CN112831502B

CN112831502B - Metallothionein DaMT3a and application of encoding gene thereof

Info

Publication number: CN112831502B
Application number: CN202011323894.XA
Authority: CN
Inventors: 黄亚成; 刘林娅; 苏龙兴; 黄东益; 王才全; 曾林
Original assignee: Liupanshui Normal University
Current assignee: Liupanshui Normal University
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-08-26
Anticipated expiration: 2040-11-23
Also published as: CN112831502A

Abstract

The invention belongs to the technical field of biology, and discloses application of metallothionein DaMT3a and a coding gene thereof. The full-length cDNA of the gene DaMT3a of the metallothionein of the ginseng potatoes is cloned for the first time, and gene expression analysis shows that the gene is specifically expressed in leaves, is up-regulated under the induction of ethephon and abscisic acid, is up-regulated under low temperature and high stress, and is down-regulated under the stress of mechanical injury, so that the gene is possibly closely related to the stress resistance of the ginseng potatoes and can be used as an important target gene for transgenic breeding of the ginseng potatoes. Further transferring the gene into prokaryotic expression strain, and transferring the transgenic strain to heavy metal ions (Cd) under normal conditions ²⁺ ) The growth under the stress of NaCl and hydrogen peroxide is obviously improved, so the gene can be used as an important gene resource and can be applied to stress resistance and heavy metal stress resistance genetic engineering of other plants or microorganisms except the ginseng potatoes.

Description

Metallothionein DaMT3a and application of encoding gene thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to metallothionein DaMT3a and application of a coding gene thereof.

Background

Metallothionein (MTs) is a low molecular weight (6-7kDa) polypeptide rich in cysteine (Cys) and capable of being combined with heavy metal, and is commonly present in microorganisms, higher animals and plants and human bodies. According to the distribution and arrangement mode of Cys residues of the metallothionein gene in the plant which is cloned at present, the plant can be divided into four types of Type1, Type2, Type3 and Type 4. The four metallothionein genes have the characteristic of tissue-specific expression, wherein Type1 is mainly expressed in roots, Type2 is mainly expressed in leaves, Type3 is mainly expressed in mature fruits and leaves, and the functional research on the metallothionein genes in plants by mainly expressing Type4 in seeds shows that the functions of the metallothionein genes are mainly expressed in the following parts: (1) can combine heavy metal in cells to maintain the ecological balance of heavy metal ions in the cells; (2) can be used as active oxygen scavenger, with free radical (. OH) scavenging ability about several thousand times that of SOD, and oxygen free radical (. O) scavenging ability about 25 times that of Glutathione (GSH), and has strong antioxidant activity; (3) participating in the growth and development of plants; (4) regulating the activity of enzyme and transcription factor.

The ginseng potato (Dioscorea alata L.) also called a big potato is also called purple ginseng potato (cloning and analysis of key genes in stability and synthesis route of anthocyanin of purple ginseng potato, Zhao Jing Mei, 2018), is used as a food and drug homologous grain crop and is mainly used for food, medicine, industry, fuel ethanol production and the like in China. In recent years, although the planting industry in China is developed more quickly, the growth and development of plants are seriously influenced by heavy metal pollution and abiotic stress of soil. Metallothionein, as a protein with important biological functions, not only can effectively reduce the damage of heavy metals to plants, but also can improve the tolerance of the plants to adverse circumstances by removing active oxygen in the plants. Therefore, the research on the heavy metal sulfur protein in the sweet potatoes is helpful for understanding the action mechanism of the heavy metal sulfur protein for improving the stress resistance of the sweet potatoes. At present, the function of the metallothionein gene in the ginseng and the potato is not reported.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of metallothionein DaMT3a and a coding gene thereof.

The first aspect of the invention provides a metallothionein gene which is named as DaMT3a gene, and the nucleotide sequence of the metallothionein gene is shown as SEQ ID NO. 1.

The second aspect of the invention provides a protein, which is metallothionein DaMT3a, and is the protein coded by the metallothionein gene of the first aspect of the invention, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant expression vector comprising an original vector and the metallothionein gene or its open reading frame (nucleotide sequences 66-254 shown in SEQ ID NO: 1) according to the first aspect of the present invention.

As the original vector, there may be used vectors commonly used in the field of genetic recombination, such as viruses, plasmids, etc. The invention is not limited in this regard. In one embodiment of the present invention, the original vector is pGEX-4T-1 vector plasmid or pMD18-T vector plasmid, but it is understood that other plasmids, viruses, or the like may be used in the present invention.

Preferably, the original vector is pGEX-4T-1 vector plasmid, and the nucleotide sequence at positions 66-254 shown in SEQ ID NO:1 is positioned between the EcoR I and Xho I restriction enzyme sites of the pGEX-4T-1 vector plasmid.

In a fourth aspect, the invention provides the use of a metallothionein gene according to the first aspect of the invention, or a protein according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention, for increasing the growth rate of a prokaryotic expression strain.

The prokaryotic expression strain may be a prokaryotic expression strain commonly used in the field of gene recombination, but the invention is not limited thereto. In a specific embodiment of the invention, the prokaryotic expression strain is e.coli BL21(DE 3).

In a fifth aspect, the invention provides the use of the metallothionein gene according to the first aspect of the invention, or the protein according to the second aspect of the invention, or the recombinant expression vector according to the third aspect of the invention, for improving the resistance of prokaryotic expression strains to cadmium heavy metal stress, NaCl stress and/or hydrogen peroxide stress.

The prokaryotic expression strain may be a prokaryotic expression strain commonly used in the field of gene recombination, which is not limited in the present invention. In a particular embodiment of the invention, the prokaryotic expression strain is e.coli BL21(DE 3).

The sixth aspect of the present invention provides a primer pair, the nucleotide sequence of which is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

The seventh aspect of the present invention provides a primer pair, the nucleotide sequence of which is shown as SEQ ID NO. 6 and SEQ ID NO. 7.

An eighth aspect of the present invention provides a primer set, characterized in that the nucleotide sequences thereof are shown as SEQ ID NO. 8 and SEQ ID NO. 9.

The full-length cDNA of the gene DaMT3a of the metallothionein of the ginseng potatoes is cloned for the first time, and gene expression analysis shows that the gene DaMT3a is specifically expressed in leaves and is subjected to up-regulated expression induced by ethephon and abscisic acid, meanwhile, the gene is subjected to up-regulated expression under low temperature and high stress, and is subjected to down-regulated expression under mechanical injury stress, so that the gene is possibly closely related to stress resistance of the ginseng potatoes, can be used as an important target gene for transgenic breeding of the ginseng potatoes, and is expected to promote the growth and development of the ginseng potatoes by regulating the expression of the gene, thereby maximally excavating the production potential of the ginseng potatoes. The gene is further transferred into prokaryotic expression strain, and the transgenic strain is subjected to heavy metal ion (Cd) under normal conditions ²⁺ ) The growth under the stress of NaCl and hydrogen peroxide is obviously improved, so the gene can be used as an important gene resource and can be applied to stress resistance and heavy metal stress resistance genetic engineering of other plants or microorganisms except the ginseng potatoes.

Drawings

FIG. 1 shows the results of tissue-specific expression analysis of the DaMT3a gene (tuber, bulbil, leaf, root, stem, flower).

FIG. 2 shows the effect of cold treatment on the expression of DaMT3a gene.

FIG. 3 shows the effect of high temperature treatment on the expression of DaMT3a gene.

FIG. 4 is a graph showing the effect of mechanical injury treatment on the expression of DaMT3a gene.

FIG. 5 is a graph showing the effect of ABA treatment on DaMT3a gene expression.

FIG. 6 is a graph showing the effect of ethephon treatment on DaMT3a gene expression.

FIG. 7 shows prokaryotic expression analysis of DaMT3a (the band shown in black box is the specifically expressed target protein produced after IPTG induction).

FIG. 8 shows the growth of transgenic E.coli on medium (A: OD grown under normal conditions) ₆₀₀ Measuring; b: OD grown under heavy metal cadmium ion stress ₆₀₀ Measuring; c: OD grown under sodium chloride stress ₆₀₀ Measuring; d: OD grown under Hydrogen peroxide stress ₆₀₀ Measurement).

Detailed Description

We clone the full-length cDNA of the gene of the metallothionein of the ginseng potato for the first time, and carry out gene expression analysis and function research, and name the gene as DaMT3 a. The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, but are all conventional products available from companies.

Example 1 acquisition of metallothionein and Gene encoding the same

Analyzing nucleotide sequences of metallothionein of arabidopsis thaliana, cassava, sweet potato and the like which are logged in at NCBI, screening and splicing a sequence (contig) after assembling of about 400bp of metallothionein genes of the sweet potato by searching EST sequence databases of tubers and leaves of the sweet potato, and designing a pair of specific primers for amplification to obtain a cDNA full-length sequence containing a complete reading frame.

The specific method comprises the following steps:

specific primers were designed as follows:

f (5' -end): 5'-ACAACACAAGCCATTCAATCTAT-3', respectively;

r (3' -end): 5'-TTCATTAAAATAAAAGAGAGGCAG-3' are provided.

The cDNA of the leaves of a Dioscorea opposita (Dioscorea alata L.; Yam germplasm resource garden of university of Hainan delirium, number Da 56; hereinafter referred to as Shen-Shu Da56) was used as a template (obtained by reverse transcription with a random primer), F and R were used as primers, the final concentration was 0.5. mu. mol/L, and PCR amplification was performed in a 25. mu.l reaction system. The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s, and 30 cycles; extension at 72 ℃ for 10 min.

The obtained nucleotide fragment of about 300bp is connected to a pMD18-T vector (TaKaRa) for sequencing, and the sequencing shows that the obtained fragment is the metallothionein gene of the invention, the fragment has the nucleotide sequence of SEQ ID NO:1 in a sequence table, the full length of the SEQ ID NO:1 in the sequence table is 345 nucleotides, the fragment comprises an Open Reading Frame (ORF) with the length of 189 nucleotides (66-254 th nucleotide sequence from the 5' end of the SEQ ID NO: 1), a 5' -UTR with the length of 65 nucleotides (1-65 th nucleotide sequence from the 5' end of the SEQ ID NO: 1) and a 3' -UTR with the length of 91 nucleotides (255-345 th nucleotide sequence from the 5' end of the SEQ ID NO: 1), and the fragment encodes a protein with the length of 62 amino acids (SEQ ID NO:2 in the sequence table) and the molecular weight of about 7.2KDa, namely the metallothionein, and the metallothionein gene is named as DaMT3a gene. The above pMD18-T recombinant vector containing the nucleotide of SEQ ID NO. 1 was named pMD18-DaMT3 a.

Meanwhile, the genomic DNA of the leaves of the ginseng potato Da56 is taken as a template to carry out PCR amplification (the program is as described above), and the sequencing result shows that the metallothionein genomic sequence is obtained, the sequence has the nucleotide sequence of SEQ ID NO:3 in the sequence table, and the total of 441 nucleotides comprises 1 intron (the intron is from the nucleotide sequence 113-208 at the 5' end of the sequence 3).

Example 2 analysis of Gene expression Pattern of the Gene of the Polymnia sonchifolia DaMT3a

<1> tissue-specific expression of DaMT3a gene of Stichopus japonicus

Using cDNA of RNA random reverse transcription of tuber, bulbil, leaf, root, stem and male flower of ginseng potato Da56 as template, using DaMT3a gene specific primer(F: 5'-ATGGGATGGTGATTGTTGAGG-3'; R:5'-CCCATCATTCTCAGCAGCAGT-3') real-time fluorescent quantitative PCR was performed. The total reaction system was 20. mu.L, containing 2. mu.L of template, 10. mu.L of 2 XSSYBR Premix and 10. mu. mol. L ^-1 0.3. mu.L of each of the upstream and downstream primers of (4); the amplification procedure was pre-denaturation at 95 ℃ for 30 s; 94 ℃ for 5s, 60 ℃ for 20s, 72 ℃ for 20s, 45 cycles. The results showed that the gene was expressed in large abundance between tissues, mainly concentrated in the stem, followed by leaves, roots and flowers, and not substantially expressed in tubers and bulbels (FIG. 1).

<2> Effect of Low temperature on the expression of DaMT3a Gene

Selecting 6-8 completely-unfolded leaves, basically consistent growth vigor and no plant diseases and insect pests, carrying out low temperature (4 ℃) on the ginseng and potato Da56 tissue culture seedlings, respectively sampling at 0h, 3h, 6h and 9h, taking down the seedlings, placing the seedlings in liquid nitrogen for freezing and storing, and extracting RNA. The real-time fluorescent quantitative PCR (tissue-specific expression of the gene DaMT3a from the same <1> ginseng potato) was carried out by using the reverse transcribed cDNA as a template and using a DaMT3a gene-specific primer, and the results show that the expression change of the gene DaMT3a is not obvious within 6 hours and is increased only within 9 hours when the tissue culture seedlings are treated at low temperature (FIG. 2).

<3> Effect of high temperature on the expression of DaMT3a Gene

Selecting 6-8 completely-unfolded leaves, basically consistent growth vigor and no plant diseases and insect pests, carrying out high temperature (45 ℃) on the ginseng and potato Da56 tissue culture seedlings, respectively sampling at 0h, 3h, 6h and 9h, taking down the seedlings, placing the seedlings in liquid nitrogen for freezing and storing, and extracting RNA. The reverse transcription cDNA is taken as a template, and a DaMT3a gene specific primer is used for carrying out real-time fluorescence quantitative PCR (like the tissue specific expression of <1> ginseng potato DaMT3a gene), and the result shows that the expression quantity of the DaMT3a gene has obvious down-regulation expression under the high-temperature treatment. (FIG. 3).

<4> Effect of injury treatment on the expression of DaMT3a Gene

Selecting 6-8 pieces of completely unfolded leaves, basically consistent growth vigor and no plant diseases and insect pests, and carrying out injury treatment on the tissue culture seedlings of the ginseng and the sweet potato Da 56. The third leaf, counting down from the apical meristem, was subjected to a single crush injury (Rajendran, Lin et al 2014) with forceps, taken down at 0h, 0.5h, 6h and 12h, frozen in liquid nitrogen and RNA extracted. The real-time fluorescent quantitative PCR (tissue-specific expression of the gene DaMT3a from the same "< 1> ginseng potato) was performed using the cDNA reverse transcribed as a template and using a DaMT3a gene-specific primer, and the results showed that the gene DaMT3a was stressed by injury and was up-regulated in the initial stage, and then the expression level began to be significantly down-regulated (FIG. 4).

<5> Effect of plant growth substances on the expression of DaMT3a Gene

Selecting plant growth substances of abscisic acid ABA and ethephon, respectively spraying 6-8 completely-unfolded leaves of the tissue culture seedlings of the ginseng potato Da56 which have basically consistent growth vigor and no plant diseases and insect pests on the whole plant until the leaves drip water, stopping spraying, respectively sampling at 0h, 3h, 6h and 9h, taking down the leaves, placing the leaves in liquid nitrogen for freezing and storing, and extracting RNA. The real-time fluorescent quantitative PCR (tissue-specific expression of the gene DaMT3a from the same <1> ginseng potato) is carried out by using the reverse transcribed cDNA as a template and using a DaMT3a gene specific primer, and the result shows that the expression of the DaMT3a gene is obviously induced by abscisic acid ABA and ethephon, the expression level reaches the peak value within 6 hours and reaches more than 2 times of the initial level, and then the down-regulation expression is started (figure 5 and figure 6).

Example 3 prokaryotic expression and functional verification of the DaMT3a Gene

The prokaryotic expression vector of the DaMT3a gene is constructed by utilizing pGEX-4T-1 expression vector (the expression vector is only exemplified in the embodiment, other expression plasmids, virus vectors and the like can be adopted in the invention), and meanwhile, Escherichia coli expression strain E.coli BL21(DE3) (competence is purchased from Tiangen Biotechnology science and technology Co., Ltd.) is adopted to induce recombinant protein, and the influence of the recombinant protein on the growth of BL21 strain is determined, and the specific method is as follows:

<1> preparation of recombinant vector containing coding region of DaMT3a Gene

Design of DaMT3a gene coding region primer

F:5'-CGCGAATTC(EcoR I cleavage site) ATGTCTAGCTGTGGCAACTGTG-3',

R:5'-CCCCTCGAG(Xho I cleavage site) TCACTTGCCGCAGTTGC-3',

performing PCR amplification by taking pMD18-DaMT3a as a template, wherein the amplification procedure is as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 30s, and 30 cycles; extension at 72 ℃ for 8 min. Carrying out double enzyme digestion on the amplification product and pGEX-4T-1 expression vector by using restriction enzymes EcoR I and Xho I, connecting the restriction enzymes EcoR I and Xho I through T4 DNA Ligase to obtain a recombinant vector, and carrying out PCR identification by using vector primers pGEX-5(5'-GGGCTGGCAAGCCACGTTTGGTG-3') and pGEX-3(5'-CCGGGAGCTGCATGTGTCAGAGG-3') to ensure that the metallothionein coding fragment is positively cloned to the expression vector. The recombinant vector is subjected to sequencing identification, and the recombinant expression vector which is identified to be correct and contains the 66 th-254 th nucleotide sequence of SEQ ID NO. 1 in the sequence table and has accurate reading frame is named as pGEX-4T-1-DaMT3 a.

<2> prokaryotic expression of DaMT3a gene

The obtained recombinant vector pGEX-4T-1-DaMT3a was introduced into E.coli BL21(DE3) to obtain recombinant expression bacteria, the correctly identified recombinant bacteria were cultured in LB medium containing 50. mu.g/mL ampicillin until OD600 became 0.4-0.6, IPTG (isopropyl-. beta. -D-thiogalactoside) was added to a final concentration of 1mM, induction culture was carried out at 30 ℃ for 4 hours, the same recombinant bacteria cultured in the medium without IPTG were used as a control, cells were collected by centrifugation, and mycoprotein was subjected to 15% SDS-PAGE electrophoresis. The results show that the gene DaMT3a realizes high-efficiency heterologous expression under IPTG induction, the recombinant protein comprises DaMT3a and cytoplasmic fusion protein GST (glutathione S transferase), and the apparent molecular weight of the expressed protein is similar to the theoretical molecular weight and is about 31kDa (figure 7).

<3> Effect of DaMT3a recombinant protein on Strain growth

The strains containing pGEX-4T-1-DaMT3a and pGEX-4T-1 were cultured to the same OD ₆₀₀ Adding IPTG to a final concentration of 1mM, inducing and culturing at 30 deg.C for 1h, 2h, 3h, 4h, 5h, 6h and 7h, and determining OD ₆₀₀ The value is obtained. The results are shown in FIG. 8A: the increase of the strain containing pGEX-4T-1-DaMT3a was higher than that of the strain containing pGEX-4T-1, indicating that the protein encoded by the DaMT3a gene can enhance the growth of the strain under normal conditions. In addition, the heavy metal ion (Cd) of the strain containing pGEX-4T-1-DaMT3a and pGEX-4T-1 was determined ²⁺ 500. mu.M), salt (NaCl, 500mM) and hydrogen peroxide (H) ₂ O ₂ 1mM) under stress for 1h, 2h, 3h, 4h, 5h, 6h and 7h ₆₀₀ The value is obtained. The results show that: the growth of the strain containing pGEX-4T-1-DaMT3a is highIn the strain containing pGEX-4T-1, it was demonstrated that the protein encoded by the DaMT3a gene can enhance the growth of the strain under the stress of heavy metal cadmium ions, sodium chloride and hydrogen peroxide (FIGS. 8B-8D). The result of the experiment conducted by introducing the DaMT3a gene into other prokaryotic expression bacteria (e.g., e.coli Rosetta, etc.) is the same as e.coli BL21(DE3), which indicates that the protein encoded by the DaMT3a gene can improve the growth of other prokaryotic strains under normal conditions, heavy metal stress, sodium chloride stress and hydrogen peroxide stress.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

<110> six-coil water college

<120> metallothionein DaMT3a and application of coding gene thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 345

<212> DNA

<213> Artificial

<400> 1

acaacacaag ccattcaatc tattaactct ttcttcttct tcttctccac aacatcatta 60

caaacatgtc tagctgtggc aactgtgact gtgctgacaa gaaccagtgt gtgaagaagg 120

ggaactcata tgggatggtg attgttgagg agaaggaggt tgttgaggtt gtggaaatgg 180

caactgctgc tgagaatgat gggtgcaagt gtgctgctaa ttgcacttgt gctggttgca 240

actgcggcaa gtgatcatat attaattacc ctttaattat gtagtaatta ataagatgta 300

agccagagtt acactggctt tctgcctctc ttttatttta atgaa 345

<210> 2

<211> 62

<212> PRT

<213> Artificial

<400> 2

Met Ser Ser Cys Gly Asn Cys Asp Cys Ala Asp Lys Asn Gln Cys Val

1 5 10 15

Lys Lys Gly Asn Ser Tyr Gly Met Val Ile Val Glu Glu Lys Glu Val

20 25 30

Val Glu Val Val Glu Met Ala Thr Ala Ala Glu Asn Asp Gly Cys Lys

35 40 45

Cys Ala Ala Asn Cys Thr Cys Ala Gly Cys Asn Cys Gly Lys

50 55 60

<210> 3

<211> 441

<212> DNA

<213> Artificial

<400> 3

acaacacaag ccattcaatc tattaactct ttcttcttct tcttctccac aacatcatta 60

caaacatgtc tagctgtggc aactgtgact gtgctgacaa gaaccagtgt gtgtaagtag 120

ttcttgtgta cttaaaacta gatttatata tatatgtttg tgtttaatta tgtatatgaa 180

atggtgtttt tggattaatg gaatgcagga agaaggggaa ctcatatggg atggtgattg 240

ttgaggagaa ggaggttgtt gaggttgtgg aaatggcaac tgctgctgag aatgatgggt 300

gcaagtgtgc tgctaattgc acttgtgctg gttgcaactg cggcaagtga tcatatatta 360

attacccttt aattatgtag taattaataa gatgtaagcc agagttacac tggctttctg 420

cctctctttt attttaatga a 441

<210> 4

<211> 23

<212> DNA

<213> Artificial

<400> 4

acaacacaag ccattcaatc tat 23

<210> 5

<211> 24

<212> DNA

<213> Artificial

<400> 5

ttcattaaaa taaaagagag gcag 24

<210> 6

<211> 21

<212> DNA

<213> Artificial

<400> 6

atgggatggt gattgttgag g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial

<400> 7

cccatcattc tcagcagcag t 21

<210> 8

<211> 31

<212> DNA

<213> Artificial

<400> 8

cgcgaattca tgtctagctg tggcaactgt g 31

<210> 9

<211> 26

<212> DNA

<213> Artificial

<400> 9

cccctcgagt cacttgccgc agttgc 26

Claims

1. A metallothionein gene which is namedDaMT3aThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A metallothionein, which is metallothionein DaMT3a, is a protein encoded by the metallothionein gene according to claim 1, and has an amino acid sequence shown as SEQ ID NO. 2.

3. A recombinant expression vector obtained by inserting the metallothionein gene or an open reading frame thereof according to claim 1 into an original vector.

4. The recombinant expression vector of claim 3, wherein the original vector is pGEX-4T-1 vector plasmid, and the nucleotide sequence at position 66-254 shown in SEQ ID NO:1 is inserted between EcoR I and Xho I restriction endonuclease sites of pGEX-4T-1 vector plasmid.

5. The recombinant expression vector of claim 3, wherein the primer pair for amplifying the metallothionein gene has nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5.

6. The recombinant expression vector of claim 3, wherein the primer pair for amplifying the open reading frame has the nucleotide sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9.

7. Use of the metallothionein gene in claim 1, or the metallothionein in claim 2, or the recombinant expression vector in claim 3 or 4 for increasing the growth rate of a prokaryotic expression strain; the prokaryotic expression strain is escherichia coliE. coli BL21（DE3）。

8. Use of the metallothionein gene in claim 1, or the metallothionein in claim 2, or the recombinant expression vector in claim 3 or 4 for increasing the resistance of prokaryotic expression strains to cadmium ion stress, and/or NaCl stress, and/or hydrogen peroxide stress; the prokaryotic expression strain is escherichia coliE. coli BL21（DE3）。