CN105505950B

CN105505950B - The new opplication of five tobacco metallothionein genes

Info

Publication number: CN105505950B
Application number: CN201610065122.8A
Authority: CN
Inventors: 张美�; 莫辉; 郭艳
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2016-01-29
Filing date: 2016-01-29
Publication date: 2019-03-29
Anticipated expiration: 2036-01-29
Also published as: CN105505950A

Abstract

The invention discloses five tobacco metallothioneins NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 and its encoding gene to improve the application in tolerance or enriching of the plant to heavy metal cadmium.The gene is expressed in engineering bacteria saccharomyces cerevisiae can be improved yeast to the tolerance of cadmium, while improve the content of cadmium in yeast, show the ability that five tobacco metallothioneins have heavy metal cadmium removing toxic substances；In addition, the yeast of five tobacco metallothionein genes of expression improves the tolerance of oxidative stress.If the gene is carried out transgene expression in engineering bacteria, on the one hand, engineering bacteria be can change to heavy metal cadmium tolerance, on the other hand can also be improved accumulation of the transgenic engineered bacteria to heavy metal cadmium, so that the engineering bacteria to be applied to the microorganism remediation engineering of heavy metal cadmium in environmental pollution.The gene has the potentiality of the genetic engineering breeding applied to plant Cadmium resistance, by regulating and controlling expression of the gene in plant, obtains the tired genetically modified plants of heavy metal cadmium ultraproduct, the phytoremediation engineering for heavy metal pollution.

Description

The new opplication of five tobacco metallothionein genes

Technical field

The invention belongs to biological gene engineering fields, and in particular to five tobacco metallothioneins (Nicotianatabacum MetallotHionein, NtMT) and its encoding gene and regulation yeast cadmium tolerance and accumulation in terms of application.

Background technique

Metallothionein is the small molecular protein being widely present in organism, is rich in cysteine, and structure height is conservative.It grinds Study carefully and shows that metallothionein there can be heavy metal detoxification in conjunction with the contents of many kinds of heavy metal ion including cadmium, zinc, copper in the cell Function, while may also participate in the storage, transhipment and metabolism of microelement.Simultaneously as metallothionein contains multiple go back The cysteine residues of originality, have stronger oxidation resistance, and the active oxygen having more than needed in energy scavenger-cell is reduced by various inverse The oxidative stress injury that border stress is shone.It can be seen that the powerful biochemical function of metallothionein determines that the albuminoid has Biggish application potential.By cloning the different intracorporal metallothionein genes of biology, and the application value of the genoid is developed, It is of great significance.The invention discloses the clones of five metallothionein genes in tobacco, and disclose the genoid Application value in terms of for the plant genetic engineering and microbiological genetic engineering of heavy metal cadmium.

Heavy metal from industry, mining industry and chemical fertilizer has become the important environmental problem in the world today to the pollution of environment One of.And cadmium pollution ranks the first in all soil pollutants in China, it is total that the exceeded area of Cadmium Pollution in Soils has reached China territory The 7% of area.One important feature of heavy metal pollution cannot exactly be degraded, can be with long-term existence in environment, and pass through Food chain enters human body, is finally detrimental to health.Heavy metal clearance technique for environment includes microorganism remediation and plant Metallothionein gene is applied to bioengineering and improves it to heavy metal for microorganism and plant to be transformed by recovery technique Accumulation ability has great importance.Currently, whether the intracorporal metallothionein gene of document report tobacco can influence not yet Heavy metal (especially cadmium) in vivo, tolerance or accumulation especially in tobacco.

In the present invention, we essentially disclose intracorporal five metallothionein genes of tobacco, and the genoid is in tobacco Intracorporal expression is to heavy metal cadmium stress response.The expression of metallothionein gene can influence heavy metal cadmium in vivo Accumulation is provided the genoid applied to engineering bacteria --- the application of the genetic modification of resistance to cadmium of saccharomyces cerevisiae can also be applied to For the genetic modification of cadmium pollution microorganism remediation engineering bacteria；The present invention also elaborates the heredity that the gene can be applied to plant The transgenosis heavy metal hyperaccumulative plant for being used for heavy metal cadmium phytoremediation is cultivated in transformation.

Summary of the invention

An object of the present invention be to provide five tobacco metallothioneins NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5；And its new opplication of encoding gene NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5.

Realize that the technical solution of above-mentioned purpose is as follows:

NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 gene are in improvement plant to the tolerance or enrichment of heavy metal cadmium Property in application, the NtMT1, NtMT2, NtMT3, the cDNA of NtMT4 and NtMT5 gene be respectively such as SEQ ID NO.1, Nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9, or be respectively with The nucleosides of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9 complementary pairing Acid sequence, or be respectively coding SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID The nucleotide sequence of tobacco metallothionein shown in NO.10.

Amino acid sequence is respectively such as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ Tobacco metallothionein NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 shown in ID NO.10 is in improvement plant to heavy metal Cd Tolerance or enriching in application.

Five tobaccos metallothionein NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 of the invention, amino acid sequence Respectively such as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10 institute in sequence table Show, the cDNA nucleotide sequence of encoding gene NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 are respectively such as SEQ in sequence table Shown in ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9.It should be understood that, it is contemplated that The degeneracy of codon modifies to the nucleotide sequence of above-mentioned encoding gene under the premise of not changing amino acid sequence, It also belongs in protection scope of the present invention.

Above-mentioned five tobacco metallothionein genes are applied in plant genetic engineering by the present invention, for changing transgenosis A possibility that plant is to the tolerance and enrichment of heavy metal cadmium.The invention discloses five tobacco metallothionein genes in Cd stress Under inducing expression feature, show the gene participate in heavy metal cadmium response and removing toxic substances.The present invention uses real time RT- The method of PCR identifies expression characteristic of five tobacco metallothionein genes under heavy metal Cd stress, demonstrates the gene For the important gene of response heavy metal stress in tobacco body, further extend the gene as influence tobacco heavy metal accumulation and The candidate functional gene of stress.

It is another object of the present invention to disclose one group of saccharomyces cerevisiae recombinant expression carrier and corresponding saccharomyces cerevisiae and its Using the recombinant vector inserts five tobacco metallothionein genes.Recombinant vector conversion is entered in saccharomyces cerevisiae, is led to It crosses galactolipin and induces overexpression of the gene in yeast, tolerance of the yeast to heavy metal cadmium can be improved under Cd stress, and Change Cd accumulation in yeast cells.

Specific technical solution is as follows.

Inserted with above-mentioned NtMT1, NtMT2, NtMT3, the wine brewing ferment of the cDNA reading frame sequence of NtMT4 and NtMT5 gene Female recombinant expression carrier.

Above-mentioned saccharomyces cerevisiae recombinant expression carrier is in improvement engineered strain saccharomyces cerevisiae to the tolerance or richness of heavy metal cadmium Application in collection property.

A kind of saccharomyces cerevisiae, conversion have above-mentioned saccharomyces cerevisiae recombinant expression carrier.The saccharomyces cerevisiae is in environment Application in the microorganism remediation engineering of heavy metal cadmium.

It is a further object of the present invention to provide the tolerance of heavy metal cadmium in a kind of improvement tobacco or the biology systems of enriching Agent.

Realize that the technical solution of above-mentioned purpose is as follows.

The biological agent of heavy metal cadmium tolerance or enriching in a kind of improvement tobacco, active ingredient derive from above-mentioned wine Brewer yeast recombinant expression carrier or saccharomyces cerevisiae or its active ingredient contain NtMT1, NtMT2, NtMT3, NtMT4 and/or NtMT5 gene, the NtMT1, NtMT2, NtMT3, the cDNA of NtMT4 and NtMT5 gene are respectively such as SEQ ID NO.1, SEQ Nucleotide sequence shown in ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9, or be respectively and SEQ ID The nucleotide sequence of NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9 complementary pairing, or Respectively encode cigarette shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10 The nucleotide sequence of careless metallothionein.

It is a further object of the present invention to provide a kind of methods of the tolerance or enriching of heavy metal cadmium in regulation tobacco.

A method of regulating and controlling the tolerance or enriching of heavy metal cadmium in tobacco, includes regulation tobacco in this method step In above-mentioned NtMT1, NtMT2, NtMT3, NtMT4 and/or NtMT5 gene expression.

Beneficial effects of the present invention are as follows: in the present invention, we pass through five tobacco metals to the acquisition of screening library The carrying out of metallothionein gene NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 elaborates that the genoid or less is answered using sex exploration With mode: (1) five tobacco metallothioneins are provided with the feature of heavy metal Cd inducing expression in intracorporal express of tobacco, show The genoid has the basic function of heavy metal stress response, the genoid can be applied to plant (crop) and be directed to heavy metal cadmium Genetic breeding, improvement plant (crop) is to the tolerance or enriching of heavy metal.(2) five tobacco metallothionein genes exist Overexpression in saccharomyces cerevisiae can be improved yeast to the tolerance of heavy metal cadmium, the genoid can be applied to engineering acicula To the tolerance genetic breeding of heavy metal cadmium；Overexpression energy of (3) five tobacco metallothionein genes in saccharomyces cerevisiae Accumulation of the yeast cells to heavy metal cadmium is enough improved, enrichment of the yeast to cadmium in external environment is promoted.It can be by the genoid application In the genetic modification of the microorganism remediation engineering bacteria for heavy metal cadmium.

The present invention may extend to microorganism and the phytoremediation field of heavy metal cadmium, pass through turning with the genoid Gene genetic transformation, obtains the microorganism or plant of heavy metal cadmium super enrichment.Meanwhile the present invention also may extend to agro-ecology skill Art field regulates and controls tolerance of the crops to heavy metal cadmium by changing expression of the tobacco metallothionein gene in crops And enrichment, and crops are participated in the adaptability reform of cadmium pollution environment.The present invention can also by regulate and control the NtMT1, The expression of NtMT2, NtMT3, NtMT4 and/or NtMT5 in plant obtains the tired genetically modified plants of heavy metal cadmium ultraproduct, is used for The phytoremediation engineering of heavy metal pollution.

Detailed description of the invention

Fig. 1 shows five tobacco metallothionein genes in expression by heavy metal cadmium, the tune of jasmonic JA and mannitol Control.

Fig. 2 shows saccharomyces cerevisiae recombinant expression carrier NtMT1-pYES260, NtMT2-pYES260, NtMT3- that building is completed PYES260, NtMT4-pYES260 and NtMT5-pYES260 schematic diagram.

Fig. 3 show conversion NtMT1-pYES260, NtMT2-pYES260, NtMT3-pYES260, NtMT4-pYES260 and The transgenic yeast Δ ycf1 of NtMT5-pYES260 improves the tolerance of cadmium, can be in the CdCl containing 0.075mM₂Consolidate It is grown on body culture medium.

Fig. 4 show conversion NtMT1-pYES260, NtMT2-pYES260, NtMT3-pYES260, NtMT4-pYES260 and CdCl of the transgenic yeast WT and Δ ycf1 of NtMT5-pYES260 at 15 μM of addition₂Solution culture fermentation 24 hours after, The intracorporal heavy metal cadmium content of yeast increases, and shows that expression of the tobacco metallothionein gene in yeast can influence heavy metal Accumulation of the cadmium in yeast cells.

Specific embodiment

The present invention describes such NtMT1, NtMT2, NtMT3, NtMT4 and NtMT5 gene answering in terms of bioengineering With.Present invention is initially set forth five tobacco metallothionein genes by the feature of heavy metal Cd inducing expression.To grow 2 weeks Tobacco SR1 seedling is material, stress-inducing is carried out with heavy metal Cd, JA and mannitol respectively, using real time RT-PCR Method detects expression characteristic of above-mentioned five genes in tobacco young root and spire respectively.In the present invention, disclosed in us five Expression of a tobacco metallothionein gene in tobacco is by the induction of heavy metal Cd, and the inducing expression feature in root Obviously, show that this five genes have both participated in the stress of tobacco response heavy metal Cd, have and carry out the removing toxic substances of tobacco heavy metal cadmium Essential characteristic, in the present invention, plant (crop) can be applied to for the potentiality of the genetic breeding of heavy metal by having.

By this five metallothionein gene overexpressions in saccharomyces cerevisiae, yeast can be improved to the resistance to of heavy metal cadmium By property；Adding low cadmium (15 μM of CdCl simultaneously₂) culture medium in grow, be transferred in the yeast of five metallothionein genes Cadmium content increase, can be improved enrichment of the yeast to heavy metal cadmium in external environment.And so on, due to such base It can also be by this kind of bases by controlling this expression of five metallothionein genes in plant because deriving from plant (tobacco) Because being applied to plant genetic engineering, for cultivating the plant transgene kind of resistance to cadmium, it is also possible to which Lai Peiyu heavy metal cadmium ultraproduct is tired Genetically modified plants, the phytoremediation engineering applied to environment heavy metal cadmium.

SEQ ID NO.1

CDNA nucleotide sequence (underscore part is reading frame sequence)

>NtMT1

CTTTCTTTCACAACAAATTAAATAGCTTATAATCTCCAAACAACAAATTAAGAAAACATG TCTTGTT GCGGAGGAAGTTGTGGCTGTGGATCTGGCTGCAAGTGCGGCAGCGGCTGCGGAGGATGTGGGATGTACCCAGACTTG GAGAAGTCCACTACCTTTACCATCGTTGATGGTGTTGCTCCCATGAAGAGCTTTGAGGAATTTGGAGAGAAAGCAGC AGAAGGAGGAAATGGCTGCAAATGCGGATCAAACTGCACCTGTGACCCTTGCAATTGTTAAGATAATTCTCTTGTGA TTCCACAATAATGTGTGTGTTTTCTGTAATAATAAGGATAAAACTACAGCTAGCCATGGAACTGATTGTCAGTTCTT AGGTTTGTTTGTTCTGATCACTTTCCTGTTTGTTTCTCCATCTGATATATATTTCTGTAGTGTGATTTGGGGTGTAA CAGAAACGTATCATATATAAATTATAAGTTTGTTAATTTGTTCAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.2

Amino acid sequence

>NtMT1protein

MSCCGGSCGCGSGCKCGSGCGGCGMYPDLEKSTTFTIVDGVAPMKSFEEFGEKAAEGGNGCKCGSNCTC DPCNC

SEQ ID NO.3

CDNA nucleotide sequence (underscore part is reading frame sequence)

>NtMT2

ACTCTTTTTGATAAATTGATTCCTAGTCTTTTTCCAAGAAAAAAAAAACATGTCTTGCTGTGGAGGAAG CTGTGGCTGTGGATCTGGTTGCAAATGCGGCAGTGGCTGTGGAGGATGTGGGATGTACCCCGACTTGGAGAACACCA CCACCTTTACCATCATTGAGGGAGTTGCACCTATGAAGAACTACGAGGGATCTGCTGAGAAAGCAACAGAAGGAGGA AATGGCTGCAAGTGCGGAGCAAACTGCAAATGTGACCCTTGCAACTGCTGATGAACATGGTCATTAGCTGGCCCAAA AATATATTATGTGTTTTAAAAGCTTTGTAGTATTAATAAAACTACAACCATGAAACTGACCATCTCCAAATCTTTTT GACAAGGTTTGTTTGATGTTGTTTCATGGTTTTTCTGTTTGGTTTTCTAATATGATATTTGTATGAAAGGAAGCTGA TTTGGGCTGTAACACGAACTTATCAATATATAGAATACAAGTTGGCTTCTCCTTTTATTATGTAAAGAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.4

Amino acid sequence

>NtMT2protein

MSCCGGSCGCGSGCKCGSGCGGCGMYPDLENTTTFTIIEGVAPMKNYEGSAEKATEGGNGCKCGANCKC DPCNC

SEQ ID NO.5

CDNA nucleotide sequence (underscore part is reading frame sequence)

>NtMT3

CAACAAAGAAAATGTCTTGCTGTGGAGGAAACTGTGGTTGTGGATCTGGCTGCAAGTGCGGCAACGGC TGTGGCGGATGCAAGATGTACCCAGATTTGAGCTACAACGAGAGCACCACAACCGAGACTTTGGTGCTTGGGGTGG GACCTGAGAAGACAAGCTTCGGCACTATGGAAATGGGTGAATCTCCTGCTGCTGAAAATGGTTGCAAATGTGGATC TGACTGCAAGTGTAACCCCTGCACTTGTTCTAAATGAACAAATTAAATCTTATTTAAGCAGAGATGGATGTTTGCC TTTATTGGCTAAGACAAAAAATAACTATAGTTTTAAAAATGTCTAGTTTATAGTTCTTGTGTCTGTTGAAAACAGG GGAAATTATGTGTTTTTTCCTCTCTAATAAGAAATAGTATGGAGTCTGAACAATGTTTGGGTTCTTGTTATTGTAT ACAAGTCGTTGTGTGGTTTGTAACATG AGCATCTCTGCTCCTGGTATTTTAACCTATGTTTTGGAAGTGGTTGTT GTTTGGTTTCAAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.6

Amino acid sequence

>NtMT3protein

MSCCGGNCGCGSGCKCGNGCGGCKMYPDLSYNESTTTETLVLGVGPEKTSFGTMEMGESPAAENGCKCG SDCKCNPCTCSK

SEQ ID NO.7

CDNA nucleotide sequence (underscore part is reading frame sequence)

>NtMT4

GGTTTATTATCTCCCAACAACATAAGAAAACAATGTCTGGCTGCGGATCAAACTGTGGATGTGGATCTA GCTGCAATTGCGGCAGTGGCGGAGGATGTGGGATGTACCCAGACTTGGAGAAATCCACTAGCTTTACTATCATTCAG GGTGTTGCTCCCATGAAGAGCTTTGAGGAATTCGGAGAGAAAGCAGCAGAAGGAGGAAATGGGTGCAAGTGTGGATC AAACTGCACCTGTGACCCTTGCAACTGTTAAGAATATTCTCACTACAAAAACTGCATTGTGTCTCTGTATTAAGTGT GTGTTCTAAAAGTTATTGTAATTATAACAATAAAACTACAACCATGGAACAGATTATGATCAGTTCTTAGGTTTGTG TGTTACTCGTGTGACGTGGCTTTTCTGTTTGTTAATTGGGTTGTGTATCAATATACTACATAAGTTTGTTTGTTTGT TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.8

Amino acid sequence

>NtMT4protein

MSGCGSNCGCGSSCNCGSGGGCGMYPDLEKSTSFTIIQGVAPMKSFEEFGEKAAEGGNGCKCGSNCTCD PCNC

SEQ ID NO.9

CDNA nucleotide sequence (underscore part is reading frame sequence)

>NtMT5

CCAACTTTTTTGTACAAAGTTGTCCCCCCAACTTTTTTGTACAAAGTTGTCCCCCCAACTTTTTTGTAC AAAGTTGTCCCCTTCTTTGGCTATTTTGCTAATTGTAATATCCTCACAAGCTGATGCAAGGGAGACGTCTAAGGCAA CTGCTCCAATTACCCAAGAAATGAATTCAAACAACACTACTGATCAGAAGATACCAAAACGTCCAAAACCAGGAGGC AATATCTTCGGCAAAGCTTGTAAAATTTGCCCATGTAAATACCAGATTTGCAGCAAATGTCCTAAATGTGATGACCA AAATATCGCCGGCAAATTTTGTAAAATTTGCTCATGTAAGACTCAGATTTGCAGTAAATGTCCTAAATGTCATAACC AAAATTAGGCCTCAGAGACTATGTACTTGTGCTGGTGTGAGTTTAGTTTTGAGAATAAAAGGAAAGTTATGAATAGC CTAATATAATTCTATTCACTTTCCTCTAGTTAATTTCTCTTAGTTTGTGTTTTGTTTTGTTAGTAGTTCCTATTGTT GCAACTTGCAACAAGTCTTGGGGTCAACATGTACCTCTTGTCTTGTAGTCTTTCGACTGTATGATATTGTACCGTAT TGTATTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO.10

Amino acid sequence

>NtMT5protein

MNSNNTTDQKIPKRPKPGGNIFGKACKICPCKYQICSKCPKCDDQNIAGKFCKICSCKTQICSKCPKCH NQN

It to facilitate the understanding of the present invention, below will be to invention is more fully described.The present invention can be with many not With form realize, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes to this The understanding of disclosure of the invention content is more thorough and comprehensive.

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc. People, molecular cloning: institute in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) The condition stated, or according to the normal condition proposed by manufacturer.Used various common chemical reagent, are commercially available in embodiment Product.

Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed Any and all combinations of project.

1: five tobacco metallothionein gene of embodiment in tobacco body by the detection of heavy metal Cd inducing expression, simultaneously Also the regulation by jasmonic and mannitol

The present invention uses tobacco seedling to detect the expression characteristics of this 5 tobacco metallothioneins first.It is used in the present invention Tobacco bred be safflower tobacco SR1 (Nicotiana tabacum L.cv.SR1), seed is protected by Chinese Academy of Sciences South China Botanical Garden Offer is provided.Tobacco seed is sprouted using 1/2 liquid MS medium, grows up to seedling after two weeks, tobacco seedling is shifted into 1/2MS liquid Culture medium continues culture 2 weeks.Then respectively with containing CdCl₂The 1/2MS liquid of (50 μM), JA (90 μM) and mannitol (300mM) Body medium treatment tobacco seedling, collect Stress treatment 0h, 6h and for 24 hours after tobacco spire and each 0.2g of young root, for extracting Total serum IgE.The extraction of RNA is carried out according to the specification of Magen company HiPure PlantRNA Kits (R4151).Using two steps Method is using total serum IgE as template reverse transcription cDNA.The synthesis of cDNA chain is according to Quan Shi King Company TransScript One-Step gDNA The specification of Removal and cDNA Synthesis SuperMix carries out.

Drawn by website NCBI (http://www.dtd.nlm.nih.gov/) Photographing On-line real-time RT-PCR Object.Primer sequence is as follows.

NtMT1RTF:5 '-CGGAGGATGTGGGATGTACC-3 ' and NtMT1RTR:5 '- TTTGATCCGCATTTGCAGCC-3 ' is used to detect the expression quantity of NtMT1 gene；(SEQ ID NO.11,SEQ ID NO.12)

NtMT2RTF:5 '-GTGGGATGTACCCCGACTTG-3 ' and NtMT2RTR:5 '- ATCAGCAGTTGCAAGGGTCA-3 ' is used to detect the expression quantity of NtMT2 gene；(SEQ ID NO.13,SEQ ID NO.14)

NtMT3RTF:5 '-CACAACCGAGACTTTGGTGC-3 ' and NtMT3RTR:5 '- GGGGTTACACTTGCAGTCAGA-3 ' is used to detect the expression quantity of NtMT3 gene；(SEQ ID NO.15,SEQ ID NO.16)

NtMT4RTF:5 '-TCATTCAGGGTGTTGCTCCC-3 ' and NtMT4RTR:5 '- AGAAAAGCCACGTCACACGA-3 ' is used to detect the expression quantity of NtMT4 gene；(SEQ ID NO.17,SEQ ID NO.18)

NtMT5RTF:5 '-ACGTCTAAGGCAACTGCTCC-3 ' and NtMT4RTR:5 '- TGCCGGCGATATTTTGGTCA-3 ' is used to detect the expression quantity of NtMT5 gene；(SEQ ID NO.19,SEQ ID NO.20)

The tobacco reference gene of selection is tobacco ribosomal protein gene NtEF-1a/NtL25 (NCBI Accession Number:L18908), primer is as follows: NtL25F (5 '-CCCCTCACCACAGAGTCTGC-3 ') and NtL25R (5 '- AAGGGTGTTGTTGTCCTCAATCTT-3’)。(SEQ ID NO.21、SEQ ID NO.22)

Real time RT- is prepared with reference to the specification of BIO-RAD company Universal SYBR Green Supermix PCR reaction system (operates) on ice.It is reacted using two-step method PCR, amplification standardization program is as follows:

All detections are all made of two biological samples and repeat, and each biological sample carries out repeating detection reaction three times. Addition program Stage3 detects solubility curve when primer uses for the first time, confirms the specificity of primer.

As shown in Figure 1, in tobacco seedling, the expression of five tobacco metallothionein genes is all under Cd Stress treatment By the induction that heavy metal Cd is coerced, the induction in root is more obvious；The expression of five tobacco metallothionein genes also by To the induction of jasmonic JA and hyperosmosis mannitol.

It is a kind of extensive heavy metal cadmium stress that disclosure above, which shows this five tobacco metallothionein genes, Response gene participates in heavy metal cadmium removing toxic substances in tobacco body.The genoid, which has, is applied to be directed to what heavy metal cadmium was coerced and was enriched with The potentiality of tobacco and the improvement of other crop genetic engineerings, also can be used as corresponding face heavy-metal contaminated soil phytoremediation gene work The alternative functional gene of journey.

The clone of 2:5 tobacco metallothionein gene of embodiment and the building of yeast recombinant expression carrier

The seedling leaves for taking tobacco SR1 (South China Botanical Garden preservation), extract the RNA of spire, and reverse transcription is at cDNA, with CDNA is template, design primer NtMT1YEF:5 '-TTTCAGGGCGCCATGTCTTGTTGCGGAGGAAGTTG-3 ' and NtMT1YER:5’-CGTTACTAGTGGATCTTAACAATTGCAAGGGTCAC-3’(SEQ ID NO.23、SEQ ID NO.24), for expanding the cDNA reading frame of NtMT1, and the DNA fragmentation of 252bp is obtained；

Design primer NtMT2YEF:5 '-TTTCAGGGCGCCATGTCTTGCTGTGGAGGAAGCTG-3 ' and NtMT2YER: 5 '-CGTTACTAGTGGATCTCAGCAGTTGCAAGGGTCAC-3 ' (SEQ ID NO.25, SEQ ID NO.26), for expanding The cDNA reading frame of NtMT2, and obtain the DNA fragmentation of 252bp；

Design primer NtMT3YEF:5 '-TTTCAGGGCGCCATGTCTTGCTGTGGAGGAAACTG-3 ' and NtMT3YER: 5 '-CGTTACTAGTGGATCTTAGCCAATAAAGGCAAACA-3 ' (SEQ ID NO.27, SEQ ID NO.28), for expanding The cDNA reading frame of NtMT3, and obtain the DNA fragmentation of 324bp；

Design primer NtMT4YEF:5 '-TTTCAGGGCGCCATGTCTGGCTGCGGATCAAACTG-3 ' and Nt MT4YER:5 '-CGTTACTAGTGGATCTTAACAGTTGCAAGGGTCAC-3 ', (SEQ ID NO.29, S EQ ID NO.30) For expanding the cDNA reading frame of NtMT4, and obtain the DNA fragmentation of 249bp；

Design primer NtMT5YEF:5 '-TTTCAGGGCGCCATGAATTCAAACAACACTACTGA-3 ' and Nt MT5YER:5 '-CGTTACTAGTGGATCCTAATTTTGGTTATGACATT-3 ' (SEQ ID NO.31, SEQ ID NO.32), For expanding the cDNA reading frame of NtMT5, and obtain the DNA fragmentation of 246bp.

The DNA fragmentation that above-mentioned amplification obtains is carried out according to Magen company HiPure Gel Pure DNA Kits specification Agarose gel electrophoresis recycling.Obtained segment is recycled for being inserted into Yeast expression carrier pYES60.Saccharomyces cerevisiae expression Carrier p YES260 is handled through NcoI and BamHI double digestion, recycles linearization plasmid.Five tobacco metallothioneins after the recovery CD NA reading frame segment and linearisation pYES260 plasmid are through Nanodrop company ultraviolet specrophotometer measurement concentration, using Ta The In- of KaRa (Clontech) companyHD Cloning Kit carries out DNA fragmentation and connects with the homologous recombination of carrier. Reaction product is converted e. coli jm109 competence bacterial strain by method to specifications.Picking monoclonal extracts plasmid, warp After sequencing is accredited as correct positive colony, it is spare to save plasmid.Its correctness is identified through sequencing analysis.Five cigarettes built Careless metallothionein the present saccharomyces cerevisiae recombinant expression carrier is as shown in Figure 2.

Expression of 3: the five tobacco metallothionein genes of embodiment in saccharomyces cerevisiae improves yeast to heavy metal cadmium Tolerance

Wine brewing yeast strain wild type WT and cadmium sensitizing mutation strain Δ ycf1 are cultivated (purchased from European yeast research center Euroscarf, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/, strain number are respectively Y00000 and Y04069), with pYE S260 plasmid and recombination five pYES260 plasmids containing tobacco metallothionein (such as Shown in Fig. 2) above-mentioned yeast is converted.It is lured since the external source tobacco metallothionein gene of insertion is placed in yeast galactolipin Under the promoter PGAL1 regulation led (as shown in Figure 2), the saccharomyces cerevisiae monoclonal that conversion obtains is placed in addition galactolipin It grows, can lure in growth selection synthetic media (Sel ective Growth Synthetic Medium, SD medium) Lead the tobacco metallothionein gene of the external source heterologous overexpression in yeast.

The method that yeast conversion uses is lithium acetate transformation method, the specific steps are as follows:

1) single bacterium for being inoculated with yeast strain WT and Δ ycf1 to be transformed is fallen in 5mL YPD fluid nutrient medium, in 30 DEG C Constant-temperature table (200rpm), overnight incubation reach saturation.

2) above-mentioned culture is shifted into 20mL YPD fluid nutrient medium in 30 DEG C of constant-temperature tables according to 1:100 ratio (200rpm) continues to cultivate, and shakes 3-5h to bacterium solution OD₆₀₀It is worth up to 0.5.

3) 4000g is centrifuged 5min to culture solution at room temperature, collects cell.Cell is resuspended with the sterile ultrapure water of 10mL, 5min, sedimentation cell are centrifuged then at 5000-6000g at room temperature.

4) cell 2mL lithium salt solution (1 × TE buffer, pH 7.5；1 × lithium acetate) it is resuspended.

5) it will be mixed in single-minded addition 1.5mL centrifuge tube of 2 μ L plasmid to be transformed and 10 μ L denaturated salmons.

6) cell suspension that 200 μ L are resuspended with lithium salt solution is added into each centrifuge tube, adds 1mL Fresh PEG solution (40%PEG；1 × TE buffer, pH 7.5；1 × lithium acetate).30 DEG C are swayed incubation 30min.

7) in 42 DEG C of heat shock 15min, be centrifuged 5s at room temperature, cell precipitation with 200 μ 1 × TE of L-1mL buffers (from 10 × Liquid storage Fresh) be resuspended, and take wherein 200 μ L be coated in the SD solid medium tablets for being added to galactolipin.30 DEG C of trainings It supports 2-5 days, until there is transformant.

Picking yeast strain WT and Δ ycf1 conversion empty carrier pYES260 and 4 tobacco metallothionein gene overexpressions Carrier (OsMT-I-1a-pYES260, OsMT-I-2a-pYES260, OsMT-I-3a-pYES260 and OsMT-I-4c- PYES260 monoclonal) is inoculated in 2ml and is added in the SD fluid nutrient medium of galactolipin, 30 DEG C of constant-temperature table (200rpm) trainings It supports to bacterium solution OD₆₀₀It is worth up to 2.Bacterium solution is diluted step by step according to 1:1,1:10,1:100,1:1000, draws 2 μ l respectively step by step Diluted bacterium solution drops in the SD added with 0.075mM Cd (addition galactolipin) solid medium tablets.30 DEG C are cultivated 7 days, are seen Examine yeast growth situation.

As shown in figure 3, the transgenic yeast of five tobacco metallothionein genes of overexpression is compared to conversion empty carrier For the Δ ycf1 yeast mutant of pYES260, it can be grown in the SD culture medium flat plate of addition 0.075mM Cd, and non-table Δ ycf1 yeast strain (conversion empty carrier pYES260) up to foreign gene cannot then grow, and show five that the present invention announces Expression of the tobacco metallothionein gene in yeast can be improved yeast to the tolerance of heavy metal cadmium.

Expression of 4: the five tobacco metallothionein genes of embodiment in saccharomyces cerevisiae improves Cadmium accumulation in yeast

Empty carrier pYES260 is converted in Example 3 respectively and containing there are five tobacco metallothionein gene recombinant vectors PYES260 (as shown in Figure 2) yeast strain wild type WT and Δ ycf1 uses 12 primary yeast of sterile toothpick picking in super-clean bench (yeast strain wild type WT and Δ ycf1 convert pYES260 and five tobacco metallothionein recombinant vector pYE to transformant respectively S260) to 2mL be added to galactolipin growth selection synthetic media (Selective Growth Synthetic Medium, S D medium) in fluid nutrient medium, 30 DEG C of constant-temperature tables (200rpm) cultures to bacterium solution OD₆₀₀It is worth up to 2.Later by 12 kinds Yeast is inoculated in 600ml according to the volume ratio of 1:500 and is added in the SD fluid nutrient medium of galactolipin, 30 DEG C of shaking table (200- About 12 hours 250rpm) are cultivated to OD₆₀₀Value adds CdCl up to 2 respectively later₂To 20 μM of final concentration.After continuing culture 24 hours Thalline were collected by centrifugation.After being cleaned thallus 3 times using distilled water, by drying 3-5 days of 65 DEG C of thallus, dry yeast cutting carries out microwave After resolution, using Cadmium accumulation in flame method Atomic Absorption Spectrometry yeast cells.

In the present invention, the two primary yeast bacterial strain (WT of five tobacco metallothionein gene recombinant vector pYES260 are converted 15 μM of CdCl is being added with Δ ycf1)₂Culture medium in, the cadmium of cylinder accumulation is above conversion pYES260 empty carrier Yeast strain (as shown in Figure 4), this shows that tobacco metallothionein gene improves yeast cells pair in the intracorporal expression of yeast The enrichment of heavy metal cadmium promotes absorption of the engineering bacteria yeast to cadmium.Five tobacco metallothionein genes that the present invention announces It can be applied to the engineering bacteria genetic modification for heavy metal cadmium microorganism remediation, for obtaining heavy metal hyperaccumulative engineering Bacterium, the removing for heavy metal in environmental engineering.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

SEQUENCE LISTING

<110>South China Botanical Garden Chinese Academy of Sciences

The new opplication of<120>five tobacco metallothionein genes

<160> 32

<170> PatentIn version 3.3

<210> 1

<211> 520

<212> DNA

<213>NtMT1 gene

<400> 1

ctttctttca caacaaatta aatagcttat aatctccaaa caacaaatta agaaaacatg 60

tcttgttgcg gaggaagttg tggctgtgga tctggctgca agtgcggcag cggctgcgga 120

ggatgtggga tgtacccaga cttggagaag tccactacct ttaccatcgt tgatggtgtt 180

gctcccatga agagctttga ggaatttgga gagaaagcag cagaaggagg aaatggctgc 240

aaatgcggat caaactgcac ctgtgaccct tgcaattgtt aagataattc tcttgtgatt 300

ccacaataat gtgtgtgttt tctgtaataa taaggataaa actacagcta gccatggaac 360

tgattgtcag ttcttaggtt tgtttgttct gatcactttc ctgtttgttt ctccatctga 420

tatatatttc tgtagtgtga tttggggtgt aacagaaacg tatcatatat aaattataag 480

tttgttaatt tgttcaaaaa aaaaaaaaaa aaaaaaaaaa 520

<210> 2

<211> 74

<212> PRT

<213> NtMT1 protein

<400> 2

Met Ser Cys Cys Gly Gly Ser Cys Gly Cys Gly Ser Gly Cys Lys Cys

1 5 10 15

Gly Ser Gly Cys Gly Gly Cys Gly Met Tyr Pro Asp Leu Glu Lys Ser

20 25 30

Thr Thr Phe Thr Ile Val Asp Gly Val Ala Pro Met Lys Ser Phe Glu

35 40 45

Glu Phe Gly Glu Lys Ala Ala Glu Gly Gly Asn Gly Cys Lys Cys Gly

50 55 60

Ser Asn Cys Thr Cys Asp Pro Cys Asn Cys

65 70

<210> 3

<211> 554

<212> DNA

<213> NtMT2

<400> 3

actctttttg ataaattgat tcctagtctt tttccaagaa aaaaaaaaca tgtcttgctg 60

tggaggaagc tgtggctgtg gatctggttg caaatgcggc agtggctgtg gaggatgtgg 120

gatgtacccc gacttggaga acaccaccac ctttaccatc attgagggag ttgcacctat 180

gaagaactac gagggatctg ctgagaaagc aacagaagga ggaaatggct gcaagtgcgg 240

agcaaactgc aaatgtgacc cttgcaactg ctgatgaaca tggtcattag ctggcccaaa 300

aatatattat gtgttttaaa agctttgtag tattaataaa actacaacca tgaaactgac 360

catctccaaa tctttttgac aaggtttgtt tgatgttgtt tcatggtttt tctgtttggt 420

tttctaatat gatatttgta tgaaaggaag ctgatttggg ctgtaacacg aacttatcaa 480

tatatagaat acaagttggc ttctcctttt attatgtaaa gaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaa 554

<210> 4

<211> 74

<212> PRT

<213> NtMT2 protein

<400> 4

Met Ser Cys Cys Gly Gly Ser Cys Gly Cys Gly Ser Gly Cys Lys Cys

1 5 10 15

Gly Ser Gly Cys Gly Gly Cys Gly Met Tyr Pro Asp Leu Glu Asn Thr

20 25 30

Thr Thr Phe Thr Ile Ile Glu Gly Val Ala Pro Met Lys Asn Tyr Glu

35 40 45

Gly Ser Ala Glu Lys Ala Thr Glu Gly Gly Asn Gly Cys Lys Cys Gly

50 55 60

Ala Asn Cys Lys Cys Asp Pro Cys Asn Cys

65 70

<210> 5

<211> 575

<212> DNA

<213>NtMT3 gene

<400> 5

caacaaagaa aatgtcttgc tgtggaggaa actgtggttg tggatctggc tgcaagtgcg 60

gcaacggctg tggcggatgc aagatgtacc cagatttgag ctacaacgag agcaccacaa 120

ccgagacttt ggtgcttggg gtgggacctg agaagacaag cttcggcact atggaaatgg 180

gtgaatctcc tgctgctgaa aatggttgca aatgtggatc tgactgcaag tgtaacccct 240

gcacttgttc taaatgaaca aattaaatct tatttaagca gagatggatg tttgccttta 300

ttggctaaga caaaaaataa ctatagtttt aaaaatgtct agtttatagt tcttgtgtct 360

gttgaaaaca ggggaaatta tgtgtttttt cctctctaat aagaaatagt atggagtctg 420

aacaatgttt gggttcttgt tattgtatac aagtcgttgt gtggtttgta acatgagcat 480

ctctgctcct ggtattttaa cctatgtttt ggaagtggtt gttgtttggt ttcaagtaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 575

<210> 6

<211> 81

<212> PRT

<213> NtMT3 protein

<400> 6

Met Ser Cys Cys Gly Gly Asn Cys Gly Cys Gly Ser Gly Cys Lys Cys

1 5 10 15

Gly Asn Gly Cys Gly Gly Cys Lys Met Tyr Pro Asp Leu Ser Tyr Asn

20 25 30

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

35 40 45

Thr Ser Phe Gly Thr Met Glu Met Gly Glu Ser Pro Ala Ala Glu Asn

50 55 60

Gly Cys Lys Cys Gly Ser Asp Cys Lys Cys Asn Pro Cys Thr Cys Ser

65 70 75 80

Lys

<210> 7

<211> 498

<212> DNA

<213>NtMT4 gene

<400> 7

ggtttattat ctcccaacaa cataagaaaa caatgtctgg ctgcggatca aactgtggat 60

gtggatctag ctgcaattgc ggcagtggcg gaggatgtgg gatgtaccca gacttggaga 120

aatccactag ctttactatc attcagggtg ttgctcccat gaagagcttt gaggaattcg 180

gagagaaagc agcagaagga ggaaatgggt gcaagtgtgg atcaaactgc acctgtgacc 240

cttgcaactg ttaagaatat tctcactaca aaaactgcat tgtgtctctg tattaagtgt 300

gtgttctaaa agttattgta attataacaa taaaactaca accatggaac agattatgat 360

cagttcttag gtttgtgtgt tactcgtgtg acgtggcttt tctgtttgtt aattgggttg 420

tgtatcaata tactacataa gtttgtttgt ttgttaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaa 498

<210> 8

<211> 73

<212> PRT

<213> NtMT4 protein

<400> 8

Met Ser Gly Cys Gly Ser Asn Cys Gly Cys Gly Ser Ser Cys Asn Cys

1 5 10 15

Gly Ser Gly Gly Gly Cys Gly Met Tyr Pro Asp Leu Glu Lys Ser Thr

20 25 30

Ser Phe Thr Ile Ile Gln Gly Val Ala Pro Met Lys Ser Phe Glu Glu

35 40 45

Phe Gly Glu Lys Ala Ala Glu Gly Gly Asn Gly Cys Lys Cys Gly Ser

50 55 60

Asn Cys Thr Cys Asp Pro Cys Asn Cys

65 70

<210> 9

<211> 679

<212> DNA

<213>NtMT5 gene

<400> 9

ccaacttttt tgtacaaagt tgtcccccca acttttttgt acaaagttgt ccccccaact 60

tttttgtaca aagttgtccc cttctttggc tattttgcta attgtaatat cctcacaagc 120

tgatgcaagg gagacgtcta aggcaactgc tccaattacc caagaaatga attcaaacaa 180

cactactgat cagaagatac caaaacgtcc aaaaccagga ggcaatatct tcggcaaagc 240

ttgtaaaatt tgcccatgta aataccagat ttgcagcaaa tgtcctaaat gtgatgacca 300

aaatatcgcc ggcaaatttt gtaaaatttg ctcatgtaag actcagattt gcagtaaatg 360

tcctaaatgt cataaccaaa attaggcctc agagactatg tacttgtgct ggtgtgagtt 420

tagttttgag aataaaagga aagttatgaa tagcctaata taattctatt cactttcctc 480

tagttaattt ctcttagttt gtgttttgtt ttgttagtag ttcctattgt tgcaacttgc 540

aacaagtctt ggggtcaaca tgtacctctt gtcttgtagt ctttcgactg tatgatattg 600

taccgtattg tattgtaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaa 679

<210> 10

<211> 72

<212> PRT

<213> NtMT5 protein

<400> 10

Met Asn Ser Asn Asn Thr Thr Asp Gln Lys Ile Pro Lys Arg Pro Lys

1 5 10 15

Pro Gly Gly Asn Ile Phe Gly Lys Ala Cys Lys Ile Cys Pro Cys Lys

20 25 30

Tyr Gln Ile Cys Ser Lys Cys Pro Lys Cys Asp Asp Gln Asn Ile Ala

35 40 45

Gly Lys Phe Cys Lys Ile Cys Ser Cys Lys Thr Gln Ile Cys Ser Lys

50 55 60

Cys Pro Lys Cys His Asn Gln Asn

65 70

<210> 11

<211> 20

<212> DNA

<213>artificial sequence

<400> 11

cggaggatgt gggatgtacc 20

<210> 12

<211> 20

<212> DNA

<213>artificial sequence

<400> 12

tttgatccgc atttgcagcc 20

<210> 13

<211> 20

<212> DNA

<213>artificial sequence

<400> 13

gtgggatgta ccccgacttg 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence

<400> 14

atcagcagtt gcaagggtca 20

<210> 15

<211> 20

<212> DNA

<213>artificial sequence

<400> 15

cacaaccgag actttggtgc 20

<210> 16

<211> 21

<212> DNA

<213>artificial sequence

<400> 16

ggggttacac ttgcagtcag a 21

<210> 17

<211> 20

<212> DNA

<213>artificial sequence

<400> 17

tcattcaggg tgttgctccc 20

<210> 18

<211> 20

<212> DNA

<213>artificial sequence

<400> 18

agaaaagcca cgtcacacga 20

<210> 19

<211> 20

<212> DNA

<213>artificial sequence

<400> 19

acgtctaagg caactgctcc 20

<210> 20

<211> 20

<212> DNA

<213>artificial sequence

<400> 20

tgccggcgat attttggtca 20

<210> 21

<211> 20

<212> DNA

<213>artificial sequence

<400> 21

cccctcacca cagagtctgc 20

<210> 22

<211> 24

<212> DNA

<213>artificial sequence

<400> 22

aagggtgttg ttgtcctcaa tctt 24

<210> 23

<211> 35

<212> DNA

<213>artificial sequence

<400> 23

tttcagggcg ccatgtcttg ttgcggagga agttg 35

<210> 24

<211> 35

<212> DNA

<213>artificial sequence

<400> 24

cgttactagt ggatcttaac aattgcaagg gtcac 35

<210> 25

<211> 35

<212> DNA

<213>artificial sequence

<400> 25

tttcagggcg ccatgtcttg ctgtggagga agctg 35

<210> 26

<211> 35

<212> DNA

<213>artificial sequence

<400> 26

cgttactagt ggatctcagc agttgcaagg gtcac 35

<210> 27

<211> 35

<212> DNA

<213>artificial sequence

<400> 27

tttcagggcg ccatgtcttg ctgtggagga aactg 35

<210> 28

<211> 35

<212> DNA

<213>artificial sequence

<400> 28

cgttactagt ggatcttagc caataaaggc aaaca 35

<210> 29

<211> 35

<212> DNA

<213>artificial sequence

<400> 29

tttcagggcg ccatgtctgg ctgcggatca aactg 35

<210> 30

<211> 35

<212> DNA

<213>artificial sequence

<400> 30

cgttactagt ggatcttaac agttgcaagg gtcac 35

<210> 31

<211> 35

<212> DNA

<213>artificial sequence

<400> 31

tttcagggcg ccatgaattc aaacaacact actga 35

<210> 32

<211> 35

<212> DNA

<213>artificial sequence

<400> 32

cgttactagt ggatcctaat tttggttatg acatt 35

Claims

1.NtMT4 and/or NtMT5 gene is improveing the application in tolerance or enriching of the tobacco to heavy metal cadmium, described The cDNA of NtMT4, NtMT5 gene is respectively the nucleotide sequence as shown in SEQ ID NO.7, SEQ ID NO.9, or is respectively With the nucleotide sequence of SEQ ID NO.7, SEQ ID NO.9 complementary pairing, or respectively coding SEQ ID NO.8, SEQ The nucleotide sequence of tobacco metallothionein shown in ID NO.10.

2. the amino acid sequence tobacco metallothionein NtMT4 and NtMT5 as shown in SEQ ID NO.8, SEQ ID NO.10 respectively Application in tolerance or enriching of the improvement tobacco to heavy metal cadmium.

3. the saccharomyces cerevisiae of the reading frame nucleotide sequence inserted with NtMT4, NtMT5 gene described in claim 1 recombinates Expression vector, the cDNA of NtMT4, NtMT5 gene are respectively the nucleotide as shown in SEQ ID NO.7, SEQ ID NO.9 Sequence, or be respectively the nucleotide sequence with SEQ ID NO.7, SEQ ID NO.9 complementary pairing, or be respectively coding SEQ The nucleotide sequence of tobacco metallothionein shown in ID NO.8, SEQ ID NO.10.

4. saccharomyces cerevisiae recombinant expression carrier as claimed in claim 3 is in improvement engineered strain yeast to the tolerance of heavy metal cadmium Or the application in enriching.

5. a kind of saccharomyces cerevisiae, characterized in that its convert have the right to require 4 described in saccharomyces cerevisiae recombinant expression carrier.

6. application of the saccharomyces cerevisiae described in claim 5 in environment in the microorganism remediation engineering of heavy metal cadmium.

7. the tolerance of heavy metal cadmium or the biological agent of enriching in a kind of improvement tobacco, characterized in that its active ingredient contains There is NtMT4 and/or NtMT5 gene, NtMT4, NtMT5 gene is respectively as shown in SEQ ID NO.7, SEQ ID NO.9 Nucleotide sequence, or be respectively nucleotide sequence with SEQ ID NO.7, SEQ ID NO.9 complementary pairing, or be respectively to compile The nucleotide sequence of tobacco metallothionein shown in code SEQ ID NO.8, SEQ ID NO.10.

8. a kind of method of the tolerance or enriching of heavy metal cadmium in regulation tobacco, characterized in that include in this method step The expression of NtMT4 and/or NtMT5 gene in regulation tobacco, NtMT4, NtMT5 gene respectively such as SEQ ID NO.7, Nucleotide sequence shown in SEQ ID NO.9, or be respectively the nucleotide with SEQ ID NO.7, SEQ ID NO.9 complementary pairing Sequence, or be respectively the nucleotide sequence of tobacco metallothionein shown in coding SEQ ID NO.8, SEQ ID NO.10.