CN108192915A - Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of ciliate desert-grass PvACR2 genes - Google Patents

Abstract

The present invention discloses a kind of initiative for the heavy metal super-enriched transgenic engineering rape for turning ciliate desert-grass PvACR2 genes, including：Clone PvACR2 genes in ciliate desert-grass；Based on plant expression vector, the PvACR2 genes are inserted into the conversion carrier by methods of homologous recombination, obtain overexpression binary vector by conversion carrier of the structure comprising strong promoter, screening-gene；Gained overexpression binary vector is transferred to by Agrobacterium tumefaciems in rape hypocotyls segment, cultivates transgenic seedling and screening transgenic positive plant；Wherein, transgene receptor plant is cabbage type rape K407 kinds.Breeding method provided by the invention can solve the problems such as wild heavy metal accumulation plant patience is limited, growth is slow, biomass is small, growing environment is special and heavy metal returns to.In addition, the present invention also provides in a kind of initiative for the heavy metal super-enriched transgenic engineering rape for turning ciliate desert-grass PvACR2 genes gained heavy metal super-enriched transfer-gen plant heavy metal-polluted soil reparation in application.

Description

Turn the initiative of the heavy metal super-enriched transgenic engineering rape of ciliate desert-grass PvACR2 genes And application

Technical field

The invention belongs to transfer-gen plants and heavy metal pollution regulation field, and in particular to turn ciliate desert-grass PvACR2 genes The initiative and application of heavy metal super-enriched transgenic engineering rape.

Background technology

Heavy metal pollution problem has become a worldwide great environmental problem, heavy metal pollution not only threaten animals and plants, Micro-organisms are developed, and also threaten human health or even life by food chain etc..Lead to the heavy metal of soil pollution mainly to have As, Cd, Co, Cr, Cu, Hg etc., the combined pollution of generally several heavy metal species.Heavy metal mining area, the weight in rapid economic development area Metallic pollution situation is particularly acute, and many agricultural product in China is caused to be denied access to international market due to heavy metals exceeding standard.

Various countries increasingly pay attention to the ecological effect of heavy metal pollution and the research of prevention, have explored a variety of improvement skills Art.However traditional heavy metal-polluted dyeing technique such as soil solidification, vitrifying, leaching, wash local method, heavy metal pollution of soil, electrochemical process etc. Physico-chemical process, it is not only with high costs, it is difficult to large-scale use, and frequently result in soil texture destruction, geobiont work Property decline and soil fertility degeneration.Therefore, it is inexpensively permanent effective to seek one kind, and can be with the replacement method of maintenance of soil fertility The difficult point and hot spot always studied in the world.

The strategy to be cleared the pollution off using green plants, i.e. phytoremediation technology have it is at low cost, it is environmental-friendly and can be again The features such as utilization is a kind of very promising environmental pollution original position governance way.The phytoremediation of broad sense refers to be extracted by plant It takes, convert, fixing, volatilizing, the process that degradation removes the pollutants such as soil and Organic Pollutants In Water, heavy metal. By plant of the plantation with heavy metal accumulation ability, by plant harvesting and deal carefully with and (be such as ashed), to reach a huge sum of money Belong to and remove soil environment, remove the purpose of heavy metal pollution of soil.

Gene is to determine most important factor of the plant to heavy metal-polluted soil absorption and accumulation characteristic, and heavy metal accumulation plant is resistance to heavy In terms of the mechanism of metal mainly includes three, first, Rejection mechanism hinders the absorption or transport in vivo of heavy metal, after absorption again It excretes；Second, localization mechanism makes heavy metal be accumulated in privileged sites such as the vacuoles, epidermal hair, cell wall of plant, from And be isolated with the other components in cell, achieve the effect that removing toxic substances；Mechanism, plant substance in vivo and heavy metal network is complexed in third It closes, including being complexed to form sulfide with inorganic matter, after small organic molecule such as GSH, oxalic acid, histidine and citric acid complexing Assemble in vacuole, be complexed with the metal chelating albumen of macromolecular.But at present, there are patience to have for wild heavy metal accumulation plant The problems such as limit, growth are slowly, biomass is small, growing environment is special and heavy metal returns to.Technique for gene engineering is current soil Therefore the most High biotechnology of prospect in heavy metal repairing research, by the heavy metal super-enriched gene cloning of plant and turns Move on to biomass is big, on the rapid plant of production to improve the ability of phytoremediation, be to solve the problems, such as heavy metal pollution of soil Effective ways.

Invention content

The purpose of the present invention is to provide a kind of heavy metal super-enriched transgenic engineering oil for turning ciliate desert-grass PvACR2 genes The initiative and application of dish, solving wild heavy metal accumulation plant, there are patience is limited, growth is slow, biomass is small, growing environment The problems such as special and heavy metal returns to.

To achieve the above object, the present invention provides a kind of heavy metal super-enriched transgenosis work for turning ciliate desert-grass PvACR2 genes The initiative of journey rape refers specifically to the construction method of heavy metal super-enriched transgenic engineering rape, including：

(1) clone of heavy metal super-enriched gene clones PvACR2 genes in ciliate desert-grass, ciliate desert-grass (Pteris Vittata) it is a kind of heavy metal super-enriched plant for being found in south China mining area.Because it has superpower resistance to heavy metal And accumulation ability, be current heavy-metal contaminated soil repairing test a kind of main rehabilitation plant.PvACR2 is responsible for trivalent The important albumen of arsenic ion reduction, result of study shows that PvACR2 is the key gene that ciliate desert-grass carries out arsenic accumulation and removing toxic substances, right Plant maintains normal growth and development to play an important roll in soil environment containing arsenic；

(2) structure of overexpression binary vector, based on plant expression vector, structure includes strong promoter, screening The PvACR2 genes that step (1) clone obtains are inserted into the conversion carrier by the conversion carrier of gene by methods of homologous recombination, Obtain overexpression binary vector；

(3) heavy metal super-enriched transfer-gen plant structure, passes through crown gall by the overexpression binary vector obtained by step (2) Agrobacterium is transferred in rape hypocotyls segment, carries out screening and culturing with screening and culturing medium, embryo under the transgenic resistance rape of acquisition Axis segment is by inducing, breaking up, taking root, the exercise and transplanting of transgenic seedling, screens heavy metal super-enriched transgenic positive plant；

Wherein, the PvACR2 gene nucleotide series such as SEQ ID NO：Shown in 1, the rape variety is Wild cabbage type oil Dish K407.

Preferably, in the step (1), the method for cloning PvACR2 genes in ciliate desert-grass is：

Using the cDNA of ciliate desert-grass as template, with such as SEQ ID NO：2 and SEQ ID NO：Sequence shown in 3 for primer pair into Row PCR amplification obtains PvACR2 genes.

Preferably, the plant expression vector is pCAMBIA1301 carriers, the strong promoter starts for Ubiquitin Son, the screening-gene are Bar screening-genes, and the conversion carrier further includes 3 × Flag labels.Using including Ubiquitin The conversion carrier of promoter, destination gene expression amount is high, and genetic stability is good, and actual application prospect protrudes, and enhances target The high conversion of gene is expected to obtain the transgenic line of target gene Efficient Conversion.In addition, 3 × Flag labels of addition, it can be square Just transformation efficiency and genetic stability analysis are carried out in protein level, is expected to filter out with lasting stability heredity for application practice Transgenic line.

Preferably, the construction method of conversion carrier described above includes the following steps：

(a) 3 × Flag marks are added between pCAMBIA1301 vector multiple cloning sites Kpn I and the Sac I Label；

(b) the Bar screening-genes on the pCAMBIA3301 carriers are cloned using pcr amplification reaction；

(c) using the carrier obtained in restriction endonuclease Xho I digestion steps (a), and will be after the Bar screening-genes and digestion Carrier carry out homologous recombination；

(d) using the Ubiquitin promoters on Hind III and BamH I digestion pUN1301 carriers；

(e) using the recombinant vector obtained in restriction endonuclease Hind III and BamH I digestion steps (c), and by described in Ubiquitin promoters are connect with the recombinant vector after digestion.

Specifically, the nucleotide sequence of the Ubiquitin promoters such as SEQ ID NO：Shown in 4, the Bar screens base Because of nucleotide sequence such as SEQ ID NO：Shown in 5, the nucleotide sequence such as SEQ ID NO of 3 × Flag labels：Shown in 6.

Preferably, in step (b), the PCR primer of the Bar screening-genes on clone's pCAMBIA3301 carriers Sequence such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.

Preferably, the screening and culturing medium includes glufosinate-ammonium.

Preferably, the plant expression vector is pCAMBIA1301 carriers, the strong promoter starts for 2 × CaMV35S Son, the screening-gene are HYG screening-genes.Using the conversion carrier for including 2 × CaMV35S promoters, destination gene expression Amount is high, and genetic stability is good, and actual application prospect protrudes, and enhances the high conversion of target gene, is expected to obtain target gene The transgenic line of Efficient Conversion.

Preferably, the HYG screening-genes are the screening-gene that the pCAMBIA1301 carriers carry, the conversion carries The construction method of body includes the following steps：

(a) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and Pst I digestions；

(b) it using pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions, obtains CaMV35S and starts Son；

(c) restriction endonuclease Hind III and Pst I restriction enzyme sites are contained according to the design of the nucleotide sequence of CaMV35S promoters Primer, and carry out pcr amplification reaction by template of CaMV35S promoters, obtain containing Hind III and Pst I restriction enzyme sites CaMV35S promoters, and the CaMV35S promoters progress digestion obtained using restriction endonuclease Hind III and Pst I to PCR；

(d) digestion products obtained in step (c) are recycled, and the digestion carrier with being obtained in step (a) is connect.

Specifically, the nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO：Shown in 7, the HYG screenings The nucleotide sequence of gene such as SEQ ID NO：Shown in 8.

Preferably, the screening and culturing medium contains hygromycin.

Preferably, in step (2), the structure of the overexpression binary vector includes the following steps：

PvACR2 genes are cloned using pcr amplification reaction, the homologous recombination PCR primer of clone's PvACR2 genes includes BamH I restriction enzyme sites.

Preferably, the homologous recombination PCR primer sequence such as SEQ ID NO of clone's PvACR2 genes：12 and SEQ ID NO：Shown in 13.

Preferably, the Agrobacterium tumefaciems is GV3101.

The present invention also provides a kind of initiatives for the heavy metal super-enriched transgenic engineering rape for turning ciliate desert-grass PvACR2 genes Application of the heavy metal super-enriched transfer-gen plant of middle gained in heavy metal-polluted soil reparation.

Compared with prior art, the heavy metal super-enriched transgenic engineering provided by the invention for turning ciliate desert-grass PvACR2 genes The initiative of rape has the advantages that following several respects：Firstth, the target gene definite functions of selection conversion, come from a wild huge sum of money Belong to super enriching plant ciliate desert-grass, coding albumen to the heavy metals such as arsenic transhipment absorb it is efficient, therefore be expected to obtain for Put into practice the super enrichment transfer-gen plant used；Secondth, structure includes the conversion carrier of strong promoter, and destination gene expression amount is high, Genetic stability is good, and actual application prospect protrudes；Third starts target gene using strong promoter, enhances target gene Height conversion, therefore it is expected to obtain the transgenic line of target gene Efficient Conversion；4th, acceptor material variety selection is special, this The rape variety that invention is selected is cabbage type rape K407, and genetic transformation effect is good, and biomass is big, wide adaptability, can be Southern and northern wide geographic area uses, therefore having a extensive future in soil remediation.

Description of the drawings

Fig. 1 is PvACR2 gene electrophoretograms；

Fig. 2 is clone's PvACR2 gene bacterium colony PCR electrophoretograms；

Fig. 3 is PvACR2 gene overexpression binary vector digestion verification figures；

Fig. 4 is pUb-PvACR2-Bar overexpression binary vector figures；

Fig. 5 is p2 × 35S-PvACR2-HYG overexpression binary vector figures；

Fig. 6 schemes for the PvACR2 transgene genetic transformation stages；

Fig. 7 is PvACR2 transgenic positive plant qualification figures；

Fig. 8 is the horizontal qualification figure of PvACR2 transfer-gen plant heavy metal adsorptions.

Specific embodiment

For the purpose of the present invention, technical solution and advantageous effect is better described, below in conjunction with attached drawing and specific implementation The invention will be further described for example.It should be noted that following the methods of implementing are explained further to what the present invention was done It is bright, it should not be taken as limitation of the present invention.If material used in the embodiment of the present invention, reagent all can be from without specified otherwise Commercial sources obtain.

Embodiment 1PvACR2 gene clonings

Ciliate desert-grass seedling is extracted its RNA using Trizol methods, inverted using TOYOBO into powdered using liquid nitrogen grinding Kit is recorded by its reverse transcription into cDNA, it is designed using Primer5 according to the cDNA sequence of the NCBI PvACR2 genes provided Upstream and downstream primer, and synthetic primer, the nucleotides sequence for designing primer are classified as：

Upstream sequence：5 ' TAGAAGACTAGAAGAACG, 3 ' (such as SEQ ID NO：Shown in 2)；

Downstream sequence：5 ' AGCGACGACTAGACGAAAGC, 3 ' (such as SEQ ID NO：Shown in 3).After primer synthesis, make It is dissolved with 1 × TE, then carries out PCR amplification, using ciliate desert-grass cDNA as template, use high-fidelity enzyme GXL Premix expand it, and amplified production is carried out electrophoresis using 1% Ago-Gel, target stripe is cut, It is recycled using plastic recovery kit, amplified production electrophoretogram is as shown in Figure 1, arrow meaning band is purpose band.Then Recovery product and PMD19-T (simple) carrier are carried out to stay overnight connection, connection product is converted into Escherichia coli using heat shock method Competence Top10 chooses monoclonal and is bacterium solution PCR, after amplification, by amplified production into row agarose gel electrophoresis, electrophoretogram As shown in Fig. 2, arrow meaning 1,2, No. 3 bacterium solutions are positive bacterium solution in figure.Positive strain is sequenced, by bacterium after sequencing is correct Liquid access LB-Amp culture mediums shake bacterium, and upgrading grain is spare.

The structure of the conversion carrier of 2 heavy metal super-enriched genetically modified plants of embodiment

(1) structure comprising Ubiquitin promoters, Bar screening-genes and the label converting carriers of 3 × Flag is based on experiment PCAMBIA1301-3 × Flag carriers that room is had by oneself, obtain after being transformed by following steps：

1. obtain Bar screening-genes：PCAMBIA3301 carriers contain Bar screening-genes and CaMV35S promoters, at this In embodiment, start Bar screening-genes using the CaMV35S promoters that pCAMBIA3301 carriers carry, target gene can be enhanced Efficient Conversion, the nucleotide sequence such as SEQ ID NO of CaMV35S promoters：Shown in 9.Use Xho I inscribe cleavages PCAMBIA3301 carriers, the Ago-Gel that digestion products are carried out to 1% carry out electrophoresis, recycle small band (608bp).According to PCAMBIA1301 carrier sequences and Bar gene orders design homologous recombination primer, the nucleotides sequence for designing primer are classified as：

Upstream sequence：5‘ACAAATCTATCTCTCTCGAGTCTACCATGAGCCCAGAACG3’；

Downstream sequence：5‘TTATTATGGAGAAACTCGAGTCAAATCTCGGTGACGGGCA3’.

Wherein, upstream sequence such as SEQ ID NO：Shown in 10, downstream sequence such as SEQ ID NO：Shown in 11.Then with recycling Segment for template, carries out PCR amplification with Bar homologous recombinations primer, it is spare after recycling segment.With Xho I inscribe cleavages PCAMBIA1301-3 × Flag carriers, the Ago-Gel that digestion products are carried out to 1% carry out electrophoresis, recycle big band.By it Before the small band that is recovered to and big band carry out homologous recombination, the product after homologous recombination is converted into Escherichia coli using heat shock method Competence Top10 chooses monoclonal and is bacterium solution PCR, then sequencing company sent to be sequenced positive strain, the nucleotides sequence of Bar genes Row such as SEQ ID NO：Shown in 5, bacterium solution access LB-Kan culture mediums are shaken into bacterium after sequencing is correct, upgrading grain is spare, intermediate carrier PCAMBIA1301-3 × Flag-Bar is built successfully.

2. obtain Ubiquitin promoters：By in the culture medium containing the bacterial strain of pUN1301 carriers access LB-Kan, shake Bacterium extracts plasmid.Using Hind III and BamH I digestion pUN1301, the Ago-Gel that digestion products carry out 1% carries out electricity Swimming, recycles small band (about 2000bp).Small band is Ubiquitin promoters.

3. build conversion carrier：By the pCAMBIA1301-3 × Flag-Bar built before using Hind III and BamH I carry out digestion, and the Ago-Gel that digestion products are carried out to 1% carries out electrophoresis, recycles big band.It will be ready to before Ubiquitin promoters be attached therewith using T4 ligases, by connection product using heat shock method convert Escherichia coli sense By state Top10, picking monoclonal carries out bacterium solution PCR, positive strain access LB-Kan culture mediums is shaken bacterium, upgrading grain uses BamH I and Hind III carry out digestion verification again.Spare, the pUb-3 × Flag- that will verify that correct bacterium shake bacterium extraction plasmid Bar conversion carriers structure is completed.

(2) what the structure of the conversion carrier comprising 2 × CaMV35S promoters and HYG screening-genes was had by oneself based on laboratory PCAMBIA1301 carriers obtain after being transformed by following steps：

1. using Hind III and BamH I digestions pCAMBIA1301 by digestion products carry out 1% Ago-Gel into It is spare to recycle small band (877bp) for row electrophoresis.

2. III containing Hind and Pst I restriction endonuclease primers are designed according to CaMV35S promoter sequences, with previous step recycling Band is template, and PCR amplification is carried out using high-fidelity enzyme, and the Ago-Gel that PCR product is carried out to 1% carries out electrophoresis, recycling Band.And using Hind III and Pst I restriction endonucleases to recovery product digestion, and glue recycling is carried out to digestion products.

3. using Hind III and Pst I restriction endonucleases to pCAMBIA1301 digestions, and glue recycling is carried out to digestion products.

4. by the recovery product in 2. with 3. in recovery product be attached using T4 ligases.Connection product is used Heat shock method converts E. coli competent Top10, and picking monoclonal carries out bacterium solution PCR, by positive strain access LB-Kan cultures Base shakes bacterium, and upgrading grain carries out digestion verification again using BamH I and Hind III.It will verify that correct bacterium carries out shaking bacterium extraction Plasmid is spare, and p2 × CaMV35S-HYG conversion carriers structure is completed.The nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO：Shown in 7, HYG screening-genes nucleotide sequence such as SEQ ID NO：Shown in 8.

The PvACR2 gene overexpression binary vectors of 3 heavy metal super-enriched transfer-gen plant of embodiment

Homologous recombination primer, the nucleotide of the homologous recombination primer of design are designed according to the ORF areas both ends of PvACR2 genes Sequence is：

Upstream sequence (such as SEQ ID NO：Shown in 12)：

5‘TTCTGCAGGTCGACTCTAGAGGATCCATGGCCTCGCTTCCATCCCTCT 3’；

Downstream sequence (such as SEQ ID NO：Shown in 13)：

5‘TTATTATGGAGAAACTCGAGTCAAATCTCGGTGACGGGCA 3’。

Using the 19T carriers containing PvACR2 as template PCR amplification is carried out using high-fidelity enzyme.PCR product is carried out 1% Ago-Gel carries out electrophoresis, recycles purpose band, spare.Respectively by the pUb-3 × Flag-Bar conversion carriers built and P2 × CaMV35S-HYG conversion carriers use BamH I single endonuclease digestions, and digestion products are directly carried out glue recycling, spare.It will be homologous Recombinant fragment carries out homologous recombination with the carrier after digestion.Homologous recombination product is converted into E. coli competent using heat shock method Top10, picking monoclonal carry out bacterium solution PCR, and positive strain access LB-Kan culture mediums are shaken bacterium, upgrading grain using BamH I into Row single endonuclease digestion verifies that digestion verification figure is as shown in figure 3, the digestion in figure for pUb-PvACR2-Bar overexpression conversion carriers is tested Card figure, wherein arrow meaning band are purpose band, i.e. plasmid 1-3 is positive plasmid.It will verify that correct plasmid send sequencing public Department's sequencing.Correct plasmid will be sequenced, Agrobacterium tumefaciems GV3101 is transferred to by electric robin, the bacterium solution after conversion is coated on On Yep-Kan tablets, when its longer monoclonal bacterium colony, picking monoclonal shakes bacterium, is bacterium solution PCR, positive strain is preserved It is spare.Build successful PvACR2 genes overexpression binary vector, pUb-PvACR2-Bar conversion carriers collection of illustrative plates such as Fig. 4 institutes Show, p2 × 35S-PvACR2-HYG conversion carrier collection of illustrative plates is as shown in Figure 5.

4 transfer-gen plant genetic transformation of embodiment

Full, uniform, the normal K407 rape seeds of color and luster are chosen, after ethyl alcohol and hypochlorite disinfectant, are placed in battalion It supports on culture medium, makes its germination.The hypocotyl of rape is cut after 5-7 days, is cut into 5-8 millimeters of segment, it is accurate before use The Agrobacterium tumefaciems GV3101 bacterial strains got ready are infected, and being placed on for infecting is co-cultured on culture medium 2 days, is placed it in later Glufosinate-ammoniumpesticideng is (a concentration of：5mg/L) or hygromycin is (a concentration of：On screening and culturing medium 35mg/L), wherein, pUb-PvACR2- The screening and culturing medium using Glufosinate-ammoniumpesticideng of Bar conversion carriers, the use of p2 × 35S-PvACR2-HYG conversion carriers are mould containing tide The screening and culturing medium of element.Then, primary screening culture medium was replaced after 15-20 days until callus differentiates regeneration bud. Regeneration bud is cut, is placed on root media, a root media was replaced after 15-20 days, until it sends out roots, so Regrowth is transplanted on Nutrition Soil afterwards and carries out earth culture, obtains positive transgenic plant.PvACR2 transgene rape genetic transformations Stage diagram is as shown in fig. 6, A to infect hypocotyl figure, B is evoked callus figure in figure, and C is taken root figure for evoked callus, D Transfer-gen plant figure for acquisition.

5 positive transgenic plant of embodiment is identified and adsorption levels measure

Transfer-gen plant is identified, transgenic positive plant is obtained, by the transgenic positive plant arsenic weight of acquisition Metal is handled, and is detected wherein content of beary metal, and being compared with control treatment, is analyzed its adsorption capacity.The positive turns base Because of plant qualification figure as shown in fig. 7, in figure, M：Molecular weight marker；WT：WT lines；1-22：Transfer-gen plant.From figure As can be seen that can't detect purpose band in WT lines, and in addition to 10 and 15 in the transgenic line detected, remaining strain Destination gene expression band is respectively provided with, and is transgenic positive plant.According to positive plant qualification result, Partial Conversion water is selected Transgenic line flat high and that expression is stable carries out heavy metal adsorption level identification, and the results are shown in Figure 8.In figure, WT：Wild type Plant, other are positive transgenic plant.It can be seen from the figure that the accumulation of positive transgenic plant pair heavy metal arsenic is higher than Wild type control plants, wherein, secondly the accumulation highest of transfer-gen plant OE-4 is transfer-gen plant OE-2.Integrated data As a result it obtains, the initiative of the heavy metal super-enriched transgenic engineering rape provided by the invention for turning ciliate desert-grass PvACR2 genes is solution The certainly effective ways of huge sum of money soil contamination problem.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention And range.

Sequence table

<110>Fertile land pollution remediation technology Co., Ltd of Shenzhen

<120>Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of ciliate desert-grass PvACR2 genes

<141> 2018-01-30

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<211> 66

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

gactacaaag accatgacgg tgattataaa gatcatgaca tcgactacaa ggatgacgat 60

gacaag 66

<210> 7

<211> 1700

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

gtccccagat tagccttttc aatttcagaa agaatgctaa cccacagatg gttagagagg 60

cttacgcagc aggtctcatc aagacgatct acccgagcaa taatctccag gaaatcaaat 120

accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc atcaagaaca 180

cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg attcaaggct 240

tgcttcacaa accaaggcaa gtaatagaga ttggagtctc taaaaaggta gttcccactg 300

aatcaaaggc catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga 360

ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca 420

acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag 480

accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc 540

attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca 600

aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc 660

ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 720

cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc 780

actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac 840

gggggacctg caggtcccca gattagcctt ttcaatttca gaaagaatgc taacccacag 900

atggttagag aggcttacgc agcaggtctc atcaagacga tctacccgag caataatctc 960

caggaaatca aataccttcc caagaaggtt aaagatgcag tcaaaagatt caggactaac 1020

tgcatcaaga acacagagaa agatatattt ctcaagatca gaagtactat tccagtatgg 1080

acgattcaag gcttgcttca caaaccaagg caagtaatag agattggagt ctctaaaaag 1140

gtagttccca ctgaatcaaa ggccatggag tcaaagattc aaatagagga cctaacagaa 1200

ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag 1260

aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat 1320

acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac 1380

ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa 1440

ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct 1500

gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 1560

gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 1620

gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 1680

ttggagagaa cacgggggac 1700

<210> 8

<211> 1026

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780

cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020

aaatag 1026

<210> 9

<211> 781

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa atgaaatgaa 60

cttccttata tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt 120

cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 180

ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 240

gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta 300

ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata 360

ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat 420

tcttggagta gacgagagtg tcgtgctcca ccatgttatc acatcaatcc acttgctttg 480

aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 540

tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 600

atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 660

aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 720

ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 780

t 781

<210> 10

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

acaaatctat ctctctcgag tctaccatga gcccagaacg 40

<210> 11

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

ttattatgga gaaactcgag tcaaatctcg gtgacgggca 40

<210> 12

<211> 48

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

ttctgcaggt cgactctaga ggatccatgg cctcgcttcc atccctct 48

<210> 13

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

ttattatgga gaaactcgag tcaaatctcg gtgacgggca 40

Claims (15)

1. A kind of 1. initiative for the heavy metal super-enriched transgenic engineering rape for turning ciliate desert-grass PvACR2 genes, which is characterized in that packet It includes：

   (1) clone of heavy metal super-enriched gene

   Clone PvACR2 genes in ciliate desert-grass；

   (2) structure of overexpression binary vector

   Based on plant expression vector, conversion carrier of the structure comprising strong promoter, screening-gene clones step (1) To PvACR2 genes the conversion carrier is inserted by methods of homologous recombination, obtain overexpression binary vector；

   (3) heavy metal super-enriched transfer-gen plant structure

   Overexpression binary vector obtained by step (2) is transferred to by Agrobacterium tumefaciems in rape hypocotyls segment, with screening Culture medium carries out screening and culturing, and the transgenic resistance rape hypocotyls segment of acquisition is by inducing, breaking up, taking root, transgenic seedling Exercise and transplanting, screen heavy metal super-enriched transgenic positive plant；

   Wherein, the PvACR2 gene nucleotide series such as SEQ ID NO：Shown in 1, the rape variety is cabbage type rape K407。
2. 2. the initiative of the heavy metal super-enriched transgenic engineering rape as described in claim 1 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, in the step (1), the method for cloning PvACR2 genes in ciliate desert-grass is：

   Using the cDNA of ciliate desert-grass as template, with such as SEQ ID NO：2 and SEQ ID NO：Sequence shown in 3 is carried out for primer pair PCR amplification obtains PvACR2 genes.
3. 3. the initiative of the heavy metal super-enriched transgenic engineering rape as described in claim 1 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the plant expression vector be pCAMBIA1301 carriers, the strong promoter be Ubiquitin promoters, institute Screening-gene is stated as Bar screening-genes, the conversion carrier further includes 3 × Flag labels.
4. 4. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 3 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the construction method of the conversion carrier includes the following steps：

   (a) 3 × Flag labels are added between pCAMBIA1301 vector multiple cloning sites Kpn I and the Sac I；

   (b) the Bar screening-genes on the pCAMBIA3301 carriers are cloned using pcr amplification reaction；

   (c) using the carrier obtained in restriction endonuclease Xho I digestion steps (a), and by the load after the Bar screening-genes and digestion Body carries out homologous recombination；

   (d) using the Ubiquitin promoters on Hind III and BamH I digestion pUN1301 carriers；

   (e) using the recombinant vector obtained in restriction endonuclease Hind III and BamH I digestion steps (c), and by described in Ubiquitin promoters are connect with the recombinant vector after digestion.
5. 5. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 4 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the nucleotide sequence of the Ubiquitin promoters such as SEQ ID NO：Shown in 4, the Bar screening-genes core Nucleotide sequence such as SEQ ID NO：Shown in 5, the nucleotide sequence such as SEQ ID NO of 3 × Flag labels：Shown in 6.
6. 6. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 4 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, in step (b), the PCR primer sequence of the Bar screening-genes on clone's pCAMBIA3301 carriers Such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.
7. 7. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 3 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the screening and culturing medium includes glufosinate-ammonium.
8. 8. the initiative of the heavy metal super-enriched transgenic engineering rape as described in claim 1 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the plant expression vector is pCAMBIA1301 carriers, the strong promoter is 2 × CaMV35S promoters, The screening-gene is HYG screening-genes.
9. 9. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 8 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the HYG screening-genes are the screening-gene that the pCAMBIA1301 carriers carry, the conversion carrier Construction method includes the following steps：

   (a) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and Pst I digestions；

   (b) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions, CaMV35S promoters are obtained；

   (c) drawing containing restriction endonuclease Hind III and Pst I restriction enzyme sites is designed according to the nucleotide sequence of CaMV35S promoters Object, and pcr amplification reaction is carried out by template of CaMV35S promoters, it obtains containing Hind III and Pst I restriction enzyme sites CaMV35S promoters, and digestion is carried out to the CaMV35S promoters that PCR is obtained using restriction endonuclease Hind III and Pst I；

   (d) digestion products obtained in step (c) are recycled, and the digestion carrier with being obtained in step (a) is connect.
10. 10. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 8 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO：Shown in 7, the HYG screening-genes Nucleotide sequence such as SEQ ID NO：Shown in 8.
11. 11. the initiative of the heavy metal super-enriched transgenic engineering rape as claimed in claim 8 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the screening and culturing medium contains hygromycin.
12. 12. the wound of the heavy metal super-enriched transgenic engineering rape for turning ciliate desert-grass PvACR2 genes as described in claim 3 or 8 System, which is characterized in that in step (2), the structure of the overexpression binary vector includes the following steps：

    PvACR2 genes are cloned using pcr amplification reaction, the homologous recombination PCR primer of clone's PvACR2 genes includes BamH I restriction enzyme sites.
13. 13. the wound of the heavy metal super-enriched transgenic engineering rape as claimed in claim 12 for turning ciliate desert-grass PvACR2 genes System, which is characterized in that the homologous recombination PCR primer sequence such as SEQ ID NO of clone's PvACR2 genes：12 and SEQ ID NO：Shown in 13.
14. 14. the initiative of the heavy metal super-enriched transgenic engineering rape as described in claim 1 for turning ciliate desert-grass PvACR2 genes, It is characterized in that, the Agrobacterium tumefaciems is GV3101.
15. 15. as claim 1~14 any one of them turns the heavy metal super-enriched transgenic engineering of ciliate desert-grass PvACR2 genes Application of the heavy metal super-enriched transfer-gen plant of gained in heavy metal-polluted soil reparation in the initiative of rape.