synthase effect, lack stilbene synthase gene.

Commercial at present is raw material with the higher relatively Pericarpium Vitis viniferae of content, Semen Vitis viniferae and giant knotweed mainly with trans-resveratrol, adopt methyl alcohol, ethanol, ethyl acetate etc. as extracting solvent, obtain high-load trans-resveratrol with method separation and purification such as high performance liquid chromatography, silica gel column chromatographies, but the trans-resveratrol that exists in the natural plant is micro-after all, and extraction step is various, extract component is complicated, must just can obtain finished product through separation and purification, so this technology is faced with problems such as resource-constrained and throughput are little.Catalyzer in the chemosynthesis process and reaction reagent then limit its application greatly to the hazardness of environment and human body.Therefore, newly ward off production approach efficiently, solve the consumers demand problem that increases day by day and become very urgent.

Main research now concentrates in the research of biosynthesizing trans-resveratrol: utilize genetic engineering technique, the biosynthetic pathway of control, improvement trans-resveratrol obtains its high yield plant strain system, utilize aspect such as mutagenesis approach breeding high-yield cell strain.But up to now, the industrial production from mass-producing requires also far.

Endogenetic fungus (endophytic fungi) be meant those in its life history certain period or all stages life plant tissue is not obviously caused a class fungi of disease symptom within plant tissue.In recent years, from multiple medicinal plant endogenetic fungus, filter out various physiologically active substances both at home and abroad and see that successively report is arranged, there is great diversity (as: antitumor, antiviral, antibacterium, antimycotic, pest-resistant etc. material) in the biologically active substance that endogenetic fungus produces, show that plant endogenesis epiphyte can produce novel structure, the special secondary metabolite of function, be the potential resources of new compound, novel drugs, they have important application prospects in fields such as medicine, industry, agriculturals.

The present invention clones from the endogenetic fungus of parthenocissus (Caulis Parthenocissi Tricuspidatae) and has obtained trans-resveratrol synthetic key gene-stilbene synthase gene (sts), the nucleotide sequence similarity of sts gene is 95.25% in this gene and the parthenocissus, amino acid sequence similarity is 97.45%, identify that through the 18S of rDNA sequence alignment this bacterium is Cladosporium (Cladosporium sp.).HPLC and mass spectrum (ESI-MS) analysis revealed, this bacterium also can synthesize secondary metabolite---the trans-resveratrol the same with its host plant under isolated culture.In addition, this provides a new approach for producing trans-resveratrol by the large scale fermentation that exsomatizes, and also is the growth by the promotion endogenetic fungus, and the interaction that reaches fungi and host plant improves Resveratrol content provides new method.

Three, summary of the invention:

The present invention relates to be separated to from the endogenetic fungus of parthenocissus a Cladosporium fungi-Cladosporium caulis, this fungi contains stilbene synthase gene (sts gene) crucial in the trans-resveratrol route of synthesis that the coding region is 1179bp.This fungi also can synthesizing resveratrol under the condition of isolated culture for high performance liquid chromatography (HPLC) and mass spectrum (ESI-MS) analysis revealed.We are with LeGDP/VHb gene and LeGDP/ipt gene afterwards, and this bacterial strain of LeGDP/iaaM gene transformation carries out the genetically engineered improvement, found that the fungi of carrying LeGDP/VHb is significantly increased than the accretion rate and the increment of not genetically modified contrast bacterium under the limited condition of oxygen, LeGDP/ipt gene and LeGDP/iaaM gene have then been accelerated fungal growth speed.The present invention provides clone parthenocissus endophyte sts the related data of gene simultaneously, finds that the sts gene of it and host plant has a very high homology.But the regulator control system difference of this gene is quite big in fungi and plant.In addition, the invention allows for and utilize endogenetic fungus to improve the method for trans-resveratrol output, comprise the method for fungi fermentation and utilize fungi to promote the host to produce the method for trans-resveratrol.

1, the isolation identification of parthenocissus endogenetic fungus

Gather fresh parthenocissus stem section, aseptic technique is carried out getting its phloem after the surface sterilization and is transferred on the PDA plate culture medium routinely, cultivates 3-7 days for 28 ℃, and the mycelia of the single bacterium colony that grows around the picking tissue block is received purification storage on the CYM slant medium.

2, clone's stilbene synthase (sts) gene from the parthenocissus endogenetic fungus

Utilize the similarity zone of stilbene synthase gene in the known parthenocissus (Caulis Parthenocissi Tricuspidatae), we designed comprise sts gene initiation codon and termination codon a pair of primer, be respectively STSFP and STSRP.Utilize Plant genome DNA Reagent reagent to extract the DNA of parthenocissus endogenetic fungus, obtain special segment about 1228bp with PCR method amplification, be connected on the pMD-18 carrier and check order, the complete coding region that shows this gene is 1179bp (12bp～1192bp), 392 amino acid of encoding.Sequencing result and other sequence are carried out similarity relatively, corresponding gene has 95.25% similarity among sts gene in the discovery parthenocissus endogenetic fungus and its host, carry out similarity relatively after translating into aminoacid sequence, both similaritys also have 97.45%.The special amino-acid residue fragment IPNSAGAIAGN that in the aminoacid sequence of sts, comprises the stilbene synthase, and the signal sequence GVLFGFGPGLT that shows stilbene synthase family characteristic.In addition, also there is the 164th halfcystine (Cys in the stilbene synthase coding region of parthenocissus endogenetic fungus ¹⁶⁴) active centre.

The complete coding region of this sts gene is:

1 ATGGCTTCAG TTGAGGAATT TAGAATCGCT CAACGTGCCA AGGGTCCGGC CACCATCCTA

61 GCCATTGGCA CTGCTACTCC AGACAACTGC GTCTACCAGT CTGATTACGC TGATTTCTAT

121 TTCAGAGTCA CAAAGAGCGA GCACATGACT GAGTTGAAGA AGAAGTTCAA TCGCATATGT

181 GAGAAATCAA TGATCAAGAA GCGTTATATT CATTTGACTG AAAAGATGCT TGAGGAGCAC

241 CCAAACATTG GTGCTTATAT GGCTCCATCT CTTAACATAC GCCAAGAGAT TATCACTGCC

301 GAGGTACCCA AGCTTGGTAA AGAAGCAGCA TTGAAGGCTC TAAAAGAGTG GGGCCAACCC

361 AAGTCCAAGA TCACCCATCT TGTATTTTGT ACAACCTCCG GTGTAGAAAT GCCTGGTGCA

421 GATTACAAAC TCGCTAATCT CTTAGGGCTT GAAACATCGG TCAGAAGAGT GATGTTGTAC

481 CATCAAGGGT GCTATGCAGG TGGAACTGTC CTCCGAACTG CTAAGGATCT TGCAGAGAAT

541 AATGCAGGAG CACGAGTTCT TGTGGTGTGC TCTGAGATCA CTGTTGTCAC ATTCCGTGGA

601 CCTTCCGAAA CTGCTTTGGA CTCTTTAGTT GGCCAAGCCC TTTTTGGTGA TGGGTCTGCA

661 GCTGTGATCG TTGGATCAGA TCCAGATATC TCGATTGAAC AACCACTTTT TCAACTCGTC

721 TCAGCAGCCC AAACATTTAT TCCTAATTCA GCAGGTGCCA TTGCCGGGAA CTTACGTGAG

781 GTGGGACTCA CATTTCATTT GTGGCCCAAT GTGCCAACTT TAATTTCTGA GAACATAGAG

841 AAATGCTTGA CTCAGGCTTT TGACCCACTT GGTATTAGCG ATTGGAACTC GTTATTTTGG

901 ATTGCTCACC CAGGTGGCCC TGCAATTCTT GATGCGGTTG AAGCAAAACT CAATTTAGAC

961 AAAAAGAAAC TTGAAGCAAC GAGCCATGTG TTAAGTGAGT ATGGCAACAT GTCAAGTGCA

1021 TGTGTGTTGT TTATTTTGGA TGAGATGAGA AAGAAATCAC TTAAGGGGGA GAAGGCCACC

1081 ACAGGTGAAG GATTGGATTG GGGAGTATTA TTTGGCTTTG GACCAGGCTT GACTATTGAG

1141 ACTGTTGTGT TGCATAGCAT TCCTATGGTT ACAAATTAA

3, the clone of sts gene 5 ' ending regulating sequence in the endogenetic fungus

Use a kind of karyomit(e) walking technology, from the genomic dna of parthenocissus and endogenetic fungus thereof, cloned 5 ' ending regulating sequence of sts gene based on 5 ' RACE technology.Its way is design primer GSP1 about known sts gene 5 ' end 280bp, locates to design primer GSP2 about 180bp; The first step is earlier done single primer linear amplification with GSP1, runs agarose gel electrophoresis with the PCR product of 1/10 volume, detects to carry out second behind the no specific band and go on foot, promptly with the PCR product tailing of dCTP to the last step; The 3rd step was then carried out nest-type PRC with GSP2 and AAP as primer, and its electrophoresis result should be uniform smear shape, and the glue that downcuts greater than 500bp reclaims, connects and transform DH5a; The 4th step was the screening of transformant: make primer (making single primer respectively with AAP and GSP2 simultaneously contrasts) with GSP2 and AAP and screen, the bigger and sample that do not have single primer amplification of picking checks order.Part 5 ' the control region of this sts gene is:

1 ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA

61 TTGATGTGAT ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG

121 ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT

181 CACTTCATCA AAAGGACAGT AGGAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT

241 AAAGGAAAGG CTATCGTTCA AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA

301 CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT

361 TGATGTGATA TCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCCGACG

421 GATCCTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA

481 TTTACAATAA CAATGGACTG TCTAGAGGAT CCGCC

4, the clone of parthenocissus sts gene intron

Primer according to parthenocissus sts gene order design clone intron:

STS-E (upstream): 5 '-CAC TAA GAG CGA GCA CAT GAC TGAG-3 '

STS-E (downstream): 5 '-ACG AGT TCC AAT CGC TAA TAC CAAG-3 '

From the parthenocissus genomic dna, we have been cloned into the intron of its sts gene 361bp, and its sequence is:

1 TCTAATTTTA ACATCCTTTG CGTGCATATA ATTGTGTATA CATATGATAA AGCTTTTAGA

61 TTCACCTCAG AGTACCGAAA CATCTTTTTC AAGCTTTCTG TGTACTCATT TTTAAATTAA

121 TACAATGCAT CCGTGACTGA TGCTCAGAGC AGGTGCTCTT TCAATCATAC GGTATAAAGC

181 CTGGGGTCAT ATCATTAATG TAATAAGTAA TAAGAACAAG CTTTTATATT CTATTAAGAT

241 GAATCATTTT ATACTCACTG GAACAACAAA AACTATATAC ATATTATTGA GTACTACTTG

301 TGTTTTACTT GCAATGCCTT GAGCTCACAT ATTACTGTTT TTTAATTCTT ATACAGGTGA

361 C

5, the mensuration of endogenetic fungus synthesizing resveratrol

With liquid CYM culture medium culturing 10 days, taking-up pressed dry with the above-mentioned bacterial strain that obtains and parthenocissus blade, control strain (bacterial strain that does not contain the sts gene), put in the low-temperature bake oven baking (40-55 ℃) again and spent the night.With pestle it is ground into fine-powder, and anhydrous methanol (1: 40, the w/v) lixiviate of spending the night, the liquid behind the suction filtration is 40 ℃ of rotation evaporates to dryness again, and residue is dissolved in 5mL water and the 10mL ethyl acetate.Fully after mixing and the layering, the organic phase evaporate to dryness heavily is dissolved in the 0.5mL acetone.Quantitatively point sample is in activatory silica-gel plate Silufol (UV 254), and plate is used chloroform successively: methyl alcohol (25: 1) exhibition layer three times.After the drying, reference standard sample Rf value is determined trans-resveratrol position onboard under ultraviolet lamp, scrapes and gets the silica gel that contains trans-resveratrol, reclaims trans-resveratrol with dissolve with methanol silica gel again.Extracting solution is resuspended in the second eyeball in 40 ℃ of evaporated in vacuo behind the suction filtration.The all operations process is all carried out under strict lucifuge condition, with oxidation or the decomposition that prevents trans-resveratrol.

HPLC (high performance liquid chromatography) shows with the analytical results of mass spectrum (ESI-MS): the endogenetic fungus of parthenocissus can produce the secondary metabolite the same with host plant---trans-resveratrol.

6, the biology of isolated endogenetic fungus classification

We classify it in conjunction with its morphological specificity with the 18SrDNA sequence of fungi.With reference to the extracting method of Wattier (2000), extract total DNA of fungi, and carry out the 18S Sequence Identification of rDNA the total DNA of red algae (Rhodophyta).

This 18S rDNA fragments sequence is:

1 TTAGCGAAAC TGCGAATGGC TCATTAAATC AGTTATCGTT TATTTGATAG TACCTTACTA

61 CATGGATAAC CGTGGTAATT CTAGAGCTAA TACATGCTAA AAACCCCGAC TTCGGAAGGG

121 GTGTATTTAT TAGATAAAAA ACCAATGCCC TTCGGGGCTC CTTGGTGAAT CATAATAACT

181 TAACGAATCG CATGGCCCTG CGCCGGCGAT GGTTCATTCA AATTTCTGCC CTATCAACTT

241 TCGATGGTAG GATAGTGGCC TACCATGGTA TCAACGGGTA ACGGGGAATT AGGGTTCGAC

301 TCCGGGGAGG GAGCCTGAGA AACGGCTACC ACATCCAAGG AAGGCAGCAG GCGCGCAAAT

361 TACCCAATCC CGACACGGGG AGGTAGTGAC AATAAATACT GATACAGGGC TCTTTTGGGT

421 CTTGTAATTG GAATGAGTAC AATTTAAATC CCTTAACGAG GAACAATTGG AGGGCAAGTC

481 TGGTGCCAGC AGCCGCGGTA ATTCCAGCTC CAATAGCGTA TATTAAAGTT GTTGCAGTTA

541 GAAAGCTCGT AGTTGAACCT TGGGCCTGGC TGGCCGGTCC GCCTCACCGC GTGTACTGGT

601 CCGGCCGGGC CTTTCCTTCT GGGGAACCTC ATGCCCTTCA CTGGGCGTGC TGGGGAACCA

661 GGACTTTTAC TTTGAAAAAA TTAGAGTGTT CAAAGCAGGC CTTTGCTCGA ATACATTAGC

721 ATGGAATAAT AGAATAGGAC GTGTGGTTCT ATTTTGTTGG TCTCTAGGAC CGCCGTAATG

781 ATTAATAGGG ATAGTCGGGG GCATCAGTAT TCAAGCGTCA GAGGTGAAAT TCTTGGATTG

841 CTTGAAGACT AACTACTGCG AAAGCATTTG CCAAGGATGA AT

The fungi that this strain of qualification result can be produced trans-resveratrol belongs to Ascomycota (Asconycota), seat capsule Gammaproteobacteria (Dothideomycetes et.), ball chamber Cordycepps (Mycosphaerellaceae), belongs to (Cladosporium sp.).

7, the genetically engineered of isolated cladosporium sp improvement

In one embodiment of the invention, we import Vitreoscilla hemoglobin gene in the cell of multiple symbiosis fungi the pale yellow coring false truffle (Rhizopogen luteolus) as eucalyptus.Improved the speed of growth of these fungies under hypoxia condition.The oxyphorase of the Vitreoscilla that is separated to from cow dung (Vitreoscilla stercoraria) has improved the interior available oxygen concentration of cell under the limit oxygen condition, thereby has changed the oxidasic relative reactivity of terminal, has promoted oxygen transfer efficiency in the cell.Thereby promote the efficient and the growth of aerobic respiration.The promotor that application is adopted us in fungi is the glyceraldehyde 3-phosphate dehydrogenase LeGDP promotor from mushroom.Made up the plasmid that is suitable in fungi, expressing.Connect and help electrization, or the PEG method, or agrobacterium mediation method has changed the VHb gene under the LeGDP driving over to fungi.These symbiosis fungies (fungi of endosymbiosis and ectosymbiosis) still can grow under non-symbiotic condition, under manually operated condition, are more convenient for studying their metabolic characteristic.The fungi that LeGDP/VHb is carried in discovery has obviously increased accretion rate and increment than not genetically modified contrast bacterium under the limited condition of oxygen.

We once utilized the rhizospheric microorganism of genetic improvement, improved growth and development of plant.This symbiotic rhizospheric microorganism and plant have more confidential relation.It has become common recognition to the improvement of the nutritional status of plant and the raising of resistance.The genetic improvement of endophyte will help setting up the relation between it and the host plant.In a similar embodiment of the present invention, we are with a kind of gene of growth hormones, and promptly (isopentanyl transferase ipt) imports fungi with growth hormone synthetic key gene LeGDP/iaaM gene to isopentenyl transferase genes.Used promotor also is LeGDP, and the result shows that the fungi of LeGDP/ipt gene transformation under culture condition, accelerated fungal growth speed.IaaM is the plant-growth plain gene, and LeGDP/iaaM can promote the growth of endophyte equally.Can on the basis that obtains endophyte, can carry out the genetic improvement of endophyte in a word.

Beneficial effect of the present invention: we have isolated the endogenetic fungus that can produce trans-resveratrol in the vitaceae parthenocissus, this fungi is a Cladosporium, owing to contain the stilbene synthase of trans-resveratrol route of synthesis, therefore this fungi can produce trans-resveratrol under isolated culture condition.The innovative point of the present invention endogenetic fungus of finding Vitaceae that is also to have no talent before this can produce trans-resveratrol under isolated culture condition, and trans-resveratrol exists only in the minority plant and content is very low, thereby wants the scale operation trans-resveratrol very difficult.We not only can produce trans-resveratrol by isolating this endogenetic fungus under isolated condition, and we have also confirmed that from molecular biological angle the stilbene synthase gene sts that contains just because of this fungi makes it can produce trans-resveratrol, by the large scale fermentation of this fungi is cultivated and can be opened up a new approach for the production of trans-resveratrol, and be the genetic improvement by this endogenetic fungus (for example: import VHb gene that under hypoxia condition, promotes its growth and ipt gene and the iaaM gene that promotes cell fission and growth), thereby make endogenetic fungus with the interaction of host plant in the promotion plant produce more trans-resveratrol.

Four, Brief Description Of Drawings:

The lithograph that separates fungi in Fig. 1 parthenocissus

The red circle mark be purpose bacterial strain of the present invention

The electrophorogram of the sts gene that Fig. 2 increases from endogenetic fungus

Our purpose band amplified band size is 760bb among the figure, and electrophoresis band is from left to right numbered and is followed successively by M, 1,2,3,4,5,6,7,8 ,+,-.M represents molecule marker Marker ,+be over against photograph ,-be that negative contrast, 1-8 are the genomic dna of different parthenocissus endogenetic fungus.The band for amplifying in the purpose bacterium of the present invention of arrow mark.

The HPLC of trans-resveratrol measures figure in Fig. 3 endogenetic fungus

The condition of HPLC is as follows:

HPLC：Agela Bonchrom-C18，4.6*150mm，5um

mobilephase：ACN∶H2O(5mmol ammonium formate)＝35∶65

flow rate：0.6mL/min

Detection wavelength＝306nm

The mass spectrum of trans-resveratrol (ESI-MS) figure in Fig. 4 endogenetic fungus

The ESI-MS condition is as follows:

HPLC：Agela Bonchrom-C18，4.6*150mm，5um

mobilephase：ACN∶H2O(5mmol ammonium formate)＝35∶65

flow rate：0.6mL/min

Detection wavelength＝306nm

MS：negative mode

nebulizer：50psi

dry gas：8L/min

dry temperature：325du

scan range：150-250

skimmer：-40v，Cap Exit：-92.7v

high voltage：4000v

Wherein (1) figure is trans-resveratrol finished product (purity 99.99%, available from sigma company, molecular weight is 228.3), and (2) figure is a molecular weight analyte, and the arrow mark is the sample peak among (3) figure

Fig. 5 is the diagrammatic sketch of the structure flow process of expression expressed in fungi carrier LeGDP/VHb

Fig. 6 is the diagrammatic sketch of the structure flow process of expression expressed in fungi carrier LeGDP/ipt

Fig. 7 is the diagrammatic sketch of the structure flow process of expression expressed in fungi carrier LeGDP/iaaM

Fig. 8 transgenosis and transgenosis strain growth comparison diagram not

The fungi of LeGPD/ipt gene transformation is compared than unconverted control group bigger increment under identical time and similarity condition.

Wherein CK is not transgenosis contrast, and 1-5 is a transgenic line

Fig. 9 is inoculated in the transgenosis of 50mlCYM liquid nutrient medium and transgenosis strain growth amount comparison diagram not

Carry the fungi of LeGPD/iaaM gene and accelerated the speed of growth.Left side figure is not genetically modified contrast; In, the right side is respectively genetically modified two strains system, their growth is obviously accelerated

Figure 10 is the growing state figure of fungi under the limit oxygen condition that changes the LeGPD/VHb gene

1 is not genetically modified contrast among the figure; 2-4 is for changeing the bacterial strain of LeGPD/VHb gene.

Five, embodiment:

All (Jin Dongyan etc. translate with reference to " molecular cloning: laboratory operation guide " for microbial culture among all embodiment and DNA operation, Science Press, Beijing (1993)) and " fine works molecular biology guide " (Yan Ziying etc. translate, Science Press, Beijing (1998)).Toolenzyme in the molecule manipulation such as restriction enzyme be all available from TaKaLa company, Promega company.

Embodiment 1: the separation of endogenetic fungus in the parthenocissus

With the parthenocissus stem section of just having gathered, rinse well with tap water, drain away the water, be cut into the segment about 2cm, following surface sterilization is carried out in aseptic technique routinely then: 75% alcohol-pickled 1 minute, 0.1% mercuric chloride soaked 30 minutes, get its phloem, be connected on the PDA flat board and cultivate, the fungi that inoculation back was grown in 4-7 days is transferred to purifying on the CYM slant medium with inoculating needle picking mycelia, be stored in 4 ℃ standby.

The endogenetic fungus that is separated to is further determined whether to contain stilbene synthase gene with the stilbene synthase primer (seeing embodiment 2) that designs.

Embodiment 2: the clone of stilbene synthase (sts) gene in the parthenocissus endogenetic fungus

Design following primer (synthetic) with reference to stilbene synthase gene in the parthenocissus (sta) by Shanghai bio-engineering corporation:

STS1 (upstream primer): 5 '＞CGG GAT CCG CCA TGG CTT CAG TTG AGAAAT TTA G＜3 ',

Contain the BamHI site

STS2 (downstream primer): 5 '＞GTG AGC TCG AAG GGTAAA CCA TTC TCT TTT AT＜3 ',

Contain the SacI site

Genome with endogenetic fungus is a template, adds in the PCR reaction system of 50 μ l: dna profiling 2 μ l, 10 * PCR buffer (MgCl ₂) 5ul, 2.5mM dNTP mix 4ul, each 2.5ul of primer (10 μ M), Taq archaeal dna polymerase (5U/ul) 0.5ul, ddH ₂O 34ul.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations; 72 ℃ are extended 10min, 10 ℃ of insulation 1min.The PCR product is run agarose reclaims glue, as a result the purpose band with over against according to consistent, with the recovery of purpose band, be connected with the pMD-18 carrier and check order.

The a small amount of preparation of fungal gene group DNA:

Extract damping fluid: contain the 68mlTEN damping fluid (0.5M NaCl, 50mM EDTA, 0.1M Tris-HCl, PH 8.0; Room temperature is deposited), 6.8ml 20%SDS and 125ul Proteinase K mother liquor (20mg/ml is dissolved in distilled water ,-20 ℃ of preservations); With using with joining.

(1) hypha,hyphae is inoculated in the CYM liquid nutrient medium, cultivated 5 days for 28 ℃, get the Effendorf pipe that 0.1g (weight in wet base) mycelia places 1.5ml, the liquid nitrogen freezing several seconds, with managing supporting glass pestle mycelia is fully ground with Effendorf, put on ice, treat respectively to add after all samples has all ground 1ml and extract damping fluid, violent mixing;

Temperature is bathed 30min in (2) 37 ℃ of shaking tables (200rpm);

(3) 4 ℃, 12, the centrifugal 15min of 000rpm shifts supernatant, ice bath 30min;

(4) 4 ℃, 12, the centrifugal 15min of 000rpm shifts supernatant, uses CI extracting albumen 1-2 time;

(5) add 600ul in the Virahol of-20 ℃ of precoolings, 4 ℃, 12, the centrifugal 15min of 000rpm abandons supernatant;

(6) precipitation is used 70% washing with alcohol, puts in the room temperature and dries;

(7) every pipe adds the dissolving of 20-40ul distilled water.

The preparation of bacteria plasmid DNA:

(1) picking list colony inoculation is in containing suitable antibiotic LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night;

(2) get 1.5ml bacterium liquid in centrifuge tube, 10, centrifugal 30 seconds of 000rpm;

(3) bacterial sediment is resuspended in the 100ul solution I (50mM sucrose, 20mM Tris.Cl (pH8.0), 10mM EDTA (pH8.0)), and the vibration mixing also left standstill several minutes;

(4) (0.2N NaCl 1%SDS), flicks mixing to the 200ul solution II of the fresh configuration of adding, places on ice 3 minutes;

(5) the 150ul solution III (60ml 5M KAc, 11.5ml glacial acetic acid, 28.5ml sterilized water) of adding precooling is flicked mixing, places on ice 5 minutes;

Centrifugal 5 minutes of (6) 12,000rpm;

(7) get supernatant liquor, add the Virahol of 1 times of volume, placed 10 minutes on ice behind the mixing;

Centrifugal 5 minutes of (8) 12,000rpm dry after the supernatant discarded a little, make it to be dissolved in the 200ul sterilized water;

(9) add the 100ul7.5M ammonium acetate, placed 10 minutes on ice behind the mixing gently; In 12, centrifugal 5 minutes of 000rpm;

(10) get supernatant liquor, add the two volumes dehydrated alcohol after, on ice or-20 ℃ placed 20 minutes, 12, centrifugal 15 minutes of 000rpm;

(11) precipitation is cleaned with 70% ethanol, is dissolved in after draining in the TE buffered soln (10mM Tris.Cl (pH8.0), 1mMEDTA (pH8.0)).

The recovery of dna fragmentation (TIANGEN company gel reclaims test kit):

(1) downcuts required dna fragmentation (volume does not surpass 100ul) from sepharose and place little centrifuge tube;

(2) add the sol solutions PN of 3 times of volumes, 50 ℃ of water-baths 10 minutes, the little centrifuge tube of jog makes glue dissolve fully for several times therebetween;

(3) the resulting solution of previous step is joined among adsorption column CA1 or the CA2 the centrifugal 1min of 12000rpm;

(4) outwell waste liquid in the collection tube, adsorption column is reentered in the collection tube;

(5) in adsorption column, add 700ul rinsing liquid PW, the centrifugal 1min of 12000rpm;

(6) outwell waste liquid in the collection tube, adsorption column is reentered in the collection tube;

(7) in adsorption column, add 500ul rinsing liquid PW, the centrifugal 1min of 12000rpm;

(8) outwell waste liquid in the collection tube, adsorption column is reentered in the collection tube, the centrifugal 2min of 12000rpm goes out rinsing liquid as far as possible;

(9) adsorption column is put in room temperature or 50 ℃ of incubator numbers minute, thoroughly dries, influence next step experiment to prevent residual rinsing liquid;

(10) adsorption column is put into a clean centrifuge tube, to the unsettled dropping in adsorption film mid-way 30ul elution buffer EB, room temperature was placed 2 minutes;

(11) the centrifugal 1min of 12000rpm is added back to the centrifugal solution that obtains in the adsorption column again, repeats 10 steps.

Connect:

Dna solution 2.4ul, the pMD-18 carrier 0.6ul, the Solution I 3ul that add above-mentioned recovery in the 6ul reaction system placed about 4 hours for 16 ℃.

The preparation of competent escherichia coli cell:

(1) the single colony inoculation of picking E.coli DH5 α is in the 100ml liquid nutrient medium, and 37 ℃ are cultured to OD ₆₀₀About 0.4;

(2) ice bath is 10 minutes, be sub-packed in the aseptic centrifuge tube, and 4 ℃, 4, centrifugal 5 minutes collecting cells of 000rpm;

(3) be resuspended in the 0.1M CaCl that 600ul ices precooling ₂In, ice bath 30 minutes;

(4) 4 ℃, 4, centrifugal 10 minutes collecting cells of 000g.0.1M CaCl with the 100ul precooling ₂Re-suspended cell, stand-by.

The conversion of competent cell:

(1) get the 100ul competent cell and add 5ul connection product, mixing is iced and was put 30 minutes gently;

(2) heat shock 90 seconds in 42 ℃ of water placed rapidly cooled on ice 1-2 minute;

(3) add 200ul LB liquid nutrient medium, 37 ℃ of shaking culture 45 minutes to 1 hour;

(4) evenly coat on the LB flat board that contains penbritin, be inverted overnight incubation for 37 ℃.

The screening of recombinant plasmid and evaluation:

A. picking list bacterium colony was cultivated 4-6 hour with the LB liquid nutrient medium of the additional penbritin of 800ul;

B.PCR detects: picking list bacterium colony is in containing suitable antibiotic 3ml LB liquid nutrient medium, and 37 ℃ of overnight incubation are extracted plasmid DNA.With the plasmid DNA is template, does pcr amplification reaction under proper condition with specific primer, and whether electrophoresis detection has the purpose band;

C. enzyme is cut evaluation: positive colony carries out enzyme with suitable restriction enzyme again and cuts further to judge whether positive clone.

Embodiment 3: the 18S of fungi identifies

18S conserved sequence according to eukaryote rDNA designs following primer:

18S upstream: 5 '-AGCGAAACTG CGAATGGC-3 ';

18S downstream: 5 '-CATCCTTGGC AAATGCTTTC-3 ';

In the PCR reaction system of 50 μ l, add: dna profiling 2 μ l, 10 * PCRbuffer (MgCl ₂) 5ul, 2.5mMdNTP mix 4ul, each 2.5ul of primer (10uM), Taq archaeal dna polymerase (5U/ul) 0.5ul, ddH ₂O 34ul.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 60s, 30 circulations; 72 ℃ are extended 10min, 10 ℃ of insulation 1min.The PCR product is run agarose reclaim glue, the purpose band is downcut reclaim, be connected with the pMD-18 carrier and check order.

Sequencing result is carried out the BLAST comparison, and determine that this endogenetic fungus is: Ascomycota (Ascomycota), seat capsule Gammaproteobacteria (Dothideomycetes et.), ball chamber Cordycepps (Mycosphaerellaceae), Cladosporium (Cladodporium sp.).

Embodiment 4: the clone of parthenocissus and endogenetic fungus sts gene 5 ' ending regulating sequence thereof

The primer that this experiment relates to:

GSP1：5’-CTTGGCGTATGTTAAGAGATGGAGC-3’

GSP2：5’-CGCTTCTTGATCATTGATTTCTC-3’

AAP：5’-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3’

AUAP：5’-GGCCACGCGTCGACTAGTAC-3’

Karyomit(e) walking based on 5 ' RACE technology

(1) single primer PCR linear amplification: extracting test kit with the Plant Genome of TIANGEN company and extract the parthenocissus genomic dna, is template with it and endogenetic fungus DNA thereof, carries out single primer PCR amplification with the GSP1 primer.Contain in the PCR reaction mixture of 40 μ l: 4.0 μ l, 10 * PCR buffer, 5.0 μ l 2.5mM dNTPmixture, 4.0 μ g dna profilings, 2.0 μ l 10mM GSP1,25.0 μ l ddH ₂O.The PCR condition is as follows: the EX TaqE (TaKaRa) that adds 2.5U behind 94 ℃ of pre-sex change 7min; Carry out 25 circulations by following condition then: 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 80s.No longer carrying out additional 72 ℃ after the loop ends extends and direct termination reaction.Get 5 μ lPCR products and walk 1% agarose gel electrophoresis, prove conclusively special nothing but amplified band after, remaining PCR product is crossed post according to the basic experiment method reclaims.

(2) tailing of single stranded DNA: in the 0.5ml centrifuge tube, add following composition: 5.0 μ, 15 * tailing buffer; 2.5 μ l2mM dCTP; 16.5 μ l ssDNA sample; 24.0 μ l ddH ₂O.Carry out following operation then: 94 ℃ of 3min, place 1min on ice, centrifugal; Add 1 μ l TdT, mixing, 37 ℃ 10 minutes; 65 ℃ of heating 10min, centrifugal, ice bath is preserved.

(3) nest-type PRC amplification, clone and order-checking:, in the 0.2ml centrifuge tube, add following composition: 5 μ l10 * PCR buffer on ice; 5.0 μ l 2.5mM dNTP; 2.0 μ l 10 μ M GSP2; 2.0 μ l 10 μ M AAP; 5.0 μ ldC-tailed ssDNA; 31.0 μ l ddH ₂O; The PCR condition is as follows: the EXTaqE (TaKaRa) that adds 2.5U behind 94 ℃ of pre-sex change 5min; Carry out 35 circulations by following condition then: 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 90s; 72 ℃ of 10min termination reactions of loop ends.The PCR product is walked 1% agarose gel electrophoresis and is detected, and 500bp-2, the smear glue in 000bp interval downcuts, with the gel recovery test kit recovery of TIANGEN company.The PCR product that reclaims is connected with the pMD-18 carrier, transforms DH5 α; Choosing 3～4 contains and longly inserts segmental positive colony and check order.

Embodiment 5: the HPLC that the parthenocissus endogenetic fungus produces trans-resveratrol detects

(1) with the above-mentioned bacterial strain that obtains and parthenocissus blade, control strain (bacterial strain that does not contain the sts gene) with liquid CYM culture medium culturing 10 days, taking-up press dry, and puts in the low-temperature bake oven baking (40-55 ℃) again and spends the night;

(2) with pestle it is ground into fine-powder, and anhydrous methanol (1: 40, the w/v) lixiviate of spending the night, the liquid behind the suction filtration is 40 ℃ of rotation evaporates to dryness again, and residue is dissolved in 5mL water and the 10mL ethyl acetate;

(3) after abundant mixing and the layering, the organic phase evaporate to dryness heavily is dissolved in the 0.5mL acetone;

(4) quantitatively point sample is in activatory silica-gel plate Silufol (UV 254), and plate is used chloroform successively: methyl alcohol (25: 1) exhibition layer three times;

(5) after the drying, reference standard sample Rf value is determined trans-resveratrol position onboard under ultraviolet lamp, scrapes and gets the silica gel that contains trans-resveratrol, reclaims trans-resveratrol with dissolve with methanol silica gel again;

(6) extracting solution is resuspended in the second eyeball in 40 ℃ of evaporated in vacuo behind the suction filtration.

^*The all operations process is all carried out under strict lucifuge condition, with oxidation or the decomposition that prevents trans-resveratrol.

(7) HPLC analyzes: suitably the extracting solution behind the thin-layer chromatography of dilution is used for sample, on the HPCHEM high performance liquid chromatograph, use Nucleosil C18 chromatographic column (4.6mm * 150mm, 5 μ), with second eyeball-aqueous solution (35: 65) is moving phase, flow velocity 0.6mL/min detects under the 306nm wavelength.

The HPLC analytical results shows: at 306nm wavelength place, sample locates to occur the crest the same with standard model about 7min.

Embodiment 6: the parthenocissus endogenetic fungus produces the electrospray ionization mass spectrometry (ESI-MS) of trans-resveratrol and analyzes

The ESI-MS condition is: HPLC:Agela Bonchrom-C18,

4.6*150mm，

5um；

Mobilephase：CAN∶H2O(5mmol ammonium formate)＝35∶65，

flow rate：0.6mL/min，

Detection wavelength＝306nm。

MS：

negative mode，

nebulizer：50psi，

dry gas：8L/min，

dry temperature：325du，

scan range：150-250，

skimmer：-40v，Cap Exit：-92.7v，

high voltage：4000v。

Result such as accompanying drawing 4, when 7.9min, same crest appears in sample and standard model, and the molecular weight of sample is 226.3, therefore, we can think this material for we required definite be trans-resveratrol.

Embodiment 7: the structure of expressed in fungi carrier LeGDP/VHb

Downcut the GUS position that pT-PV goes up the vhb fragment replacement P1221 carrier of about 0.5kb size with BamH I+Sac I, obtain recombinant plasmid pLvhb; From the p301-bG1 plasmid, downcut the Tnos-GUS-pLeGPD-bar-P1L-LB fragment of about 5.5kb size again, this fragment is inserted between the Not I site of pLvhb, thereby obtains the LeGDP/VHb expression vector with Spe I.

Wherein, pT-PV and p301-bG1 carrier are made up by protein gene engineering key lab of Peking University and preserve, and related enzyme is available from promega company.

Embodiment 8: the structure of expressed in fungi carrier LeGDP/ipt

Between the Nco of pBI321b I and SacI site, introduce the ipt fragment of the 0.7kb that downcuts from pT-ipt again, obtain the intermediate carrier pBI321b-ipt of about 8.5kb.The PLeGPD fragment of 1.3kb scaled off from carrier pL321b with Hind III+Nco I at last and replace the P35S-35S fragment between Hind III and Nco I in the pBI321b-ipt carrier, obtaining pL321b-ipt is the LeGDP/ipt expression vector.

Wherein, pBI321b and pT-ipt carrier are made up by protein gene engineering key lab of Peking University and preserve, and related enzyme is available from promega company.

Embodiment 9: the structure of expressed in fungi carrier LeGDP/iaaM

Ipt fragment on the pL321b-ipt carrier is used from the iaam fragment replacement of PGEMRT-iaam carrier with Not I and Hind III cutting-out 0.6kb, can be obtained the pL321b-ipt carrier.

Wherein, PGEMRT-iaam carrier and pL321b-ipt carrier are to have by protein gene engineering key lab of Peking University to make up preservation, and related enzyme is available from promega company.

Embodiment 10: the genetically engineered improvement of parthenocissus endogenetic fungus

The cultivation of fungi

Fungi is inoculated in the CYM solid medium, cultivated 5 days for 26 ℃; Will activation good bacterial classification inoculation in the 500mL Erlenmeyer flask that granulated glass sphere and 100mL liquid CYM substratum are housed, 26 ℃ static cultivation 4～5d days, shake bottle 1～2 time every day to break up mycelia.

The a large amount of preparations and the purifying of plasmid to be transformed

(1) with the bacterial classification inoculation of plasmid to be transformed in the 100mL liquid LB substratum of band ammonia benzyl resistance, be cultured to logarithmic phase in 37 ℃ of shaking tables;

(2) thalline is centrifugal is collected in the 50mL centrifuge tube, adds the 8mL solution I thalline is hanged fully, the vibration mixing;

(3) add the new solution II 8mL for preparing, abundant mixing, it is more clear that room temperature is placed to solution;

(4) add the 8mL solution III, mixing, room temperature is placed 10min, the centrifugal 5min of 12000rpm;

(5) add the 16mL Virahol in the supernatant, mixing, the centrifugal 5min of 12000rpm, the DNA precipitation is loaded in 2 Effendorf pipes with 1.2mL distilled water dissolving back average mark carries out purifying;

(6) add 300 μ l 7.5MNH4Ac in each Eppendorf pipe respectively, mixing is placed 10-20min for-20 ℃; The centrifugal 5min of 12000rpm gets supernatant, adds 600 μ l Virahols, the centrifugal 5min of 12000rpm after the equal-volume CI extracting;

(7) two pipe precipitations lump together, and with the dissolving of 250 μ l distilled waters, add 250 μ l 5MLiCl (precooling), place 10min, the centrifugal 5min of 12000rpm for-20 ℃;

(8) get supernatant, add the 1mL pre-cooled ethanol, mixing, the centrifugal 5min of 12000rpm;

(9) precipitation adds 300 μ l PEG solution (13%PEG8000,1.6M NaCl), mixing, the centrifugal 10min of 12000rpm with 300 μ l distilled waters dissolving back;

(10) precipitation adds 150 μ l 7.5M NH4AC with 300 μ l distilled waters dissolving back, places 10min, the centrifugal 10min of 12000rpm for-20 ℃;

(11) get supernatant, add the 1mL dehydrated alcohol, the centrifugal 5min of 12000rpm, precipitation is drained with 70% washing with alcohol final vacuum, adds the dissolving of 50 μ l aseptic double-distilled waters;

(12) with the content of agarose gel electrophoresis detection plasmid, plasmid concentration is adjusted to 1 μ g/ μ l (attention aseptic technique) ,-20 ℃ of preservations are standby.

The preparation of fungi protoplastis (aseptic technique)

(1) with 100mL mycelium culture thing with 4 layers of filtered through gauze, with aseptic water washing 2-3 time, blot mycelia with thieving paper again;

(2) mycelia is transferred in the 15-mL plastics screw socket centrifuge tube, adds 1mL lywallzyme enzymolysis solution (1.5% lywallzyme, 0.6M N.F,USP MANNITOL, 0.1M Na2HPO4-citrate buffer solution, pH5.6; Be sub-packed in the Eppendorf pipe-20 ℃ of preservations after the frozen-thawed sterilization), fully enzymolysis in the rearmounted 30 ℃ of water-baths of mixing is put upside down centrifuge tube several times to promote the release of protoplastis every 15min;

(3) examined under a microscope when a large amount of protoplastiss discharge and finish enzyme digestion reaction, added 2mL0.6M N.F,USP MANNITOL, mixing is poured the 10mL syringe inner filtration that 0.5cm absorbent cotton post is housed into, and filtrate collection is in 1.5mL Eppendorf pipe;

(4) the centrifugal 5min of 4000rpm abandons supernatant, with 1mL 0.6M N.F,USP MANNITOL centrifuge washing 2 times, the protoplastis of purifying is suspended in the 100 μ l 0.6M N.F,USP MANNITOL.

The conversion of fungi protoplastis and regeneration

(1) 1mL is shocked by electricity damping fluid (pH 7.0 for 0.6M N.F,USP MANNITOL, 10mM Na2HPO4-NaH2PO4) adds in the protoplastis (107-108) of purifying, and the centrifugal 5min of 4000rpm abandons supernatant;

(2) protoplastis is resuspended in the 200 μ l electric shock damping fluid, adds 10 μ g plasmid DNA to be transformed, change the electric shock cup behind the mixing over to, ice bath 10min;

(3) under the condition of voltage 1000v, electric capacity 25 μ F, resistance 400 Ω, apply electricimpulse one time, ice bath 10min;

(4) add 5mL CYM selectivity regeneration culture medium and (contain 0.6M N.F,USP MANNITOL, 20 μ g/mL weedicides, 1% low melting-point agarose; Fusing back keeps 37～40 ℃), mixing is poured on the flat board of completing with 30mL CYM selectivity regeneration culture medium (containing 1% agarose), treat that the upper strata substratum solidifies fully after, flat board is sealed and is put 26 ℃ of cultivations with sealing film;

(5) flat board after 5-7 days, can be seen the regeneration of transformant bacterium colony in 26 ℃ of cultivations.

The evaluation of transgenic fungus

(1) a small amount of of fungal gene group DNA preparation in the extracting genome DNA reference example 2 of fungi.

(2) PCR of transgenic fungus identifies: the genomic dna with fungi is a template, with the method screening positive plant of pcr amplification VHb, ipt and iaaM.

(3) southren of transgenic fungus detection is to use digoxigenin labeled, and concrete grammar please refer to molecular cloning experiment guide third edition 487-509 page or leaf.

(4) northern of transgenic fungus detection is to use digoxigenin labeled, and concrete grammar please refer to molecular cloning experiment guide third edition 532-552 page or leaf.

Table 1 fungi PDA culture medium prescription

Composition	Every liter
		Potato extracting solution glucose	1.0 rise 20.0 grams

^*Potato extracting solution: take by weighing potato 200 gram, clean, peeling, fragmentation, add 1000ml water, boil half an hour, get final product with gauze elimination dregs.

Table 2 fungi CYM solid culture based formulas

Composition	Every liter of content (gram)
		Maltose glucose yeast extract peptone MgSO 4.7H 2O KH 2PO 4Agar	10.0 20.0 2.0 2.0 0.5 4.6 10.0

Table 3 fungi CYM liquid culture based formulas

Composition	Every liter of content (gram)
		Maltose glucose yeast extract peptone MgSO 4.7H 2O KH 2PO 4	10.0 20.0 2.0 2.0 0.5 4.6

Six, sequence table

＜110〉Lin Zhongping

＜120〉can produce Cladosporium (Cladosporium sp.) endogenetic fungus of trans-resveratrol

<151>2006-10-24

<160>1

<210>1

<211>1179

<212>DNA

＜213〉parthenocissus branch spore mould (Cladosporium csulis)

<220>

<221>CDS

<222>(1)...(1179)

<400>1

ATGGCTTCAG TTGAGGAATT TAGAATCGCT CAACGTGCCA AGGGTCCGGC CACCATCCTA 60

GCCATTGGCA CTGCTACTCC AGACAACTGC GTCTACCAGT CTGATTACGC TGATTTCTAT 120

TTCAGAGTCA CAAAGAGCGA GCACATGACT GAGTTGAAGA AGAAGTTCAA TCGCATATGT 180

GAGAAATCAA TGATCAAGAA GCGTTATATT CATTTGACTG AAAAGATGCT TGAGGAGCAC 240

CCAAACATTG GTGCTTATAT GGCTCCATCT CTTAACATAC GCCAAGAGAT TATCACTGCC 300

GAGGTACCCA AGCTTGGTAA AGAAGCAGCA TTGAAGGCTC TAAAAGAGTG GGGCCAACCC 360

AAGTCCAAGA TCACCCATCT TGTATTTTGT ACAACCTCCG GTGTAGAAAT GCCTGGTGCA 420

GATTACAAAC TCGCTAATCT CTTAGGGCTT GAAACATCGG TCAGAAGAGT GATGTTGTAC 480

CATCAAGGGT GCTATGCAGG TGGAACTGTC CTCCGAACTG CTAAGGATCT TGCAGAGAAT 540

AATGCAGGAG CACGAGTTCT TGTGGTGTGC TCTGAGATCA CTGTTGTCAC ATTCCGTGGA 600

CCTTCCGAAA CTGCTTTGGA CTCTTTAGTT GGCCAAGCCC TTTTTGGTGA TGGGTCTGCA 660

GCTGTGATCG TTGGATCAGA TCCAGATATC TCGATTGAAC AACCACTTTT TCAACTCGTC 720

TCAGCAGCCC AAACATTTAT TCCTAATTCA GCAGGTGCCA TTGCCGGGAA CTTACGTGAG 780

GTGGGACTCA CATTTCATTT GTGGCCCAAT GTGCCAACTT TAATTTCTGA GAACATAGAG 840

AAATGCTTGA CTCAGGCTTT TGACCCACTT GGTATTAGCG ATTGGAACTC GTTATTTTGG 900

ATTGCTCACC CAGGTGGCCC TGCAATTCTT GATGCGGTTG AAGCAAAACT CAATTTAGAC 960

AAAAAGAAAC TTGAAGCAAC GAGCCATGTG TTAAGTGAGT ATGGCAACAT GTCAAGTGCA 1020

TGTGTGTTGT TTATTTTGGA TGAGATGAGA AAGAAATCAC TTAAGGGGGA GAAGGCCACC 1080

ACAGGTGAAG GATTGGATTG GGGAGTATTA TTTGGCTTTG GACCAGGCTT GACTATTGAG 1140

ACTGTTGTGT TGCATAGCAT TCCTATGGTT ACAAATTAA 1179

＜110〉Lin Zhongping

＜120〉can produce Cladosporium (Cladosporium sp.) endogenetic fungus of trans-resveratrol

<151>2006-10-24

<160>1

<210>1

<211>882

<212>DNA

＜213〉parthenocissus branch spore mould (Cladosporium caulis)

<220>

<221>CDS

<222>(1)...(882)

<400>1

TTAGCGAAAC TGCGAATGGC TCATTAAATC AGTTATCGTT TATTTGATAG TACCTTACTA 60

CATGGATAAC CGTGGTAATT CTAGAGCTAA TACATGCTAA AAACCCCGAC TTCGGAAGGG 120

GTGTATTTAT TAGATAAAAA ACCAATGCCC TTCGGGGCTC CTTGGTGAAT CATAATAACT 180

TAACGAATCG CATGGCCCTG CGCCGGCGAT GGTTCATTCA AATTTCTGCC CTATCAACTT 240

TCGATGGTAG GATAGTGGCC TACCATGGTA TCAACGGGTA ACGGGGAATT AGGGTTCGAC 300

TCCGGGGAGG GAGCCTGAGA AACGGCTACC ACATCCAAGG AAGGCAGCAG GCGCGCAAAT 360

TACCCAATCC CGACACGGGG AGGTAGTGAC AATAAATACT GATACAGGGC TCTTTTGGGT 420

CTTGTAATTG GAATGAGTAC AATTTAAATC CCTTAACGAG GAACAATTGG AGGGCAAGTC 480

TGGTGCCAGC AGCCGCGGTA ATTCCAGCTC CAATAGCGTA TATTAAAGTT GTTGCAGTTA 540

GAAAGCTCGT AGTTGAACCT TGGGCCTGGC TGGCCGGTCC GCCTCACCGC GTGTACTGGT 600

CCGGCCGGGC CTTTCCTTCT GGGGAACCTC ATGCCCTTCA CTGGGCGTGC TGGGGAACCA 660

GGACTTTTAC TTTGAAAAAA TTAGAGTGTT CAAAGCAGGC CTTTGCTCGA ATACATTAGC 720

ATGGAATAAT AGAATAGGAC GTGTGGTTCT ATTTTGTTGG TCTCTAGGAC CGCCGTAATG 780

ATTAATAGGG ATAGTCGGGG GCATCAGTAT TCAAGCGTCA GAGGTGAAAT TCTTGGATTG 840

CTTGAAGACT AACTACTGCG AAAGCATTTG CCAAGGATGA AT 882

＜110〉Lin Zhongping

＜120〉can produce Cladosporium (Cladosporium sp.) endogenetic fungus of trans-resveratrol

<151>2006-10-24

<160>1

<210>1

<211>515

<212>DNA

＜213〉parthenocissus branch spore mould (Cladosporium caulis)

<220>

<221>promoter

<222>(1)...(515)

<400>1

ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA 60

TTGATGTGAT ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG 120

ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT 180

CACTTCATCA AAAGGACAGT AGGAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT 240

AAAGGAAAGG CTATCGTTCA AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA 300

CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT 360

TGATGTGATA TCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCCGACG 420

GATCCTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 480

TTTACAATAA CAATGGACTG TCTAGAGGAT CCGCC 515

＜110〉Lin Zhongping

＜120〉can produce Cladosporium (Cladosporium sp.) endogenetic fungus of trans-resveratrol

<151>2006-10-24

<160>1

<210>1

<211>361

<212>DNA

＜213〉parthenocissus branch spore mould (Cladosporium caulis)

<220>

<221>intron

<222>(1)...(361)

<400>1

TCTAATTTTA ACATCCTTTG CGTGCATATA ATTGTGTATA CATATGATAA AGCTTTTAGA 60

TTCACCTCAG AGTACCGAAA CATCTTTTTC AAGCTTTCTG TGTACTCATT TTTAAATTAA 120

TACAATGCAT CCGTGACTGA TGCTCAGAGC AGGTGCTCTT TCAATCATAC GGTATAAAGC 180

CTGGGGTCAT ATCATTAATG TAATAAGTAA TAAGAACAAG CTTTTATATT CTATTAAGAT 240

GAATCATTTT ATACTCACTG GAACAACAAA AACTATATAC ATATTATTGA GTACTACTTG 300

TGTTTTACTT GCAATGCCTT GAGCTCACAT ATTACTGTTT TTTAATTCTT ATACAGGTGA 360

C 361

Seven, patent bacterial classification explanation:

The bacterial classification that this patent relates to is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, Zhong Guan-cun, unit address BeiJing, China, this classification of fungi name is called cladosporium sp (Cladosporium sp.), the preservation code name is PSHEn, deposit number is CGMCC No.1835, and preservation date is on October 12nd, 2006.

In a word, we have isolated the endogenetic fungus that can produce trans-resveratrol in the vitaceae parthenocissus, and this fungi is a Cladosporium, owing to contain the stilbene synthase of trans-resveratrol route of synthesis, therefore this fungi can produce trans-resveratrol under isolated culture condition.We utilize high performance liquid chromatography (HPLC) and mass spectrum (ESI-MS) analytical technology to detect it and produce the content of trans-resveratrol, this endogenetic fungus that has confirmed isolated culture by experiment also can produce the product trans-resveratrol, and with LeGDP/VHb gene and LeGDP/ipt gene, and this bacterial strain of LeGDP/iaaM gene transformation carries out genetically engineered improvement, and the result has obviously improved accretion rate and the increment of fungi under the limited condition of oxygen.This also provides new method for improving trans-resveratrol output by the interaction that improves endogenetic fungus and host plant for to come the scale operation trans-resveratrol to open up a new way by this fungi of fermentation culture.

Claims (9)

1, an isolated strain can produce the endogenetic fungus of trans-resveratrol from parthenocissus, is the Cladosporium fungi, has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the numbering of registering on the books CGMCC No.1835.

2, fungi according to claim 1 is characterized in that 18S rDNA fragment 882bp, and sequence is:

1 TTAGCGAAAC TGCGAATGGC TCATTAAATC AGTTATCGTT TATTTGATAG TACCTTACTA

61 CATGGATAAC CGTGGTAATT CTAGAGCTAA TACATGCTAA AAACCCCGAC TTCGGAAGGG

121 GTGTATTTAT TAGATAAAAA ACCAATGCCC TTCGGGGCTC CTTGGTGAAT CATAATAACT

181 TAACGAATCG CATGGCCCTG CGCCGGCGAT GGTTCATTCA AATTTCTGCC CTATCAACTT

241 TCGATGGTAG GATAGTGGCC TACCATGGTA TCAACGGGTA ACGGGGAATT AGGGTTCGAC

301 TCCGGGGAGG GAGCCTGAGA AACGGCTACC ACATCCAAGG AAGGCAGCAG GCGCGCAAAT

361 TACCCAATCC CGACACGGGG AGGTAGTGAC AATAAATACT GATACAGGGC TCTTTTGGGT

421 CTTGTAATTG GAATGAGTAC AATTTAAATC CCTTAACGAG GAACAATTGG AGGGCAAGTC

481 TGGTGCCAGC AGCCGCGGTA ATTCCAGCTC CAATAGCGTA TATTAAAGTT GTTGCAGTTA

541 GAAAGCTCGT AGTTGAACCT TGGGCCTGGC TGGCCGGTCC GCCTCACCGC GTGTACTGGT

601 CCGGCCGGGC CTTTCCTTCT GGGGAACCTC ATGCCCTTCA CTGGGCGTGC TGGGGAACCA

661 GGACTTTTAC TTTGAAAAAA TTAGAGTGTT CAAAGCAGGC CTTTGCTCGA ATACATTAGC

721 ATGGAATAAT AGAATAGGAC GTGTGGTTCT ATTTTGTTGG TCTCTAGGAC CGCCGTAATG

781 ATTAATAGGG ATAGTCGGGG GCATCAGTAT TCAAGCGTCA GAGGTGAAAT TCTTGGATTG

841 CTTGAAGACT AACTACTGCG AAAGCATTTG CCAAGGATGA AT

3, fungi according to claim 1 is characterized in that this fungi contains the stilbene synthase gene (sts) that can produce trans-resveratrol.Sts gene complete coding region is 1179bp, and sequence is:

1 ATGGCTTCAG TTGAGGAATT TAGAATCGCT CAACGTGCCA AGGGTCCGGC CACCATCCTA

61 GCCATTGGCA CTGCTACTCC AGACAACTGC GTCTACCAGT CTGATTACGC TGATTTCTAT

121 TTCAGAGTCA CAAAGAGCGA GCACATGACT GAGTTGAAGA AGAAGTTCAA TCGCATATGT

181 GAGAAATCAA TGATCAAGAA GCGTTATATT CATTTGACTG AAAAGATGCT TGAGGAGCAC

241 CCAAACATTG GTGCTTATAT GGCTCCATCT CTTAACATAC GCCAAGAGAT TATCACTGCC

301 GAGGTACCCA AGCTTGGTAA AGAAGCAGCA TTGAAGGCTC TAAAAGAGTG GGGCCAACCC

361 AAGTCCAAGA TCACCCATCT TGTATTTTGT ACAACCTCCG GTGTAGAAAT GCCTGGTGCA

421 GATTACAAAC TCGCTAATCT CTTAGGGCTT GAAACATCGG TCAGAAGAGT GATGTTGTAC

481 CATCAAGGGT GCTATGCAGG TGGAACTGTC CTCCGAACTG CTAAGGATCT TGCAGAGAAT

541 AATGCAGGAG CACGAGTTCT TGTGGTGTGC TCTGAGATCA CTGTTGTCAC ATTCCGTGGA

601 CCTTCCGAAA CTGCTTTGGA CTCTTTAGTT GGCCAAGCCC TTTTTGGTGA TGGGTCTGCA

661 GCTGTGATCG TTGGATCAGA TCCAGATATC TCGATTGAAC AACCACTTTT TCAACTCGTC

721 TCAGCAGCCC AAACATTTAT TCCTAATTCA GCAGGTGCCA TTGCCGGGAA CTTACGTGAG

781 GTGGGACTCA CATTTCATTT GTGGCCCAAT GTGCCAACTT TAATTTCTGA GAACATAGAG

841 AAATGCTTGA CTCAGGCTTT TGACCCACTT GGTATTAGCG ATTGGAACTC GTTATTTTGG

901 ATTGCTCACC CAGGTGGCCC TGCAATTCTT GATGCGGTTG AAGCAAAACT CAATTTAGAC

961 AAAAAGAAAC TTGAAGCAAC GAGCCATGTG TTAAGTGAGT ATGGCAACAT GTCAAGTGCA

1021 TGTGTGTTGT TTATTTTGGA TGAGATGAGA AAGAAATCAC TTAAGGGGGA GAAGGCCACC

1081 ACAGGTGAAG GATTGGATTG GGGAGTATTA TTTGGCTTTG GACCAGGCTT GACTATTGAG

1141 ACTGTTGTGT TGCATAGCAT TCCTATGGTT ACAAATTAA

4, stilbene synthase gene according to claim 3 (sts) is characterized in that 5 ' control region partial sequence of this gene is:

1 ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA

61 TTGATGTGAT ACTCCAAGAA TATCAAAGAT ACAGTCTCAG AAGACCAAAG GGCTATTGAG

121 ACTTTTCAAC AAAGGGTAAT ATCGGGAAAC CTCCTCGGAT TCCATTGCCC AGCTATCTGT

181 CACTTCATCA AAAGGACAGT AGGAAAGGAA GGTGGCACCT ACAAATGCCA TCATTGCGAT

241 AAAGGAAAGG CTATCGTTCA AGATGCCTCT GCCGACAGTG GTCCCAAAGA TGGACCCCCA

301 CCCACGAGGA GCATCGTGGA AAAAGAAGAC GTTCCAACCA CGTCTTCAAA GCAAGTGGAT

361 TGATGTGATA TCTCCACTGA CGTAAGGGAT GACGCACAAT CCCACTATCC TTCGCCGACG

421 GATCCTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA

481 TTTACAATAA CAATGGACTG TCTAGAGGAT CCGCC

5, fungi stilbene synthase gene according to claim 1 (sts) is compared with sts gene in the host plant and is not contained intron sequences, and the intron of a 361bp is contained at host plant sts gene 183bp place, the control methods difference of two kinds of sts genes, host's intron sequences is:

1 TCTAATTTTA ACATCCTTTG CGTGCATATA ATTGTGTATA CATATGATAA AGCTTTTAGA

61 TTCACCTCAG AGTACCGAAA CATCTTTTTC AAGCTTTCTG TGTACTCATT TTTAAATTAA

121 TACAATGCAT CCGTGACTGA TGCTCAGAGC AGGTGCTCTT TCAATCATAC GGTATAAAGC

181 CTGGGGTCAT ATCATTAATG TAATAAGTAA TAAGAACAAG CTTTTATATT CTATTAAGAT

241 GAATCATTTT ATACTCACTG GAACAACAAA AACTATATAC ATATTATTGA GTACTACTTG

301 TGTTTTACTT GCAATGCCTT GAGCTCACAT ATTACTGTTT TTTAATTCTT ATACAGGTGA

361 C

6, endogenetic fungus according to claim 1 is characterized in that product is to have medicinal and trans-resveratrol (Resveratrol) nourishing function, and it has the effect that improves disease resistance for plant self.

7, endogenetic fungus according to claim 1, the product after its fermentation is to have medicinal and trans-resveratrol (Resveratrol) nourishing function.

8, endogenetic fungus according to claim 1, it has obviously increased accretion rate and increment than not genetically modified contrast bacterium by the fungi that transforms behind the Vitreoscilla hemoglobin gene (VHb) that LeGDP is a promotor under the limited condition of oxygen.

9, endogenetic fungus according to claim 1, it is the gene of a kind of growth hormones of promotor by transforming LeGDP, import this fungi as isopentenyl transferase genes (ipt) and plant hormone genes involved (iaaM), the result has accelerated fungal growth speed.

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