CN108315348A - Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of Nipponbare rice Os Nramp5 genes - Google Patents

Abstract

The present invention discloses a kind of initiative for the heavy metal super-enriched transgenic engineering rice turning Nipponbare rice Os Nramp5 genes, including：OsNramp5 genes in cloning rice；Based on plant expression vector, the OsNramp5 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 Rice Callus, transgenic seedling and screening transgenic positive plant are cultivated；Wherein, transgene receptor plant is rice Nipponbare kind.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 application the present invention also provides a kind of heavy metal super-enriched transfer-gen plant of gained in initiative for the heavy metal super-enriched transgenic engineering rice turning Nipponbare rice Os Nramp5 genes in heavy metal-polluted soil reparation.

Description

Turn the heavy metal super-enriched transgenic engineering rice of Nipponbare rice Os Nramp5 genes Initiative and application

Technical field

The invention belongs to transfer-gen plants and heavy metal pollution regulation field, and in particular to turn Nipponbare rice Os Nramp5 The initiative and application of the heavy metal super-enriched transgenic engineering rice of gene.

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..The heavy metal of soil pollution is caused mainly to have As, Cd, Co, Cr, Cu, Hg etc., the combined pollution of generally several heavy metal species.Heavy metal mining area, rapid economic development area weight 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 deals carefully with and (be such as ashed), to reach a huge sum of money Belong to and remove soil environment, removes the purpose of heavy metal pollution of soil.

Gene is the most important factor for determining 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 is complexed to form sulfide with inorganic matter, after small organic molecule such as GSH, oxalic acid, histidine and citric acid complexing Assemble in vacuole, is complexed with the metal chelating albumen of macromolecular.But currently, 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 Most therefore the High biotechnology of foreground by the heavy metal super-enriched gene cloning of plant and turns in heavy metal repairing research 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 transgenosis turning Nipponbare rice Os Nramp5 genes The initiative and application of engineering rice, solve wild heavy metal accumulation plant there are patience limited, growth slowly, biomass it is small, raw The problems such as long environment is special and heavy metal returns to.

To achieve the above object, the present invention provides a kind of turning Nipponbare rice Os Nramp5 genes heavy metal super-enriched turn The initiative of Transgenic rice refers specifically to the construction method of heavy metal super-enriched transgenic engineering rice, including：

(1) clone of heavy metal super-enriched gene, OsNramp5 genes in cloning rice.Result of study shows OsNramp5 albumen has stronger adsorption capacity to heavy metal ion such as cadmium, manganese, is the key that the heavy metals such as rice cadmium Gene, the overexpression gene can significantly improve adsorption capacity of the rice to heavy metals such as cadmiums；

(2) structure of overexpression binary vector, based on plant expression vector, structure includes strong promoter, screening The OsNramp5 genes that rapid (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 Rice Callus, and screening and culturing, the transgenic resistance rice callus group of acquisition are carried out with screening and culturing medium It knits by induction, break up, take root, the exercise and transplanting of transgenic seedling, screen heavy metal super-enriched transgenic positive plant；

Wherein, the OsNramp5 gene nucleotide series such as SEQ ID NO：Shown in 1, the transgenic engineering rice product Kind is Nipponbare.

Preferably, in the step (1), the method for OsNramp5 genes is in cloning rice：

Using the cDNA of rice as template, with such as SEQ ID NO：2 and SEQ ID NO：Sequence shown in 3 carries out for primer pair PCR amplification obtains OsNramp5 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) carrier obtained in restriction endonuclease Xho I digestion steps (a) is used, and will be after the Bar screening-genes and digestion Carrier carry out homologous recombination；

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

(e) recombinant vector obtained in restriction endonuclease Hind III and BamH I digestion steps (c) is used, and will be described 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) pCAMBIA1301 carriers described in restriction endonuclease Hind III and Pst I digestions are used；

(b) pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions are used, CaMV35S is obtained 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 using CaMV35S promoters as template carry out pcr amplification reaction, obtain contain Hind III and Pst I restriction enzyme sites CaMV35S promoters, and the CaMV35S promoters progress digestion that PCR is obtained using restriction endonuclease Hind III and Pst I；

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

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：

OsNramp5 genes, the homologous recombination PCR primer of clone's OsNramp5 genes are cloned using pcr amplification reaction Including BamH I restriction enzyme sites.

Preferably, the homologous recombination PCR primer sequence such as SEQ ID NO of clone's OsNramp5 genes：12 and SEQ ID NO：Shown in 13.

Preferably, the Agrobacterium tumefaciems is EHA105.

The present invention also provides a kind of heavy metal super-enriched transgenic engineering rice turning Nipponbare rice Os Nramp5 genes Initiative in gained heavy metal super-enriched transfer-gen plant heavy metal-polluted soil reparation in application.

Compared with prior art, provided by the invention to turn the heavy metal super-enriched of Nipponbare rice Os Nramp5 genes and turn base Because the initiative of engineering rice has the advantages that following several respects：The first, the target gene definite functions of selection conversion, to cadmium, manganese Etc. heavy metal ion have stronger adsorption capacity, be the key gene of the heavy metals such as rice cadmium, the overexpression gene Adsorption capacity of the rice to heavy metals such as cadmiums can be significantly improved, therefore can be obtained for putting into practice the super enrichment transgenosis used Plant；The second, structure includes the conversion carrier of strong promoter, and destination gene expression amount is high, and genetic stability is good, before practical application Scape protrudes；Third starts target gene using strong promoter, enhances the high conversion of target gene, therefore is expected to obtain target The transgenic line of gene efficient conversion；4th, acceptor material variety selection is special, and the rice varieties that the present invention selects are Japan Fine rice, genetic transformation effect is good, and biomass is big, wide adaptability, can be used in southern and northern wide geographic area, therefore in soil Having a extensive future in reparation.

Description of the drawings

Fig. 1 is OsNramp5 gene electrophoretograms；

Fig. 2 is clone's OsNramp5 gene bacterium colony PCR electrophoretograms；

Fig. 3 is OsNramp5 gene overexpression binary vector digestion verification figures；

Fig. 4 is pUb-OsNramp5-Bar overexpression binary vector figures；

Fig. 5 is p2 × 35S-OsNramp5-HYG overexpression binary vector figures；

Fig. 6 is to scheme the OsNramp5 transgene genetic transformation stages；

Fig. 7 is OsNramp5 transgenic positive plant qualification figures；

Fig. 8 is the horizontal qualification figure of OsNramp5 transfer-gen plant heavy metal adsorptions.

Specific implementation mode

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.

1 OsNramp5 gene clonings of embodiment

Rice seedling is extracted its RNA using Trizol methods, use TOYOBO reverse transcriptions at powdered using liquid nitrogen grinding Its reverse transcription at cDNA, it is designed according to the cDNA sequence of the NCBI OsNramp5 genes provided by kit using Primer5 The nucleotides sequence of upstream and downstream primer, and synthetic primer, design primer is classified as：

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

Downstream sequence：5 ' GCGATAGAGAGCACAAATA, 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 rice cDNA as template, use high fidelity enzymeGXL Premix expands it, and amplified production is carried out electrophoresis using 1% Ago-Gel, target stripe is cut, is used Plastic recovery kit is recycled, and amplified production electrophoretogram is as shown in Figure 1, arrow meaning band is purpose band.Then it will return It receives product to carry out staying overnight connection with PMD19-T (simple) carrier, connection product is experienced using heat shock method conversion Escherichia coli State Top10 chooses monoclonal and is bacterium solution PCR, after amplification, by amplified production into row agarose gel electrophoresis, electrophoretogram such as Fig. 2 Shown, arrow meaning 1-5 bacterium solutions are positive bacterium solution in figure.Positive strain is sequenced, accesses bacterium solution after sequencing is correct 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 Own pCAMBIA1301-3 × Flag the carriers in room, obtain after being transformed by following steps：

1. obtaining Bar screening-genes：PCAMBIA3301 carriers contain Bar screening-genes and CaMV35S promoters, at this In embodiment, starts 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, and the nucleotides sequence of design primer is classified as：On Swim sequence：5‘ACAAATCTATCTCTCTCGAGTCTACCATGAGCCCAGAACG 3’；Downstream sequence：5 ‘TTATTATGGAGAAACTCGAGTCAAATCTCGGTGACGGGCA 3’.Wherein, upstream sequence such as SEQ ID NO：Shown in 10, Downstream sequence such as SEQ ID NO：Shown in 11.Then using the segment of recycling as template, PCR expansions are carried out with Bar homologous recombinations primer Increase, it is spare after recycling segment.With Xho I inscribe cleavage pCAMBIA1301-3 × Flag carriers, digestion products are carried out 1% Ago-Gel carry out electrophoresis, recycle big band.The small band and big band that are recovered to before are subjected to homologous recombination, it will be same Product after the recombination of source converts E. coli competent Top10 using heat shock method, chooses monoclonal and is bacterium solution PCR, then will be positive Bacterial strain send sequencing company to be sequenced, the nucleotide sequence such as SEQ ID NO of Bar genes：Shown in 5, bacterium solution is accessed after sequencing is correct LB-Kan culture mediums shake bacterium, and upgrading grain is spare, and intermediate carrier pCAMBIA1301-3 × Flag-Bar is built successfully.

2. obtaining Ubiquitin promoters：It will access in the culture medium of LB-Kan, shake containing the bacterial strain of pUN1301 carriers 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. building 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.Correct bacterium spare, the pUb-3 × Flag- that shake bacterium extraction plasmid will be verified 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 2. recovery product 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.Correct bacterium will be verified to carry 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 OsNramp5 gene overexpression binary vectors of 3 heavy metal super-enriched transfer-gen plant of embodiment

Homologous recombination primer, the nucleosides of the homologous recombination primer of design are designed according to the areas the ORF both ends of OsNramp5 genes Acid sequence is：

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

5‘TTCTGCAGGTCGACTCTAGAGGATCCATGGAGATTGAGAGAGAGAGCAGTGAGAG 3’；

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

5‘CTTTGTAGTCGGTACCCGGGGATCCCCTTGGGAGCGGGATGTCGG 3’。

Using the 19T carriers containing OsNramp5 as template PCR amplification is carried out using high fidelity enzyme.PCR product is carried out 1% Ago-Gel carry out electrophoresis, recycle purpose band, it is spare.PUb-3 × Flag-Bar the conversion carriers that will be built respectively BamH I single endonuclease digestions are used with p2 × CaMV35S-HYG conversion carriers, digestion products are directly subjected to glue recycling, it is spare.It will be same Source recombinant fragment carries out homologous recombination with the carrier after digestion.Homologous recombination product is experienced using heat shock method conversion Escherichia coli State Top10, picking monoclonal carry out bacterium solution PCR, positive strain access LB-Kan culture mediums are shaken bacterium, upgrading grain uses BamH I Single endonuclease digestion verification is carried out, digestion verification figure is as shown in figure 3, be the enzyme of pUb-OsNramp5-Bar overexpression conversion carriers in figure Proof diagram is cut, wherein arrow meaning band is purpose band, i.e. plasmid 1-4 is positive plasmid.Correct plasmid will be verified and send survey Sequence company is sequenced.Correct plasmid will be sequenced, Agrobacterium tumefaciems EHA105 is transferred to by electric robin, the bacterium solution after conversion is coated with On Yep-Kan tablets, when its longer monoclonal colonies, picking monoclonal shakes bacterium, is bacterium solution PCR, positive strain is protected It deposits spare.Successful OsNramp5 genes overexpression binary vector is built, pUb-OsNramp5-Bar conversion carriers collection of illustrative plates is such as Shown in Fig. 4, p2 × 35S-OsNramp5-HYG conversion carrier collection of illustrative plates is as shown in Figure 5.

4 transfer-gen plant genetic transformation of embodiment

Choose full, uniformly, the normal Nipponbare rice paddy seed of color and luster after hypochlorite disinfectant, is placed in more using ethyl alcohol Hinder on inducing culture, while preparing to infect required Agrobacterium.Callus is grown after 5-7 days, ready before use EHA105 Agrobacterium tumefaciems is infected, and being placed on co-cultivation culture medium for infecting uses the nothing containing cephalo after 3 days, 3 days Bacterium water cleans it, and it is (a concentration of to place it in Glufosinate-ammoniumpesticideng later：5mg/L) or hygromycin is (a concentration of：35mg/L) It is cultivated two weeks on screening and culturing medium, wherein the screening and culturing medium using Glufosinate-ammoniumpesticideng of pUb-OsNramp5-Bar conversion carriers, Screening and culturing medium of the use of p2 × 35S-OsNramp5-HYG conversion carriers containing hygromycin.In order to reduce transgenosis false positive Rate moves on to newly longer resistant calli on new screening and culturing medium, until graininess kanamycin-resistant callus tissue is grown.It will Resistant calli is transferred on RE-III culture mediums, until it grows seedling.Seedling is transferred on HF culture mediums after growing and lures It leads and takes root；Equal seedlings, which grow to bottle cap height, uncaps and sterile water is added practices seedling.It can be transplanted in Nutrition Soil after young plant grows up. For OsNramp5 transgenic paddy rice genetic transformation stage diagrams as shown in fig. 6, A is evoked callus figure in figure, B is to obtain resistance to be cured Injured tissue figure, C are that evoked callus is taken root figure, and D be the transfer-gen plant figure of acquisition.

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

Transfer-gen plant is identified, transgenic positive plant is obtained, by a transgenic positive plant huge sum of money for acquisition Belong to cadmium to be handled, detection wherein heavy metal cadmium content, and be compared with control treatment, analyzes its adsorption capacity.The positive turns For gene plant qualification figure as shown in fig. 7, in figure, WT is WT lines, and abscissa is transfer-gen plant, and ordinate is target base Because of expression, it can be seen from the figure that the gene expression of transfer-gen plant U1, U2, U7, U8, U9, U10, U11, U12, U13 Amount is high, is positive transgenic plant.Then according to positive plant qualification result, selects the horizontal height of Partial Conversion and express stabilization Transgenic line carries out heavy metal adsorption level identification, and the results are shown in Figure 8.From figure 8, it is seen that transgenic positive plant pair The accumulation of heavy metal cadmium is apparently higher than control Wild plant, wherein accumulations of the transgenic positive plant U1 to heavy metal cadmium Secondly highest is transfer-gen plant U7, U8.Integrated data result obtains, provided by the invention to turn Nipponbare rice Os Nramp5 The initiative of the heavy metal super-enriched transgenic engineering rice of gene is to solve the 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 is protected to the present invention The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled 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>Guangdong Kaiyuan Environment Technology Co., Ltd.

<120>Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of Nipponbare rice Os Nramp5 genes

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tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga 1440

tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat 1500

cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt 1560

tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc 1620

ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg 1680

tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt 1740

atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc 1800

atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt ataattattt 1860

tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc 1920

tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg 1980

tttggtgtta cttctgcagg tcgactctag a 2011

<210> 5

<211> 552

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60

gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120

caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180

gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240

aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300

ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360

agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420

ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480

gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540

accgagattt ga 552

<210> 6

<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> 55

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

ttctgcaggt cgactctaga ggatccatgg agattgagag agagagcagt gagag 55

<210> 13

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

ctttgtagtc ggtacccggg gatccccttg ggagcgggat gtcgg 45

Claims (15)

1. a kind of initiative for the heavy metal super-enriched transgenic engineering rice turning Nipponbare rice Os Nramp5 genes, feature exist In, including：

(1) clone of heavy metal super-enriched gene

OsNramp5 genes in cloning rice；

(2) structure of overexpression binary vector

Based on plant expression vector, conversion carrier of the structure comprising strong promoter, screening-gene obtains rapid (1) clone OsNramp5 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 Rice Callus, is trained with screening Support base and carry out screening and culturing, the transgenic resistance Rice Callus of acquisition by inducing, breaking up, taking root, the forging of transgenic seedling Refining and transplanting, screen heavy metal super-enriched transgenic positive plant；

Wherein, the OsNramp5 gene nucleotide series such as SEQ ID NO：Shown in 1, the transgenic engineering rice varieties are Nipponbare.

2. turning the heavy metal super-enriched transgenic engineering rice of Nipponbare rice Os Nramp5 genes as described in claim 1 Initiative, which is characterized in that in the step (1), the method for OsNramp5 genes is in cloning rice：

Using the cDNA of rice as template, with such as SEQ ID NO：2 and SEQ ID NO：Sequence shown in 3 is that primer pair carries out PCR Amplification, obtains OsNramp5 genes.

3. the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the plant expression vector is pCAMBIA1301 carriers, and the strong promoter starts for Ubiquitin Son, the screening-gene are Bar screening-genes, and the conversion carrier further includes 3 × Flag labels.

4. the heavy metal super-enriched transgenic engineering rice as claimed in claim 3 for turning Nipponbare rice Os Nramp5 genes Initiative, which 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) carrier obtained in restriction endonuclease Xho I digestion steps (a) is used, and by the load after the Bar screening-genes and digestion Body carries out homologous recombination；

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

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

5. the heavy metal super-enriched transgenic engineering rice as claimed in claim 4 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the nucleotide sequence of the Ubiquitin promoters such as SEQ ID NO：Shown in 4, the Bar screenings Gene nucleotide series such as SEQ ID NO：Shown in 5, the nucleotide sequence such as SEQ ID NO of 3 × Flag labels：Shown in 6.

6. the heavy metal super-enriched transgenic engineering rice as claimed in claim 4 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that 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.

7. the heavy metal super-enriched transgenic engineering rice as claimed in claim 3 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the screening and culturing medium includes glufosinate-ammonium.

8. the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the plant expression vector is pCAMBIA1301 carriers, and the strong promoter is that 2 × CaMV35S is opened Mover, the screening-gene are HYG screening-genes.

9. the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the HYG screening-genes are the screening-gene that the pCAMBIA1301 carriers carry, and the conversion carries The construction method of body includes the following steps：

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

(b) pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions are used, 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, acquisition contains 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 is connect with the digestion carrier obtained in step (a).

10. the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO：Shown in 7, the HYG sieves Select the nucleotide sequence such as SEQ ID NO of gene：Shown in 8.

11. the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the screening and culturing medium contains hygromycin.

12. the heavy metal super-enriched transgenic engineering water for turning Nipponbare rice Os Nramp5 genes as described in claim 3 or 8 The initiative of rice, which is characterized in that in step (2), the structure of the overexpression binary vector includes the following steps：

OsNramp5 genes are cloned using pcr amplification reaction, the homologous recombination PCR primer of clone's OsNramp5 genes includes BamH I restriction enzyme sites.

13. the heavy metal super-enriched transgenic engineering rice as claimed in claim 12 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that it is described clone OsNramp5 genes homologous recombination PCR primer sequence such as SEQ ID NO：12 Hes SEQ ID NO：Shown in 13.

14. the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning Nipponbare rice Os Nramp5 genes Initiative, which is characterized in that the Agrobacterium tumefaciems be EHA105.

15. turning base as claim 1~14 any one of them turns the heavy metal super-enriched of Nipponbare rice Os Nramp5 genes Because in the initiative of engineering rice gained heavy metal super-enriched transfer-gen plant heavy metal-polluted soil reparation in application.