CN102154317B - Application of lotus metallothionein gene MT2a in improving seed longevity and activity - Google Patents

Abstract

The invention belongs to the fields of molecular biology and biotechnology, and particularly relates to an application of a lotus metallothionein gene MT2a in improving plant seed longevity and activity. A lotus seed maturation later-stage embryo axis cDNA (complementary deoxyribonucleic acid) library is constructed, EST sequencing is performed to obtain a full-length lotus metallothionein gene which is named as MT2a, and the accession number of the full-length lotus metallothionein gene in GenBank is EF421200. A sense over-expression vector of the MT2a is further constructed, the model plant arabidopsis thaliana is transformed, and the result indicates that the transgenic arabidopsis thaliana seed has stronger capability in resisting artificial aging and germination activity under sodium chloride stress than the wild seed. Therefore, if the MT2a is transferred into important crops such as rice, wheat, corn, soybean and the like, the storage life and activity of the crop seeds can be possibly prolonged. Due to the application of MT2a, the global tremendous agricultural loss caused by seed deterioration and salt stress every year can be reduced, and the MT2a has important economic benefits and application prospects.

Description

Lotus metallothionein gene MT2a is in the application that improves on seed longeivity and the vigor

Technical field

The invention belongs to molecular biology and biological technical field, relate to concretely the lotus metallothionein gene MT2aIn the application that improves on plant seed life-span and the vigor.

Background technology

Seed is the basic of agriculture production.The life-span of seed and vigor preserve for plant propagation, crop yield, germ plasm resource and species diversity plays conclusive impact.After preserving for a long time or under the adverse environmental factor, high-quality seed still can keep high germination rate and grow healthy and strong seedling.But along with the most seed of the prolongation of storage time begins to occur bad change, gradually devitalization becomes more and more responsive to adverse circumstance when sprouting, finally can not sprout.The bad accommodation of seed often is accompanied by the variation on a series of Physiology and biochemistry, such as the fracture of DNA, the peroxidation of lipid and the damage of albumen etc.McDonald etc. think that seed longeivity is subjected to inherent gene and external environmental factors joint effect.Although carried out a large amount of research for seed longeivity and vigor, still not fully aware of to its definite mechanism.

People generally accept old and feeble free-radical oxidn theory, and active oxygen (Reactive Oxygen Species, ROS) is considered to cause an important factor of seed deterioration.In order to tackle the oxidative damage that is caused by active oxygen, plant evolution has gone out the complete antioxidant system of a cover, comprises enzymatic antioxidant system and non-enzymatic antioxidant system.The height of antioxidant system efficient affects life-span and the vigor of seed usually.For example, 2004, the proof vitamin-Es such as Sattler played vital effect for the life-span of keeping seed and vigor.And the antioxidases such as superoxide-dismutase, catalase, glutathione reductase and ascorbate peroxidase enzyme also are proved to be relevant with vigor (Bailly etc., 1996,2001 with the life-span of seed; Lee etc., 2010).

Metallothionein(MT) (Metallothioneins, MTs) is the class lower molecular weight that extensively exists of organic sphere, be rich in sulfydryl, can be in conjunction with the protein of heavy metal ion.Metallothionein(MT) plays an important role (Cobbett and Goldsbrough, 2002) in the detoxifcation of keeping heavy metal ion stable state, heavy metal ion and the aspects such as transportation, Scavenger of ROS.Existing studies show that, metallothionein(MT) and vegeto-animal aging are closely related.Yang etc. (2006) prove that turning the metallothionein(MT) mouse has the ability of the oxidative damage that stronger opposing aging induces than transgenic mice not, therefore shows the longer life-span.In the model plant Arabidopis thaliana, the metallothionein gene of the overwhelming majority raises (Guo etc., 2003) in a large number at the blade of aging and the expression in the fruit pod.In autumn, metallothionein gene can great expression in the old and feeble leaf of aspen tree, and can (Bhalerao etc., 2003) in spire.Except having the close relationship with aging, research in recent years also show metallothionein(MT) in the degeneration-resistant process of plant, bring into play in important effect.For example, upland rice is when being subject to sodium-chlor or polyethylene glycol 6000 and coercing, and the table of metallothionein(MT) is to amount can rise in a large number (Wu etc., 2005; Yang etc., 2009).

Lotus ( Nelumbo nuciferaGaertn.) be one of earth ancient plant before ice age of surviving.The lotus seed is through the most long-lived of scientific verification and one of the most resistant to elevated temperatures plant seed (Shen-Miller etc., 1995; Huang Shangzhi etc., 2003).As seen, the lotus seed provides the splendid genetic resources that excavates control seed longeivity and vigor gene for us.In view of the above, the present invention is undertaken having obtained a total length lotus metallothionein gene after the EST order-checking by making up lotus seed maturity later stage plumular axis cDNA library, MT2aFurther make up MT2aJust over-express vector, transformation mode plant Arabidopis thaliana, the result shows that the transgenic arabidopsis seed has the artificially-aged ability of stronger opposing and the sprouting vigor under NaCl Stress than wild type seeds. MT2aThe genetic transformation that can be used for plant, life-span and the sprouting vigor of raising plant seed.

Summary of the invention

The object of the invention is to overcome the defective of prior art, the lotus metallothionein gene is provided MT2aApplication aspect the application on raising plant seed life-span and the vigor and the sprouting vigor under raising plant seed salt stress.

Another object of the present invention is for providing a kind of coding such as the lotus metallothionein gene MT2aAmino acid improving the application on plant seed life-span and the vigor and improving application aspect the sprouting vigor under the plant seed salt stress.

The present invention is achieved through the following technical solutions:

The lotus metallothionein gene of nucleotide sequence shown in SEQ ID NO:1 MT2aIn the application that improves on plant seed life-span and the vigor.

The lotus metallothionein(MT) of aminoacid sequence shown in SEQ ID NO:2 is in the application that improves on plant seed life-span and the vigor.

The lotus metallothionein gene of nucleotide sequence shown in SEQ ID NO:1 MT2aIn the application that improves the sprouting vigor under the plant seed salt stress.

The lotus metallothionein(MT) of aminoacid sequence shown in SEQ ID NO:2 is in the application that improves the sprouting vigor under the plant seed salt stress.

Lotus metallothionein gene of the present invention MT2aBy making up lotus seed maturity later stage plumular axis cDNA library, then the library being carried out obtaining after EST checks order. MT2aAccession number in GenBank is EF421200, and opening code-reading frame is 243bp, 5 '-UTR is 99 bp, 3 '-UTR is 357 bp, 80 amino acid of encoding. MT2aThe sequence identity of European beechy MT2 is 74% among albumen and the GenBank of coding, shows that what obtain through above-mentioned steps is the gene of lotus metallothionein(MT) of encoding.

According to what obtain MT2aSequence information, restriction enzyme site has been added in design SmaI and SacThe primer of I amplifies from the lotus plumular axis library that makes up with the method for PCR MT2aThe opening code-reading frame fragment, then the PCR product is connected into the pGEMT-Easy carrier, make up intermediate carrier pGEMT-MT2a, transform intestinal bacteria after, the order-checking of picking mono-clonal is identified.Extract the correct clone's of order-checking plasmid, use SmaI and SacThe I enzyme is cut rear recovery small segment, connects in the pBI121 large fragment with same enzymic digestion, thereby makes up plant expression vector pBI121-MT2a.The carrier that builds is transformed intestinal bacteria, and the order-checking of picking mono-clonal is identified.Extract the correct clone's of order-checking plasmid, electric shocking method transforms agrobacterium tumefaciens EHA105, adopts agriculture bacillus mediated inflorescence infusion method to transform the wild Arabidopis thaliana of Col-0 type.Identify by kantlex screening and blade PCR, obtain genetically modified positive plant.Continue to cultivate until obtain the T3 transgenosis homozygote in generation.Utilize RT-PCR and Western Blot to prove MT2aGene has all obtained great expression on rna level and the protein level in the transgenic arabidopsis seed.

With transgenic seed and the wild type seeds that filters out, through behind the Artificial ageing, sprout respectively experiment and vigor Coloration experiment.Sprout the sprouting speed of experimental result demonstration transgenic seed with finally germination rate is all far above wild type seeds, the upgrowth situation of transgenosis seedling is significantly better than the wild-type seedling.Vigor Coloration experiment result shows that the wild type seeds major part has all been lost vigor behind the Artificial ageing, and transgenic seed still keeps higher vigor.The above results shows MT2aHas the ability that improves plant seed life-span and vigor.Transgenosis and wild type seeds are sprouted at 0.5 MS solid medium of the sodium-chlor that contains 150 mM, and the result shows that the sprouting speed of transgenic seed and final germination rate all apparently higher than the wild type seeds of contrast, show MT2aCan improve the sprouting vigor of plant seed under salt stress.

Compared with prior art, beneficial effect of the present invention is:

(1) from lotus, has been separated to metallothionein gene MT2a, confirmed by transformation mode plant Arabidopis thaliana MT2aCan improve the life-span of plant seed and the sprouting vigor under salt stress.

(2)Will MT2aBe transferred in the important farm crop such as paddy rice, wheat, corn and soybean, then may improve staging life and the vigor of important crop seeds.

(3) MT2aApplication can reduce the annual huge agricultural losses that cause because of seed deterioration and salt stress in the whole world, have important economic benefit and application prospect.

Description of drawings

Fig. 1It is the diagram of plant expression vector pBI121-MT2a.RB: right margin, LB: left margin.

Fig. 2RT-PCR and the Western Blot detected result of transgenic arabidopsis seed.

A is the detected result of RT-PCR, Actin muscle 2( Actin2) be confidential reference items; B is the detected result of Western Blot, and 20kD is molecular size range.WT represents wild-type.1,2,3 is respectively three different transgenic lines.

Fig. 3The sprouting figure as a result behind Artificial ageing wild-type and the transgenic arabidopsis seed.

X-coordinate is fate after planting, and ordinate zou is germination rate.■ represents wild-type, ● ◆ the transgenic line that ▲ expression is different.

Fig. 4The growth of seedling figure behind Artificial ageing wild-type and the transgenic arabidopsis seed.

WT represents wild-type.1,2,3 is respectively three different transgenic lines.

Fig. 5It is the vigor colored graph behind Artificial ageing wild-type and the transgenic arabidopsis seed.

WT represents wild-type.1,2,3 is respectively three different transgenic lines.The great-hearted seed of dark expression, the seed of light expression vigor decline or devitalization.Scale=1mm.

Fig. 6As a result figure of wild-type and the sprouting of transgenic arabidopsis seed under NaCl Stress.

X-coordinate is fate after planting, and ordinate zou is germination rate.■ represents wild-type, ● ◆ the transgenic line that ▲ expression is different.

Embodiment:

Embodiment 1: the lotus metallothionein gene MT2aThe clone

(1) preparation of lotus seed plumular axis: gather in the crops the lotus seed in the later stage of reaching maturity, strip plumular axis;

(2) extraction of total RNA: adopt the Trizol product of Invitrogen company to extract total RNA;

(3) cDNA library makes up: adopt the SMART cDNA library construction kit of Clontech company to make up the library;

(4) EST order-checking: take the positive monoclonal bacterium liquid that obtains as the order-checking sample, entrust Shanghai Bo Ya biotech company, the positive monoclonal after with 3730 sequenators PCR being verified carries out sequencing;

(5) homology retrieval: use the homologous gene of BLAST instrument search gained est sequence in GenBank, confirm that the gene that obtains is the lotus metallothionein gene.

Embodiment 2: the structure of plant expression vector

(1) clone of gene fragment: take embodiment 1 described lotus cDNA library as template, obtain having added restriction enzyme site by the PCR clone SmaI and SacThe PCR fragment of I.The PCR primer is as follows, and underscore partly is restriction enzyme site.

Forward primer: SEQ ID NO:3:

5′-TCC CCCGGGAATGTCTTGCTGCGGAGGA-3′

Reverse primer: SEQ ID NO:4:

5′-CTG GAGCTCGCCCTC CTCTCATTTGCAG-3′

The PCR reaction system is: 2 μ l lotus cDNA libraries, 6 μ l dNTP(2.5 mM), 1.5 μ l forward primer (10 μ M), 1.5 μ l reverse primer (10 μ M), 5 μ l 10*PCR damping fluids, 1 μ l EX Taq enzyme (Takara company product) replenishes deionized water at last, and making totally is 50 μ l.The reaction conditions of PCR is: 94 ° of C 3 minutes; Then enter following circulation: 94 ° of C 30 seconds, 58 ° of C 30 seconds, 72 ° of C 30 seconds, totally 30 circulations; Last 72 ° of C extended 7 minutes;

(2) the T carrier connects: get 2 microlitre PCR products and be connected with the pGEMT-Easy carrier, operate structure intermediate carrier pGEMT-MT2a according to the specification sheets step of Promega company;

(3) intestinal bacteria transform: will connect product and transform bacillus coli DH 5 alpha competence (day root company product), and operate according to the product description of sky root company.Coated plate on the LB solid medium that contains IPTG, X-gal and penbritin (100mg/l), after 37 ° of C incubated overnight, the single bacterium colony of picking white is grown in the LB liquid nutrient medium that contains penbritin (100mg/l), and the bacterium liquid that takes a morsel carries out PCR to be identified;

(4) preparation of bacteria plasmid DNA: collect the thalline in the above-mentioned LB liquid nutrient medium, prepare bacteria plasmid DNA according to the little extraction reagent kit specification sheets of plasmid of sky root company, the enzyme evaluation of cutting and check order.Order-checking is finished by Guangzhou Invitrogen company;

(5) structure of expression vector: extract the correct clone's of order-checking plasmid, according to the product description of Takara company, use SmaI and SacI carries out double digestion, then reclaims small segment, connects among the pBI121 with same enzymic digestion, makes up plant expression vector pBI121-MT2a, and diagram as shown in Figure 1.Then will connect product and transform the bacillus coli DH 5 alpha competence, carry out PCR, enzyme is cut and check order evaluation;

(6) conversion of agrobacterium tumefaciens: extract the correct clone's of order-checking plasmid, transform agrobacterium tumefaciens EHA105 by electric shocking method.

Embodiment 3: the genetic transformation of Arabidopis thaliana

One transformation of Arabidopsis thaliana pre-treatment

When its main tongue grows to 5-6cm, cut whole inflorescence at the inflorescence base portion, remove its apical dominance, 1 all rear 4-6 newborn side tongues that grow at the axillalry bud position, treat that its side tongue inflorescence forms that bud and part are bloomed or when forming 1-2 angle fruit, namely can be used for transforming, need cut off the angle that grown up to before the conversion really.Water sufficient moisture to plant the day before yesterday that transforms, and cover a plastics bag to keep high humidity environment.

Two contaminate the preparation of substratum

The dip-dye medium component that be used for to soak the Arabidopis thaliana titbit is the 1/2MS substratum that contains 5% sucrose, pH=5.8 (regulating with KOH), autoclaving.Time spent is added 0.02%-0.05% tensio-active agent Silwet L-77 or tensio-active agent soil temperature-20, and operation needs soft.

Three Agrobacteriums are prepared and transformation of Arabidopsis thaliana

(1) activation of bacterial classification: the agrobacterium liquid or the dull and stereotyped bacterium that preserves that store are drawn the plate activation at the YEB solid medium that contains kantlex (Km, 100mg/L) and Rifampin (Rif, 30mg/L), and the performing PCR of going forward side by side detects;

(2) the positive Agrobacterium of the mono-clonal that contains goal gene of picking activation is trained in the base to the fresh YEB liquid that contains kantlex and Rifampin of 5m1, and 28 ° of C, 180rpm shake training 24 hours;

(3) get above-mentioned bacterium liquid 5ml(1% one 2%) be connected in the fresh YEB liquid nutrient medium that contains kantlex and Rifampin of 500 m1,28 ° of C, 180rpm cultivated 18-24 hour, made OD value reach about 0.8 (usefulness YEB+Rif+Km is as blanks);

(4) above-mentioned bacterium liquid is divided install in the 100 ml centrifuge tubes, room temperature, 5000 leave the heart collected thalline in 20 minutes;

(5) with bacterial suspension in contaminating substratum, make final thalline suitable concentration OD600 value be approximately 0.8-1(and compare with the dip-dye substratum);

(6) above-mentioned dip-dyeing solution is poured in the beaker, plant to be transformed is tipped upside down on rapidly in the dip-dyeing solution, take out after making 60 seconds of inflorescence submergence.As far as possible careful during operation, do not allow the soil bits wait and fall to and contaminate in the substratum;

(7) suck too much bacterium liquid with thieving paper after the conversion, but do not need to inhale too driedly.Plant is kept flat, and cover the Arabidopis thaliana over-ground part with the black plastic bag.After moisturizing is secretly cultivated 16-24h, carefully remove plastics bag, and water sufficient water, recover normal illumination;

(8) in order to improve transformation efficiency, can after a week, repeat an infection processs;

(9) normal management, the results mature seed.Seed after the results is the MS of the Pyocianil of the kantlex that contains 50mg/l and 150mg/l solid medium screening transgenic positive plant.

Embodiment 4: the RT-PCR of transgenic seed detects

(1) extraction of total RNA: extract the total RNA of Arabidopsis that test kit specification sheets step is extracted wild-type and different transgenic line according to the general RNA of the plant of hundred Imtech;

(2) first chain cDNA's is synthetic: according to the specification sheets step operation of the PrimeScript 1st Strand cDNA Synthesis Kit of Takara company;

(3) RT-PCR reaction:

MT2aGene specific primer

Forward primer: SEQ ID NO:5:5 '-ACCCGGACTTCAGCTTCT-3 '

Reverse primer: SEQ ID NO:6:5 '-GCTCATCTCGGACCCTTC-3 '

Confidential reference items Actin2Gene primer

Forward primer: SEQ ID NO:7:5 '-ATTACCCGATGGGCAAGTCA-3 '

Reverse primer: SEQ ID NO:8:5 '-TGCTCATACGGTCAGCGATA-3 '

MT2aWith Actin2The PCR reaction system be: the first chain cDNA template that 2 μ l dilution is 5 times, 25.5 μ l PCR Mix(Dong Sheng company product), 2 μ l forward primers (10 μ M), 2 μ l reverse primers (10 μ M), replenish at last deionized water, making totally is 50 μ l.The PCR reaction conditions is: 94 ° of C 15 seconds, 58 ° of C 15 seconds, 72 ° of C 20 seconds. MT2aCycle number be 30, Actin2Cycle number is 32.The result of RT-PCR does not detect the lotus metallothionein(MT) in wild-type Arabidopis thaliana seed shown in Fig. 2 A MT2aExpression, and in three different transgenic lines, can both detect in various degree lotus metallothionein(MT) MT2aExpression.

Embodiment 5: the Western Blot of transgenic seed detects

(1) prokaryotic expression: take embodiment 2 described intermediate carrier pGEMT-MT2a as template, obtain having added restriction enzyme site by the PCR clone EcoRI and XhoIThe PCR fragment.The PCR primer is as follows, and underscore partly is restriction enzyme site.

Forward primer: SEQ ID NO:9:

5′-CCG GAATTCATGTCTTGCTGCGGAGGAA-3′

Reverse primer: SEQ ID NO:10:

5′- CCG CTCGAGGCCCTCCTCTCATTTGCAG-3′

The PCR reaction system is: 1 μ l template, 4 μ l dNTP(2.5 mM), 2 μ l forward primers (10 μ M), 2 μ l reverse primers (10 μ M), 5 μ l 10*PCR damping fluids, 0.3 μ l EX Taq enzyme (Takara company product) replenishes deionized water at last, making totally is 50 μ l.PCR response procedures: 94 ° of C 3 minutes; Then enter following circulation: 94 ° of C 30 seconds, 58 ° of C 30 seconds, 72 ° of C 30 seconds, totally 30 circulations; Last 72 ° of C extended 7 minutes.PCR product behind the purifying is used EcoRI and XhoIAfter enzyme is cut, reclaim, connect into the pET-32a(Novagen company product with same enzymic digestion) in, make up prokaryotic expression carrier Trx-6His-MT2a.Then transform e. coli bl21 (DE3), carry out PCR, enzyme is cut and the evaluation of checking order, method is identical with embodiment 2.The purifying of albumen carries out according to the His bind purification kit specification sheets of Novagen company;

(2) antibody preparation: the albumen behind the above-mentioned purifying is used for the injection rabbit and prepares specific antibody anti-MT2a;

(3) protein extraction: the protein extraction of wild-type and transgenic arabidopsis seed is with reference to the method for (1997) such as Fan;

(4) Western Blot: the immune marking is with reference to Mizzen(1996) method.The result of Western Blot does not detect the accumulation of lotus metallothionein(MT) MT2a at wild-type Arabidopis thaliana seed, and can both detect the accumulation of lotus metallothionein(MT) MT2a in various degree in three different transgenic line shown in Fig. 2 B.

Embodiment 6: the sprouting experiment of transgenic seed behind Artificial ageing

(1) temper(ing) is finished in moisture eliminator.Use first 10% commercially available SYNTHETIC OPTICAL WHITNER (bleach) to process the vessel such as moisture eliminator 30 minutes before aging, then use a large amount of flushing with clean water moisture eliminators, wash again once with aqua sterilisa at last, dry, prevent the growth of fungi during the temper(ing);

(2) inject aqua sterilisa in moisture eliminator bottom, will be positioned in the water isolation type constant incubator after the moisture eliminator sealing, temperature is made as 43 ° of C, and balance two days allows the humidity in the moisture eliminator reach 100%;

(3) wild-type and genetically modified Arabidopis thaliana seed are installed with the centrifuge tube that removes lid of 1.5 ml after (about 100 of every pipe) put into rapidly the good moisture eliminator of balance after being positioned on the centrifuge tube shelf, the good seal moisture eliminator was processed 72 hours.Try not to open the door of constant incubator during the processing;

After (4) 72 hours, the Arabidopis thaliana seed handled well is taken out, the balance sterilization that carries out disinfection to the room temperature at once, 4 ° of C laminations are processed and are sprouted two days later experiment, and the statistics germination rate reaches takes pictures to the growing state of seedling.Seed germination result after the temper(ing) as shown in Figure 3, the sprouting speed of transgenic seed and final germination rate are all far above the seed of wild-type.At after planting the 4th day, the germination rate of wild type seeds only was 6%, and the germination rate of transgenic line is up to 45-51%.Sow the growing state of the seedling after 10 days as shown in Figure 4, the cotyledon sheetage of transgenosis seedling and seedling size will significantly better than wild-type, show the overexpression gene MT2aCan improve life-span and the vigor of seed.

Embodiment 7: the vigor Coloration experiment of transgenic seed behind Artificial ageing

(1) the Artificial ageing step of wild-type and genetically modified Arabidopis thaliana seed just is made as 41 ° of C with the temperature of processing as described in Example 6 in the present embodiment;

(2) take out seed behind Artificial ageing, place on the dry sanding paper back and forth evenly soft friction several lower, make kind of skin form a cut;

The seed that (3) will rub is positioned in the centrifuge tube, adds 1% tetrazolium (2,3,5-triphenyl tetrazolium chloride the is abbreviated as TTC) aqueous solution, and the amount of solution is not can there to be seed as suitable.Then be positioned in 30 ° of C constant incubators dark lower insulation 2 days;

(4) from constant incubator, take out the seed that dyes lechery, place microscopically to take pictures.The result of vigor dyeing as shown in Figure 5, the great-hearted seed of dark expression, light expression vigor descend or the seed of devitalization.Wild-type Arabidopis thaliana seed has a large amount of seeds to be shown as at the Artificial ageing poststaining to have lost the light color behind the vigor, and transgenic seed then major part is still keeping great-hearted dark color, shows the overexpression gene MT2aCan improve life-span and the vigor of seed.

Embodiment 8: the salt stress of transgenic seed is sprouted experiment

Wild-type and transgenic arabidopsis seed are positioned over 4 ° of C laminations and processed two days behind sterilization.Then be seeded on the 1/2MS solid medium that contains 100 mM, 125 mM, 150 mM and 175 mM NaCl and sprout.Statistics Seed germination rate every day amounts to 7 days.Sprouting situation on the 1/2MS solid medium that contains 150 mM NaCl as shown in Figure 6, the sprouting speed of transgenic seed and final germination rate are all far above the seed of wild-type.At after planting the 4th day, the germination rate of wild type seeds only was 53%, and the germination rate of transgenic line shows the overexpression gene up to 71-95% MT2aCan improve the vigor that seed is sprouted under salt stress.

SEQUENCE LISTING

＜110〉Zhongshan University

＜120〉lotus metallothionein gene MT2a is in the application that improves on seed longeivity and the vigor

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Claims (4)

1. lotus metallothionein gene MT2a is in the application that improves on plant seed life-span and the vigor; Described lotus metallothionein

White gene M T2a nucleotide sequence is such as SEQ ID NO: shown in the l; Described plant is Arabidopis thaliana.

2. application as claimed in claim 1 is characterized in that lotus metallothionein gene MT2a is applied to improve plant species

The sprouting vigor of son under temper(ing) and salt stress.

3. the lotus metallothionein(MT) is in the application that improves on plant seed life-span and the vigor; Described lotus metallothionein casamino acid

Sequence is such as SEQ ID NO: shown in the of 2; Described plant is Arabidopis thaliana.

4. application as claimed in claim 3 is characterized in that the lotus metallothionein(MT) is applied to improve plant seed manually

Sprouting vigor under the aging and salt stress.

Images