CN112458098A - Cadmium-resistant gene Vvmrp1S derived from grape and application thereof - Google Patents

Abstract

A gene synthesis method is used for transforming a Vvmrp1 gene derived from grapes to obtain the Vvmrp1S gene with a nucleotide sequence shown as SEQ ID No1, a recombinant expression vector is constructed, the cadmium-resistant gene Vvmrp1S is successfully introduced into Arabidopsis thaliana, and the obtained transgenic Arabidopsis thaliana plants have strong cadmium resistance and enrichment capacity. After being treated by a cadmium solution within 5mM, the biomass of the transgenic line is more than that of the control line, and the cadmium content in the transgenic line is 3-5 times of that of the control line, which shows that the Vvmrp1S gene modified and synthesized by the invention has the capability of improving the resistance of plants and enriching cadmium, and can be used for cultivating plants enriching cadmium.

Description

Cadmium-resistant gene Vvmrp1S derived from grape and application thereof

Technical Field

The invention belongs to the technical field of cultivating cadmium-enriched plants, and particularly relates to a cadmium-resistant gene Vvmrp1S from grapes and application thereof.

Background

Cadmium (Cd) is a heavy metal element with the strongest biological toxicity, has strong chemical activity in the environment, large mobility and lasting toxicity, is easy to endanger human health through the enrichment function of a food chain, has the functions of causing diseases, carcinogenesis and mutation on a human body, and can induce diseases such as renal decay, arthritis, cancer and the like.

Cadmium easily enters human bodies through the approach of crops to cause toxicity. World health organization (2003) andthe American environmental protection agency (1994) stipulates that the maximum allowable intake (ADI value) of Cd in human bodies is 1 mu g kg^－1·d^－1. Cadmium is an accumulated extremely toxic element, the content of cadmium in certain organs of a human body increases with the age, and researches on aspects of environmental behaviors, pollution prevention and the like of cadmium are widely concerned.

With the continuous acceleration of the urbanization process of China, industries such as mining, tanning, smelting, electroplating, caustic soda manufacturing, garbage incineration, sewage irrigation and the like are vigorously developed in China at the present stage, but due to the conditions of incomplete management system, relatively backward technology, low efficient recycling efficiency and the like existing in the process, a large amount of heavy metal pollutants such as lead, mercury, cadmium, cobalt and the like enter the atmosphere, water and soil, and the problem of serious environmental pollution is caused.

Heavy metal pollution not only can cause the reduction of the production capacity of soil, but also can be absorbed by roots, transferred into roots, stems, leaves and fruits of crops and finally accumulated into human bodies through a food chain, thereby harming the health of human bodies. Since the middle of the 70 th century in the 20 th century, China began to investigate cadmium pollution of farmland soil, at present, the content of heavy metals in farmland in China is obviously increased, the cadmium pollution condition of soil is more and more serious, the cadmium-polluted soil area in China reaches 20 ten thousand square kilometers, which accounts for 1/6 of the total cultivated land area in China, the cadmium-polluted soil relates to 11 provinces and 25 regions, so that the yield of grain in China is reduced by 1000 ten thousand tons every year, the economic loss reaches 200 hundred million yuan, and in field crops, the main pollutants of agricultural products are heavy metals, especially cadmium is the most prominent.

The cadmium pollution of soil causes the quality reduction of agricultural products of rice and vegetables in China. In 2000, the environment monitoring system of the Ministry of agriculture detects 2110 samples of 14 provincial meeting cities, the pollution standard exceeding rate of heavy metal cadmium and the like in vegetables reaches 23.5%, and the leaf sample analysis of 18 detection points in the suburb of Nanjing shows that the cadmium content exceeds the food sanitation standard by 17 times at most.

How to reduce the cadmium content in the soil environment, reduce the pollution to crop products and ensure the ecological system, especially the human health, has become a leading point and a difficult point of international research in the field of crossing soil plant nutrition and ecological environment.

Because the heavy metal elements in the soil can not be degraded by the microorganisms in the soil, great difficulty is added to pollution treatment, and therefore, how to effectively and thoroughly remove the heavy metal elements in the soil is a difficult point of research. Conventional soil heavy metal pollution treatment methods, such as physical methods and chemical methods, have strict requirements on the technology, have high economic cost, are easy to cause secondary pollution to the soil, may damage the soil structure in the remediation process, and have various adverse factors limiting large-scale popularization and application.

The phytoremediation has the advantages of low remediation cost, easy operation, no damage to the soil structure and the like, is developed into the technology with the most development prospect for the heavy metal remediation of the soil at present, and opens up a new way for the soil remediation.

ABC transporters, also known as adenosine triphosphate binding cassette transporters (ATP-binding cassette transporters), are one of the largest, oldest family of proteins known to date. This protein is called ABC transporter because it contains an Adenosine Triphosphate (ATP) binding cassette in its structure and functions to transport substances.

All ABC transporters have a structure that contains a highly conserved ATP-binding domain (NBF) and transmembrane domain (TMDs), and thus, they function as a membrane-integrating protein to transport them. The transported substrates mainly comprise peptides, sugar, lipid, heavy metal chelate, polysaccharide, alkaloid, steroid, inorganic ions, glutathione conjugate and the like.

ABC transporters are widely present in eukaryotes and prokaryotes, among them, MRP (15 members) found in the organisms at present belong to ABC whole-molecule transporters, and AtMRP1 and AtMRP2 in Arabidopsis have glutathione-conjugated transport activity, and can transport glutathione compounds in cytoplasm into vacuoles.

At present, no research report of applying MRP gene to cadmium resistance of plants is found.

Disclosure of Invention

The invention aims to provide a cadmium-resistant gene Vvmrp1S derived from grapes and application thereof, which can be efficiently expressed in arabidopsis thaliana, and the cadmium-resistant gene Vvmrp1S can be transformed into arabidopsis thaliana or rice to improve the cadmium resistance of transgenic arabidopsis thaliana and transgenic rice, and has important theoretical significance and practical significance for cultivating and improving cadmium resistance and enrichment plant varieties.

In order to achieve the purpose, the invention provides the following technical scheme:

a cadmium-resistant gene Vvmrp1S derived from grape has a nucleotide sequence shown in SEQ ID No. 1.

A recombinant expression vector containing the cadmium-resistant gene Vvmrp 1S.

Preferably, the recombinant expression vector further comprises a double 35S promoter, a GUS reporter gene and an intron-containing kanamycin resistance marker gene.

The invention provides application of a cadmium-resistant gene Vvmrp1S derived from grapes in cultivation of cadmium-resistant plants.

Preferably, the cadmium-tolerant plant is arabidopsis thaliana or rice.

A method for transferring the cadmium-resistant gene Vvmrp1S derived from grapes into Arabidopsis thaliana comprises the following steps:

1) amplification of cadmium-resistant genes

Designing primers according to the sequence of the cadmium-resistant gene Vvmrp1S, wherein the inner primers are respectively 1.5ng, the outer two primers are respectively 30ng, and carrying out PCR to amplify a Vvmrp1S fragment;

2) construction of recombinant expression vectors

After PCR amplification is finished, recovering a product, connecting the product with a T/A cloning vector to obtain a DNA connecting product, and efficiently transforming into DH5 alpha competent cells;

after carrying out double enzyme digestion on a PCR product by using BamHI and SacI, recovering a Vvmrp1S gene fragment, connecting the recovered Vvmrp1S gene with a plant expression vector 1301 containing a double 35S promoter by using T4DNA ligase, carrying out enzyme digestion identification and sequence determination to obtain a recombinant plasmid AH555 containing a target gene Vvmrp1S, wherein the expression vector also comprises a GUS reporter gene and an intron kanamycin resistance marker gene;

3) transformation of Arabidopsis thaliana

The recombinant plasmid AH555 is introduced into agrobacterium tumefaciens LBA4404 by an electric shock method, the agrobacterium tumefaciens LBA4404 containing Vvmrp1S gene which is constructed by the agrobacterium transformation by a flower dipping method is transformed into arabidopsis, hygromycin is used for screening transformed plants, normal seedlings grow on a hygromycin plate for seedling transplanting, and the seedlings are harvested for positive seedling identification.

The cadmium-resistant gene Vvmrp1S is derived from a grape MRP1 gene, the encoded protein belongs to ABC transporter, the grape-derived MRP1 gene is firstly modified and then used in Arabidopsis thaliana for plant cadmium-resistant research, the cadmium-resistant performance of plants is improved, and the functions of the plant cadmium-resistant gene in the aspects of plant cadmium resistance and accumulation are verified.

Transferring the Vvmrp1S gene into arabidopsis thaliana, selfing and homozygous for 3 generations to obtain homozygous transformant, collecting seeds, sowing, continuously irrigating with cadmium aqueous solution, and respectively determining the cadmium content in the transgenic line and the control line before phenotype difference occurs, wherein the cadmium content in the transgenic line is 3-5 times that in the control line, thereby greatly improving the cadmium enrichment of arabidopsis thaliana. After three times of continuous irrigation, the transgenic plants still grow stably, have large and green leaves and can bolt and seed. However, the growth of the wild control strain is seriously influenced, and the fructification is obviously delayed, which shows that the cadmium resistance of the transgenic arabidopsis into which the Vvmrp1S gene of the invention is transferred is obviously improved.

The invention has at least the following beneficial effects:

the invention adopts a gene synthesis method to synthesize a cadmium-resistant gene Vvmrp1S from grapes, which can be successfully transformed into arabidopsis thaliana and efficiently expressed in arabidopsis thaliana to obtain transgenic arabidopsis thaliana; the Arabidopsis plant with the Vvmrp1S gene transferred thereto and a wild Arabidopsis plant have obvious difference in cadmium resistance, the transgenic Arabidopsis can tolerate continuous irrigation treatment of a cadmium aqueous solution within 5mM, after continuous irrigation is carried out for 3 times by using the 5mM cadmium aqueous solution, the cadmium enrichment capacity of the transgenic Arabidopsis is 3-5 times that of the wild Arabidopsis, the leaves of the transgenic Arabidopsis are large and green, the phenotype is obviously superior to that of the wild Arabidopsis, and the transfer of the Vvmrp1S gene is shown to improve the cadmium resistance and the cadmium enrichment capacity of the Arabidopsis plant.

Drawings

FIG. 1 is a schematic diagram showing the construction of recombinant plant plasmid AH555 containing a target gene according to the present invention.

FIG. 2 is a diagram showing the PCR amplification result of transgenic Arabidopsis positive seedlings in example 4 of the present invention, in which M is DNA mark, WT is a wild-type plant, and 555-2, 555-4, and 555-5 are three lines of transgenic Arabidopsis, respectively.

FIG. 3 is a cadmium-resistant phenotype diagram of Arabidopsis thaliana transformed with Vvmrp1S gene after cadmium irrigation and wild Arabidopsis thaliana in example 5 of the present invention, wherein: WT is wild type Arabidopsis thaliana, and 555-2, 555-4 and 555-5 are three strains of transgenic Arabidopsis thaliana respectively.

Detailed Description

The present invention will be further described with reference to the following embodiments.

The test materials and sources used in the present invention include:

wild type Arabidopsis thaliana (Arabidopsis thaliana) and ecological type Arabidopsis thaliana Columbia are cultured in an artificial climate room at 23 ℃ and are cultured under illumination for 16 h; escherichia coli (Escherichia coli) DH5 alpha by Shanghai city agricultural science institute of biotechnology research institute plant gene engineering research room preservation; cloning vector pMD-18-Simple T, various restriction enzymes, Taq polymerase, ligase, dNTP, 10 XPCR buffer and DNA marker were purchased from Bao bioengineering, Dalian, Inc., all chemicals were purchased from Sigma chemical Co., USA and Shanghai pharmaceutical chemicals, ABI PRIAM Big-Dye Terminator DNA sequencing kit was purchased from USA applied systems.

The general Molecular biology procedures used in the present invention are described in detail in Molecular cloning.2nd ed.Cold Spring Harbor Laboratory Press, 1989.

The reagents used in the present invention were purchased from Sigma-Aldrich (Sigma-Aldrich) unless otherwise specified.

Example 1

Gene synthesis method for synthesizing cadmium-resistant gene Vvmrp1S from grape

An ABC transporter gene Vvmrp1S derived from grape was cloned by using a gene synthesis method (Xiong et al, Nucl Acids Res, 2004, 32: e98), and the ABC transporter gene Vvmrp1S encoding grape was synthesized anew according to the preferred codon of Arabidopsis thaliana, while keeping the amino acid sequence of the Vvmrp1 gene unchanged.

The gene synthesis design principle comprises: according to the preferred codon of arabidopsis thaliana, PolyA tailing signals such as ATTTA and the like are avoided from appearing in the gene; 6 or more continuous A + T sequences are avoided, 5 or more G + C sequences are avoided, the proportion of G + C is 40-60%, intron cutting sequences are prevented, two-stage structural hairpins in the gene are reduced, and CG and TA double oligonucleotides (CG is easy to cause methylation in plants) at 2 and 3 positions are avoided.

Vvmrp1S fragment was amplified by PCR using 1.5ng of the inner primer and 30ng of the outer two primers in a 50. mu.l reaction system, 1. mu.l of KOD FXtaq enzyme (Toyobo Co., Japan), 5. mu.l of 10 XPCR buffer and 4. mu.l of dNTP, and sterile water was added to make a volume of 50. mu.l; the amplification conditions were: preheating at 94 deg.C for 1 min; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 1min, for 25 cycles.

After PCR, 1% agarose gel is used for recovery, 10 microliter of the recovered product is directly connected with the pMD-18-Simple T cloning vector, and the product is connected at 4 ℃ overnight to obtain a DNA connecting product which is efficiently transformed into DH5 alpha competent cells.

Extracting plasmid from DH5 alpha transformant cell, PCR amplifying by using the extracted plasmid as template to obtain a 4875bp segment, comparing by sequencing and BLAST, showing that the amino acid sequence of the gene synthesized by PCR method is completely identical to that of Vvmrp1 gene, the gene is Vvmrp1 gene from grape, the nucleotide sequence is shown as SEQ ID No1, and the coded amino acid sequence is shown as SEQ ID No 2.

EXAMPLE 2 construction of recombinant expression vector containing ABC transporter Vvmrp1S Gene derived from grape

The product of the double-restriction PCR was performed with BamHI and SacI, respectively, a DNA fragment was recovered with 1% agarose gel, the Vvmrp1S gene fragment recovered in example 1 was ligated with a plasmid containing the double 35S promoter 1301 by T4DNA ligase, and enzyme restriction identification and sequence analysis were performed to obtain a recombinant expression vector AH555 containing the glycine oxidase gene Vvmrp1S gene, which further contained the GUS reporter gene and the intron-containing kanamycin resistance marker gene, as shown in FIG. 1.

Example 3 transformation into Arabidopsis thaliana

The recombinant expression vector obtained in example 2 was introduced into Agrobacterium by electroporation.

1. Preparation of competent cells

Selecting agrobacterium tumefaciens LBA4404 single bacteria to culture in 25mL YEB culture medium (50mg/L rifampicin) overnight, taking 5mL bacteria liquid to transfer to 100mL YEB culture medium (50mg/L rifampicin), culturing until OD600 is 0.7-0.8, placing the bacteria liquid on ice for 10 minutes, centrifuging at 5000rpm for 10min, collecting the bacteria at 4 ℃, adding 100mL sterile double distilled water, and cleaning twice; the cells were suspended in 4mL of 10% glycerol and transferred to a 50mL centrifuge tube. Centrifuging at 5500rpm for 10min at 4 deg.C; the cells were collected, 500. mu.L of 10% glycerol was added to suspend the cells, and the cells were transferred to a 1.5ml centrifuge tube to obtain Agrobacterium competent cells.

2. Electric shock conversion

70 mu L of the prepared agrobacterium tumefaciens competent cells are taken, added with 1 mu L of recombinant expression vector AH555 and uniformly mixed by a decapitated yellow gun head, and then transferred into a 0.1cm electric shock cup. Electric shock parameters: 200 Ω, 1.7KV, 2.5F, 800. mu.L SOC culture broth was added immediately after the shock (see molecular cloning, A laboratory Manual, Sambrook and Russell, 2001).

After culturing for 1 hour, 100 mu L of the coated resistance plate is taken to screen transformants, and the strains successfully introduced with the recombinant expression vector AH555 are screened after culturing at 28 ℃.

Example 4 Arabidopsis floral Dipper transformation

Inoculating a single colony of an agrobacterium strain containing a target plasmid into 5mL of LB culture medium containing corresponding antibiotics for 2 days at 28 ℃, transferring 5mL of the bacterial liquid into 500mL of liquid LB culture medium for 16-24 hours at 28 ℃ (OD is 1.5-2.0), preserving the liquid at 4 ℃ for 30 days, centrifugally collecting the bacterial body at room temperature, and centrifuging at 4000g for 10 minutes; suspending with 5% of fresh sucrose solution with the same volume, adding 0.02% Silwet-77, mixing, transferring into a beaker to obtain the transformed bacterial liquid.

Inverting the wild arabidopsis thaliana, immersing the inverted wild arabidopsis thaliana into a transformation bacterium solution for 10 seconds, infecting both the rosette and the inflorescence, transforming each strain by using 300 ml, transforming into a 2-3 pot, transforming for 1 time after 7 days, air-drying the bacterium solution of the transgenic arabidopsis thaliana plant for 3-5 seconds after infection, well circling the transgenic arabidopsis thaliana plant by using a preservative film, and horizontally placing for 16-24 hours; and (3) after transformation, placing the plant in high temperature and strong light, uncovering the preservative film, keeping certain humidity, harvesting seeds after 1 month of re-growth, screening transformed plants by using 50 mu g/mL hygromycin, transplanting seedlings by growing normal seedlings on a hygromycin plate, harvesting the seedlings, and identifying positive seedlings.

After the seedlings grow to about 3 weeks, extracting RNA from arabidopsis leaves of different strains, carrying out reverse transcription to obtain CDNA, amplifying Vvmrp1S fragment by utilizing PCR, in a 50-microliter reaction system, using 2 microliter of specific primer, 1 microliter of KOD FXtaq enzyme (Toyobo company, Japan), 5 microliter of 10 XPCR buffer and 4 microliter of dNTP, adding sterile water to fix the volume to 50 microliter, wherein the amplification conditions are as follows: preheating at 94 deg.C for 1 min; the results are shown in FIG. 2, using Arabidopsis Actin gene as internal reference, with 30s at 94 ℃, 50 ℃, 30s, 72 ℃ for 1min for 30 cycles.

As can be seen from FIG. 2, transgenic plants into which Vvmrp1S gene is transferred all have very bright specific bands with the length of 280bp, and target gene fragments with the length of 280bp are not amplified in wild type plants, so that the transgenic plants are proved to be positive seedlings, which indicates that the target gene is successfully introduced into Arabidopsis thaliana and expressed at the transcription level.

EXAMPLE 5 identification of Glyphosate resistance in plants transformed with the synthetic Vvmrp1S Gene

Selfing and homozygosis 3 generations of the transgenic arabidopsis thaliana successfully transferred into the Vvmrp1S gene to obtain homozygosis transformants, and collecting seeds. After sowing, growing for 3 weeks at 22 ℃, simultaneously culturing non-transgenic arabidopsis (namely wild arabidopsis WT) as a control experiment, bagging and irrigating 5mM cadmium aqueous solution respectively for wild type, transgenic strain 555-2, transgenic strain 555-4 and transgenic strain 555-5, irrigating once every 10 days, and irrigating for three times to observe the cadmium resistance effect of the transgenic arabidopsis and the non-transgenic arabidopsis.

After the first watering with 5mM cadmium in water for 1 week, cadmium content was determined before the phenotypic difference occurred, and it was found that the content of cadmium in the transgenic lines was significantly higher than that in the wild type lines, as shown in Table 1.

TABLE 1 cadmium content of Vvmrp1S Gene transferred Arabidopsis thaliana and wild Arabidopsis thaliana after cadmium irrigation

Numbering	Cadmium content (mg/kg)
		WT	32.84
555-2	156.74
		555-4	100.47
555-5	126.68

After the second watering with 5mM cadmium aqueous solution, phenotypic differences began to appear between the transgenic lines and the wild-type lines.

After the third watering for 10 days with 5mM cadmium aqueous solution, the characters of wild type Arabidopsis and transgenic Arabidopsis are counted, the leaves of the wild type Arabidopsis are small and yellow, and the leaves of the three transgenic lines are large and green, as shown in figure 3.

Experimental results show that the difference between the cadmium resistance of wild type arabidopsis and the cadmium resistance of transgenic arabidopsis is obvious, and the cadmium resistance of the transgenic arabidopsis is obviously improved, so that the Vvmrp1 gene synthesized by the invention can improve the cadmium resistance and enrichment performance of plants.

Sequence listing

<110> Shanghai city academy of agricultural sciences

<120> cadmium-resistant gene Vvmrp1S derived from grape and application thereof

<130> 2011239

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cgatggtatg tccgatttgg agtcatttat ctcttggtag gggatgctgt gatgctcaat 480

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Met Ala Phe Glu Pro Leu Val Trp Tyr Cys Gln Pro Val Ala Asn Gly

1 5 10 15

Val Trp Ala Lys Ala Ala Glu Ser Ala Phe Gly Pro Tyr Thr Pro Cys

20 25 30

Ala Val Asp Ser Ile Val Val Cys Ile Ser His Leu Val Leu Leu Gly

35 40 45

Leu Cys Cys Tyr Arg Ile Trp Leu Ile Lys Met Asp Phe Lys Val Gln

50 55 60

Arg Phe Cys Leu Gln Ser Asn Tyr Tyr Asn Tyr Met Leu Gly Leu Leu

65 70 75 80

Ala Cys Tyr Cys Thr Ala Glu Pro Leu Phe Arg Leu Val Met Gly Val

85 90 95

Ser Ile Phe Asp Leu Asp Glu Gln Thr Gly Leu Ala Pro Tyr Glu Ile

100 105 110

Val Ser Leu Ile Ile Glu Ala Ala Thr Trp Cys Ser Met Leu Val Met

115 120 125

Ile Gly Val Glu Thr Lys Ile Tyr Ile Arg Gln Phe Arg Trp Tyr Val

130 135 140

Arg Phe Gly Val Ile Tyr Leu Leu Val Gly Asp Ala Val Met Leu Asn

145 150 155 160

Leu Ile Leu Ser Leu Lys Asp Ser Tyr Ser Arg Ser Val Leu Tyr Pro

165 170 175

Pro Ile Ser Ser Val Leu Cys Gln Val Leu Phe Gly Ile Cys Leu Leu

180 185 190

Val His Val Pro Asn Leu Asn Pro Tyr Val Gly Tyr Thr Pro Met Gln

195 200 205

Ser Asp Ser Leu Glu Asn Thr Lys Tyr Glu Val Leu Pro Gly Gly Asp

210 215 220

Gln Ile Cys Pro Glu Lys His Ala Asn Met Phe Ser Arg Ile Tyr Phe

225 230 235 240

Gly Trp Met Thr Pro Leu Met Gln Gln Gly Tyr Lys Lys Pro Ile Thr

245 250 255

Glu Lys Asp Ile Trp Lys Leu Asp Thr Trp Asp Gln Thr Glu Thr Leu

260 265 270

Ser Arg Arg Phe Gln Lys Cys Trp Ile Glu Glu Ser Gln Arg Ser Lys

275 280 285

Pro Arg Leu Leu Arg Ala Leu Asn Cys Ser Leu Gly Gly Arg Phe Trp

290 295 300

Arg Gly Gly Phe Phe Lys Ile Gly Asn Asp Leu Ser Gln Phe Val Gly

305 310 315 320

Pro Val Leu Leu Asn His Leu Leu Gln Ser Met Gln Arg Gly Asp Pro

325 330 335

Ala Trp Ile Gly Tyr Ile Tyr Ala Phe Ser Ile Phe Ile Gly Val Ser

340 345 350

Leu Gly Val Leu Cys Glu Ala Gln Tyr Phe Gln Asn Val Met Arg Val

355 360 365

Gly Phe Arg Leu Arg Ser Thr Leu Val Ala Ala Ile Phe Arg Lys Ser

370 375 380

Leu Arg Leu Thr His Glu Gly Arg Lys Asn Phe Pro Ser Gly Lys Ile

385 390 395 400

Thr Asn Met Met Thr Thr Asp Ala Asn Ala Leu Gln Gln Ile Cys Gln

405 410 415

Gln Leu His Ala Leu Trp Ser Ala Pro Phe Arg Ile Ile Ile Ala Met

420 425 430

Val Leu Leu Tyr Gln Gln Leu Gly Val Ala Ser Leu Leu Gly Ser Leu

435 440 445

Met Leu Leu Leu Met Leu Pro Ile Gln Thr Phe Ile Ile Ser Lys Met

450 455 460

Arg Lys Leu Ser Lys Glu Gly Leu Gln Arg Thr Asp Lys Arg Val Ser

465 470 475 480

Leu Met Asn Glu Ile Leu Ala Ala Met Asp Thr Val Lys Cys Tyr Ala

485 490 495

Trp Glu Lys Ser Phe Gln Ser Lys Val Gln Ser Met Arg Asn Asp Glu

500 505 510

Leu Ser Trp Phe Arg Lys Ala Gln Leu Leu Ser Ala Cys Asn Ser Phe

515 520 525

Ile Leu Asn Ser Ile Pro Val Ile Val Thr Val Thr Ser Phe Gly Ala

530 535 540

Phe Thr Leu Leu Gly Gly Asp Leu Thr Pro Ala Arg Ala Phe Thr Ser

545 550 555 560

Leu Ser Leu Phe Ala Val Leu Arg Phe Pro Leu Asn Met Leu Pro Asn

565 570 575

Leu Ile Thr Gln Val Val Thr Ala His Val Ser Ile Gln Arg Leu Glu

580 585 590

Gln Leu Phe Leu Thr Glu Glu Arg Val Leu Ala Pro Asn Pro Thr Leu

595 600 605

Glu Pro Gly Leu Pro Ala Ile Ser Ile Lys Asp Gly Tyr Phe Ser Trp

610 615 620

Asp Ser Lys Val Glu Lys Pro Thr Leu Ser Asn Ile Asn Leu Asp Ile

625 630 635 640

Pro Val Gly Ser Leu Val Ala Val Val Gly Gly Thr Gly Glu Gly Lys

645 650 655

Thr Ser Leu Ile Ser Ala Met Leu Gly Glu Leu Pro Pro Leu Ser Asp

660 665 670

Ala Ser Val Val Ile Arg Gly Thr Val Ala Tyr Val Pro Gln Ile Ser

675 680 685

Trp Ile Phe Asn Ala Thr Val Arg Gly Asn Ile Leu Phe Gly Ser Asp

690 695 700

Phe Glu Pro Ala Arg Tyr Trp Lys Ala Ile Asp Val Thr Glu Leu Gln

705 710 715 720

His Asp Leu Asp Leu Leu Pro Gly His Asp Leu Thr Glu Ile Gly Glu

725 730 735

Arg Gly Val Asn Ile Ser Gly Gly Gln Lys Gln Arg Val Ser Met Ala

740 745 750

Arg Ala Val Tyr Ser Asn Ser Asp Val Tyr Ile Phe Asp Asp Pro Leu

755 760 765

Ser Ala Leu Asp Ala His Val Ala Gln Gln Val Phe Ser Asn Cys Ile

770 775 780

Lys Glu Glu Leu Lys Gly Lys Thr Arg Val Leu Val Thr Asn Gln Leu

785 790 795 800

His Phe Leu Pro His Val Asp Arg Ile Ile Leu Val Ser Asp Gly Thr

805 810 815

Val Lys Glu Asp Gly Thr Phe Asp Asp Leu Ser Lys Asn Ser Lys Leu

820 825 830

Phe Gln Lys Leu Met Glu Asn Ala Gly Lys Met Glu Glu Gln Val Glu

835 840 845

Glu Asn Glu Cys Arg Glu Asn Leu Ser Asn Asn Lys Ser Lys Pro Thr

850 855 860

Thr Asn Gly Glu Val Asn Glu Leu Pro Lys Asn Ala Ile His Ser Asn

865 870 875 880

Lys Gly Lys Glu Gly Lys Ser Val Leu Ile Lys Gln Glu Glu Arg Glu

885 890 895

Thr Gly Ile Val Ser Trp Lys Val Leu Met Arg Tyr Lys Asp Ala Leu

900 905 910

Gly Gly Leu Trp Val Val Thr Leu Leu Phe Ala Cys Tyr Val Leu Thr

915 920 925

Glu Val Leu Arg Val Leu Ser Ser Thr Trp Leu Ser Val Trp Thr Asp

930 935 940

Gln Ser Met Ser Lys Asp Tyr Arg Pro Gly Tyr Tyr Asn Leu Ile Tyr

945 950 955 960

Ala Leu Leu Ser Phe Gly Gln Val Met Val Thr Leu Gly Asn Ser Phe

965 970 975

Trp Leu Ile Thr Ser Ser Leu His Ala Ala Lys Ile Leu His Asn Val

980 985 990

Met Leu Asn Ser Ile Leu Arg Ala Pro Met Val Phe Phe His Thr Asn

995 1000 1005

Pro Ile Gly Arg Ile Ile Asn Arg Phe Ala Lys Asp Leu Gly Asp Ile

1010 1015 1020

Asp Arg Asn Val Ala Pro Ser Ala Asn Met Phe Leu Gly Gln Val Trp

1025 1030 1035 1040

Gln Leu Leu Ser Thr Phe Val Leu Ile Ala Ile Val Ser Thr Ile Ser

1045 1050 1055

Leu Trp Ala Ile Met Pro Leu Leu Ile Leu Phe Tyr Ala Ala Tyr Leu

1060 1065 1070

Tyr Tyr Gln Ser Thr Ser Arg Glu Val Lys Arg Leu Asp Ser Ile Thr

1075 1080 1085

Arg Ser Pro Val Tyr Ala Gln Phe Gly Glu Ala Leu Asn Gly Leu Ser

1090 1095 1100

Thr Ile Arg Ala Tyr Lys Ala Tyr Asp Arg Met Ala Ser Ile Asn Gly

1105 1110 1115 1120

Lys Ser Met Asp Asn Asn Ile Arg Phe Thr Leu Ala Asn Ile Ser Ser

1125 1130 1135

Asn Arg Trp Leu Thr Ile Arg Leu Glu Thr Leu Gly Gly Leu Met Ile

1140 1145 1150

Cys Leu Thr Ala Thr Phe Ala Val Met Glu Asn Ser Arg Glu Glu Asn

1155 1160 1165

Pro Ala Ala Phe Ala Ser Thr Met Gly Leu Leu Leu Ser Tyr Thr Leu

1170 1175 1180

Asn Ile Thr Ser Leu Leu Ser Gly Val Leu Arg Gln Ala Ser Arg Ala

1185 1190 1195 1200

Glu Asn Ser Phe Asn Ala Val Glu Arg Val Gly Thr Tyr Val Asp Leu

1205 1210 1215

Pro Ser Glu Ala Pro Thr Ile Ile Glu Ser Asn Arg Pro Pro Pro Gly

1220 1225 1230

Trp Pro Ser Ser Gly Ser Ile Arg Phe Glu Asp Val Val Leu Arg Tyr

1235 1240 1245

Arg Pro Glu Leu Pro Pro Val Leu His Gly Ile Ser Phe Lys Ile Ser

1250 1255 1260

Pro Ser Glu Lys Leu Gly Ile Val Gly Arg Thr Gly Ala Gly Lys Ser

1265 1270 1275 1280

Ser Met Ile Asn Ala Leu Phe Arg Ile Val Glu Leu Glu Arg Gly Arg

1285 1290 1295

Ile Trp Ile Asp Glu Tyr Asp Ile Ala Lys Phe Gly Leu Thr Asp Leu

1300 1305 1310

Arg Lys Val Leu Ser Ile Ile Pro Gln Ser Pro Val Leu Phe Ser Gly

1315 1320 1325

Thr Val Arg Phe Asn Leu Asp Pro Phe Asn Glu His Asn Asp Ala Asp

1330 1335 1340

Leu Trp Glu Ala Leu Glu Arg Ala His Leu Lys Asp Val Ile Arg Arg

1345 1350 1355 1360

Asn Ser Phe Gly Leu Asp Ala Glu Val Ala Glu Gly Gly Glu Asn Phe

1365 1370 1375

Ser Val Gly Gln Arg Gln Leu Leu Ser Leu Ala Arg Ala Leu Leu Arg

1380 1385 1390

Arg Ser Lys Ile Leu Val Leu Asp Glu Ala Thr Ala Ala Val Asp Val

1395 1400 1405

Arg Thr Asp Ala Leu Ile Gln Lys Thr Ile Arg Glu Glu Phe Lys Thr

1410 1415 1420

Cys Thr Met Leu Val Ile Ala His Arg Leu Asn Thr Ile Ile Asp Cys

1425 1430 1435 1440

Asp Arg Ile Leu Val Leu Asp Ala Gly Gln Val Val Glu Tyr Asp Thr

1445 1450 1455

Pro Glu Glu Leu Leu Gln Asp Glu Gly Ser Ser Phe Ser Arg Met Val

1460 1465 1470

Arg Ser Thr Gly Ala Ala Asn Ala Gln Tyr Leu Arg Ser Leu Val Phe

1475 1480 1485

Gly Glu Asp Gly Gln Lys Lys Ser Gly Arg Glu Glu Ala Lys Gln Leu

1490 1495 1500

Asp Arg Gln Lys Arg Trp Leu Ala Ser Ser Arg Trp Ala Ala Ala Thr

1505 1510 1515 1520

Gln Phe Ala Leu Ser Ile Ser Leu Thr Ser Ser Gln Asn Gly Leu Gln

1525 1530 1535

Phe Leu Asp Val Glu Asp Glu Met Asn Ile Leu Lys Lys Thr Asn Asp

1540 1545 1550

Ala Val Leu Thr Leu Arg Gly Val Leu Glu Gly Thr His Asp Glu Val

1555 1560 1565

Ile Glu Glu Met Leu Lys Glu Tyr Gln Val Pro Arg Asp Arg Trp Trp

1570 1575 1580

Ser Ala Leu Tyr Lys Met Val Glu Gly Leu Ala Val Met Asn Arg Leu

1585 1590 1595 1600

Ala Arg His Arg Phe Gln Gln Ser Glu His Asp Phe Glu Asp Thr Thr

1605 1610 1615

Leu Asp Trp Asp Leu Thr Glu Met

1620

Claims (6)

1. A cadmium-resistant gene Vvmrp1S derived from grape has a nucleotide sequence shown in SEQ ID No. 1.

2. A recombinant expression vector comprising the cadmium-tolerant gene Vvmrp1S of claim 1.

3. The recombinant expression vector of claim 3, further comprising a double 35S promoter, a GUS reporter gene, and an intron-containing kanamycin resistance marker gene.

4. The use of the grape-derived cadmium-tolerant gene Vvmrp1S of claim 1 in breeding cadmium-tolerant plants.

5. The use of claim 4, wherein the cadmium-tolerant plant is Arabidopsis thaliana.

6. A method for transferring the cadmium-resistant gene Vvmrp1S derived from grapes into Arabidopsis thaliana comprises the following steps:

1) amplification of cadmium-resistant genes

Designing primers according to the sequence of the cadmium-resistant gene Vvmrp1S, wherein the inner primers are respectively 1.5ng, the outer two primers are respectively 30ng, and carrying out PCR to amplify a Vvmrp1S fragment;

2) construction of recombinant expression vectors

After PCR amplification is finished, recovering a product, connecting the product with a T/A cloning vector to obtain a DNA connecting product, and transforming into DH5 alpha competent cells;

after carrying out double enzyme digestion on a PCR product by using BamHI and SacI, recovering a Vvmrp1S fragment, connecting the recovered Vvmrp1S gene with a plant expression vector 1301 containing a double 35S promoter by using T4DNA ligase, carrying out enzyme digestion identification and sequence determination to obtain a recombinant expression vector containing a target gene Vvmrp1S gene, wherein the expression vector also comprises a GUS reporter gene and an intron-containing kanamycin resistance marker gene;

3) transformation of Arabidopsis thaliana

Introducing the recombinant expression vector obtained in the step 2) into agrobacterium tumefaciens LBA4404 by an electric shock method, transforming the constructed agrobacterium tumefaciens LBA4404 containing the Vvmrp1S gene into Arabidopsis by an agrobacterium flower dipping method, screening transformed plants by hygromycin, transplanting seedlings growing normally on a hygromycin plate, harvesting the seedlings, and identifying positive seedlings.

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