CN102206673B

CN102206673B - Application of stress-tolerant relative protein MtMYB5 of plant in cultivating stress-tolerant plant

Info

Publication number: CN102206673B
Application number: CN2011100907975A
Authority: CN
Inventors: 董江丽; 王涛; 胡晓娜; 张运芹
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2011-04-12
Filing date: 2011-04-12
Publication date: 2012-11-14
Anticipated expiration: 2031-04-12
Also published as: CN102206673A

Abstract

The invention discloses an application of a stress-tolerant relative protein MtMYB5 of a plant in cultivating a stress-tolerant plant. According to the application of the MtMYB5 protein in cultivating the stress-tolerant plant, an amino acid sequence of the MtMYB5 protein is a sequence 2 in a sequence table; and the application is as follows: an encoding gene of the MtMYB5 protein is introduced into a target plant to obtain a transgenic plant with stress tolerance higher than that of the target plant. According to the invention, experiments prove that a transcription factor MtMYB5 outstandingly increased in expression level after being induced by salt is screened, and important value is reflected on cultivating salt-tolerant drought-tolerant transgenic leguminous crops by introducing the gene into leguminous crops, i.e. cash crop alfalfa and the like.

Description

The application of plant stress tolerance correlative protein MtMYB5 in cultivating plant with adverse resistance

Technical field

The present invention relates to a kind of genetically engineered field, relate in particular to the application of plant stress tolerance correlative protein MtMYB5 in cultivating plant with adverse resistance.

Background technology

Arid, low temperature, the soil salinization and disease, insect pest are the serious problems that influence agriculture prodn.In numerous researchs in relevant plant stress-resistance border, scientist has cloned the functional gene relevant with adverse circumstance of many different sourcess and has been transformed into them in the plant materials, but the effect that obtains is unsatisfactory.The degeneration-resistant proterties of plant is the complex character of controlled by multiple genes.Plant is not often depended on a certain monofactor to the power of high salt, arid and cold tolerance, and its proterties is influenced by many factors.

Transcription factor is claimed trans-acting factor again, regulates and control transcribing of downstream gene through combining special DNA element.Some transcription factors rise transcribes activation, and some rise transcribes restraining effect, and other existing transcriptional activation activities also have transcriptional repression activity.A transcription factor can be regulated and control a plurality of expression of gene relevant with proterties, through strengthen some crucial regulatory factors be used for promote these adversity gene resources to play a role, make the resistance of plant obtain improvement comprehensive, essence.In improving the molecular breeding of crop to environment-stress; Improve the traditional method of certain resistance compares with importing or improvement discrete function gene; Set about from improvement or the transcription factor ability of regulation and control that strengthens a key, the resistance that improves thing will be a kind of more efficiently method and approach.The resistance research of therefore using transcription factor raising plant has become current research focus.

Cut that the type clover has that genome is little, chromosome number is 2 * 8 (2n=16), vegetative period weak point, self-pollination, nodule nitrogen fixation, genetic transformation efficiency are high, with characteristics such as pulse family staple crop sibship is nearer, so be selected as the pulse family model plant.Research shows that the abiotic side of body can influence the dross of plant such as salt stress, thereby influences fixed nitrogen, therefore cuts some transcription factors of type clover and possibly participate in abiotic stress and dross simultaneously.Therefore, research cuts the salt-tolerant drought-resistant genes involved (especially transcription factor) of type clover, will valuable molecule manipulation foundation be provided for cultivating good leguminous plants kind.

Summary of the invention

The purpose of this invention is to provide the application of MtMYB5 albumen in cultivating plant with adverse resistance.

The application of MtMYB5 albumen provided by the invention in cultivating plant with adverse resistance, the proteic aminoacid sequence of said MtMYB5 is the sequence 2 in the sequence table.

Said being applied as imports the proteic encoding sox of MtMYB5 in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than said purpose plant.

The nucleotides sequence of the proteic encoding sox of said MtMYB5 is classified the sequence 1 in the sequence table as.

The proteic encoding sox of said MtMYB5 imports in the said purpose plant through expression vector.

Said expression vector is the carrier that obtains between the BglII of the proteic encoding sox insertion of said MtMYB5 carrier pCAMBIA1302 and the Spe I restriction enzyme site.

Said resistance of reverse is drought tolerance and/or salt tolerance.

Said purpose plant monocotyledons or dicotyledons, said dicotyledons is preferably Arabidopis thaliana, like the environmental Arabidopis thaliana of columbia.

Sequence 2 in the above-mentioned sequence table is made up of 278 amino-acid residues.

Sequence 1 in the above-mentioned sequence table is made up of 837 Nucleotide, and the coding region is the 1-837 position Nucleotide from 5 ' end of sequence 1.

Experiment of the present invention proves; The transcription factor MtMYB5 that the salt that the present invention screens induces the back expression amount significantly to raise; This gene is imported Arabidopis thaliana; Through means such as Totomycin primary dcreening operation, Molecular Detection, salt stress and drought stresses, screen the transgenic arabidopsis strain system that anti-salt and drought-resistant ability improve, leguminous crops such as this gene importing cash crop clover will there be important value to cultivating the drought-resistant transgenic leguminous crop of salt tolerant.

Description of drawings

Fig. 1 is the transcriptional activity analysis of MtMYB5 in yeast

Fig. 2 is the Subcellular Localization analysis of MtMYB5 in onion epidermis

Fig. 3 makes up schema for carrier pCAMBIA1302-MtMYB5

Fig. 4 is T ₁Molecular level for transgenic arabidopsis detects

Fig. 5 is T ₃Molecular level for transgenic arabidopsis detects

Fig. 6 is T ₃Germination rate statistical graph under coercing for transgenic arabidopsis NaCl

Fig. 7 is T ₃For the long statistical graph of the root under the transgenic arabidopsis drought stress

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

Cut type clover (Medicago truncatula) A17: available from French INRA BRC-MTR:BiologicalResource Centre for the model species Medicago truncatula L..

Arabidopis thaliana (Arabidopsis thaliana, columbia-o is environmental, below is called the wild-type Arabidopis thaliana): available from salk company.

Agrobacterium EHA105 is documented in microbiotic and suppresses effect of Agrobacterium and the influence that yezoensis laver is grown, Wang Ping etc., and aquatic science, 200928 (7), in, the public can obtain from China Agricultural University.

The clone of embodiment 1, MtMYB5 gene

1, the acquisition of MtMYB5 gene

Find the gene that this expression amount significantly raises in the screening of chip after salt stress cuts type clover A17, in order to study the function of this gene in abiotic stress, cut type clover A17 4 age in week seedling watered the 250mMNaCL aqueous solution one hour; Extract RNA, reverse transcription obtains cDNA, is template with the cDNA that cuts type clover A17; With MtMYB5___5 ' and MtMYB5___3 ' is primer, carries out pcr amplification, obtains the PCR product; The PCR product is inserted pMD18 T-simple carrier (available from TaKaRa bio-engineering corporation), obtain recombinant vectors, order-checking; The result does, this PCR product has the Nucleotide of sequence 1 in the sequence table, and the unnamed gene of this PCR product is MtMYB5; The coding region of this gene is that sequence 1 is held the Nucleotide shown in the 1-837 position from 5 ' in the sequence table; The albumen called after MtMYB5 of this genes encoding, this proteic aminoacid sequence are the sequence 2 in the sequence table, with this recombinant vectors called after pMD18 T-simple-MtMYB5.

Right by the synthetic Auele Specific Primer of Invitrogen company:

MtMYB5___5 ': 5 '-ATGGATACTAATTACAAAACCAATA-3 ' (sequence 3);

MtMYB5___3 ': 5 '-TCATAAATCATCAGCCAATTGTTGC-3 ' (sequence 4).

Also but artificial synthesized sequence 1, adopts identical method to make up and obtains pMD18 T-simple-MtMYB5.

2, the transcriptional activity analysis of MtMYB5 in the Yeast system

With pMD18 T-simple-MtMYB5 is template, carries out pcr amplification with primer 5 '-GAATTCATGGATACTAATTACAAAAC and the 3 '-GTCGACTCATAAATCATCAGCCAATT that contains EcoR I and Sal I restriction enzyme site respectively, obtains the PCR product.This PCR product is cut through EcoR I and Sal I enzyme, reclaimed fragment, with carrier pBDGAL4 (the Liao Y that cuts through same enzyme; Zou HF, Wang HW, Zhang WK; Ma B, Zhang JS, Chen SY (2008) Soybean GmMYB76; GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res 18:1047-1060, the public can obtain from China Agricultural University.) connect, will connect product transformed yeast bacterial strain YRG2 (Liao Y, Zou HF; Wang HW, Zhang WK, Ma B; Zhang JS, Chen SY (2008) Soybean GmMYB76, GmMYB92; And GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res 18:1047-1060, the public can obtain from China Agricultural University.) in, converted product is coated in the SD substratum that lacks tryptophane, growth be positive colony.(as CK+, the carrier source is identical with pBDGAL4, is documented in Liao Y to transform pGAL4 simultaneously; Zou HF, Wang HW, Zhang WK; Ma B, Zhang JS, Chen SY (2008) Soybean GmMYB76; GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res 18:1047-1060, the public can obtain from China Agricultural University), pBDGAL4 empty carrier (as CK-) is to yeast strain YRG2.

Positive colony is coated with at the same time on the culture medium flat plate that lacks tryptophane, Histidine grows.Utilize X-Gal that the beta-galactosidase enzymes enzymic activity is detected simultaneously.The result is as shown in Figure 1; Wherein SD/-Trp-His is that SD substratum, the SD/-Trp substratum that lacks tryptophane, Histidine simultaneously is the SD substratum that lacks tryptophane; CK+ is for changeing the yeast that pGAL4 is arranged; CK-is for changeing the yeast that pBDGAL4 is arranged, and positive colony can be grown on the SD/-Trp-His culture medium flat plate, and X-Gal is to beta-galactosidase enzymes enzymic activity tests positive.Explain that MtMYB5 has transcriptional activation activity.

2, the Subcellular Localization analysis of MtMYB5 in onion epidermis

Amplification MtMYB5 full length gene ORFs (ORF), primer contains Xho I and Kpn I restriction enzyme site, 5 '-CTCGAGGATGGATACTAATTACAAAAC and 3 '-GGTACCCTAAATCATCAGCCAATTGTT respectively; Be cloned into carrier E3025 (Jin Jingchen; Depend in the tobacco NAD < '+clone and function preliminary of sorbitol dehydrogenase gene, 2008 master thesis of Agricultural University Of He'nan, 2008; 28-32, the public can obtain from China Agricultural University.) on, obtain fusion plasmid.

Utilize the method for particle gun that fusion plasmid is bombarded onion epidermis,, observe under Laser Scanning Confocal Microscope as contrast with empty carrier E3025, the result is as shown in Figure 2, and E3025 contains the GFP gene, explains that MtMYB5 is positioned nucleus.

The acquisition and the functional study thereof of embodiment 2, commentaries on classics MtMYB5 Arabidopis thaliana

1, the structure of pCAMBIA1302-MtMYB5 composing type efficient expression vector

It is right that Invitrogen company synthesizes the Auele Specific Primer that contains BglII and Spe I restriction enzyme site respectively:

5 '-AGATCTTATGGATACTAATTACAAAACCAATA-3 ' (sequence 5)

5 '-ACTAGTTCATAAATCATCAGCCAATTGTTGC-3 ' (sequence 6)

With pMD18 T-simple-MtMYB5 is template, is primer with the dna molecular shown in dna molecular shown in the sequence 5 and the sequence 6, and amplification obtains containing the fragment of BglII and Spe I restriction enzyme site; This fragment and carrier pCAMBIA1302 (the Center for the Application of Molecular Biology to International Agriclture that cut through same enzyme; Www.cambia.org) connect, connect the product transformed into escherichia coli, obtain transformant; Extract the plasmid of transformant; Order-checking, the result shows that this plasmid is the carrier that obtains between the BglII of the 1 insertion pCAMBIA1302 of the sequence in the sequence table and SpeI restriction enzyme site; With this plasmid called after pCAMBIA1302-MtMYB5, the detailed structure of this plasmid is seen Fig. 4.

2, the acquisition of Agrobacterium EHA105/pCAMBIA1302-MtMYB5

1) preparation of EHA105 Agrobacterium competent cell

The single colony inoculation of picking Agrobacterium EHA105 is in 100ml YEB liquid nutrient medium, and 28 ℃ of shaking culture of 220rpm are to OD ₆₀₀=0.5; Change aseptic centrifuge tube over to, the centrifugal 5min of 5000rpm removes supernatant, adds the CaCl of the 0.15M of 10ml precooling ₂Solution, suspension cell is placed 20min on ice gently; 4 ℃, the centrifugal 5min of 5000rpm removes supernatant, adds the CaCl of the 0.15M that contains 10% glycerine of 4ml precooling ₂Solution suspends gently; Agrobacterium suspension is sub-packed in the sterile eppendorf tubes, and every pipe 200 μ l quick-frozen 1min in liquid nitrogen is frozen in-70 ℃.

2) expression vector transforms Agrobacterium EHA105

The expression vector pCAMBIA1302-MtMYB5 that gets the above-mentioned acquisition of 1 μ g joins in the 200 μ l EHA105 competent cells mixing, static 5min; Quick-frozen 1min in the liquid nitrogen, 37 ℃ of water-bath 5min add 1ml YEB liquid nutrient medium, 28 ℃ of 150rpm shaking culture 4h; The centrifugal 3min of 5000rpm abandons supernatant, adds 0.1ml YEB liquid nutrient medium, again suspension cell; Coat on the YEB solid plate that contains 50 μ g/ml Kan and 50 μ g/ml Rifampins, cultivate about 48h, obtain transformant for 28 ℃.

Transformant is carried out bacterium liquid PCR identify that primer is the dna molecular shown in dna molecular shown in the sequence 5 and the sequence 6, obtains the positive clone of fragment of 850bp.Positive colony is extracted plasmid, and sequencing result proves that further carrier pCAMBIA1302-MtMYB5 has successfully changed in the Agrobacterium, with positive colony called after EHA105/pCAMBIA1302-MtMYB5.

3, change the acquisition of MtMYB5 Arabidopis thaliana

EHA105/pCAMBIA1302-MtMYB5 is inoculated in 10ml contains in the YEP liquid nutrient medium of 50 μ g/mlKan and 50 μ g/ml Rifampins, 28 ℃, the 220rpm shaking culture is spent the night; Transform and to be inoculated in 200ml with 1: 50 proportional concentration previous day and to contain in the identical antibiotic YEP substratum enlarged culturing to OD ₆₀₀Be 1.6,5000rpm, the centrifugal collection of 10min bacterium is resuspended in and infiltrates damping fluid (5% sucrose solution of new system), makes OD ₆₀₀Be 0.8; Can adopt the wild-type Arabidopis thaliana of Foral dip method with the Agrobacterium-mediated Transformation bud stage.Can when Arabidopis thaliana bolting 5cm, cut off terminal inflorescence; Impel the axillary inflorescence growth; To obtain more inflorescence, wound should be positioned at the highest stem leaf top when cutting, and transforms after about 5 days; The flower that soil is fully drenched and will open removes, and only keeps unopened small bud; During conversion the whole strain of Arabidopis thaliana tipped upside down in the container that fills bacterium liquid and 0.05%Silwet L-77 with flowerpot and soaks 1.5-2min, soak finish after, take out flowerpot; Be sidelong in pallet; Dark culturing 16 hours, upright again flowerpot recovers normal illumination cultivation, obtains T ₀In generation, changeed the MtMYB5 Arabidopis thaliana, from T ₀In generation, changeed MtMYB5 Arabidopis thaliana results seed, is T ₁In generation, changeed the seed of MtMYB5 Arabidopis thaliana.

4, change the screening of MtMYB5 Arabidopis thaliana

1) hygromycin selection

Results T ₁In generation, be seeded on the MS solid plate that contains 80mg/L Hyg+ after changeing the seed of MtMYB5 Arabidopis thaliana, 4 ℃ of vernalization 72 hours; Put just putting in the illumination box 22 ℃ of full suns and cultivated 5 days, observe phenotype, transformant shows as true leaf and is deep green; And, 15 T have been obtained apparently higher than non-transformant ₁For transgenic line.The transformant that screens moved to do not contain rejuvenation in the antibiotic MS substratum, forward in the soil seedling of green to numerous kind after 4 days, results T ₂In generation, changeed MtMYB5 Arabidopis thaliana seed.Same method screening T ₂In generation, obtain T ₃In generation, changeed MtMYB5 Arabidopis thaliana seed, sowing, and obtaining 15 strains is T ₃In generation, changeed the MtMYB5 Arabidopis thaliana.

2) Molecular Detection

Extract T ₁In generation, changeed the genomic dna of Arabidopis thaliana; And with it as template; (forward primer is selected the inner fragment of gene for use with 5 '-TGATCTTCATTCCCGTTGG-3 ' and 5 '-TCACACGTGGTGGTGGTG-3 '; Reverse primer is selected the fragment on the carrier for use) be primer, obtain the purpose fragment of 1293bp, the transgenic arabidopsis of MtMYB5 is described.

Adopting uses the same method changes empty carrier pCAMBIA1302 in the wild-type Arabidopis thaliana over to, obtains T ₁In generation, changeed empty carrier Arabidopis thaliana seed, sowing T ₁In generation, changeed the empty carrier Arabidopis thaliana, extracts genomic dna, is primer with 5 '-TGATCTTCATTCCCGTTGG-3 ' and 5 '-TCACACGTGGTGGTGGTG-3 ', do not have the purpose fragment, and the T that is that obtains is described ₁In generation, changeed the empty carrier Arabidopis thaliana, from T ₁Withhold and obtain T ₂For seed, from T ₂For plant results T ₃For seed, from T ₃For planting seed, obtain T ₃In generation, changeed the empty carrier Arabidopis thaliana.

Reaction system:

The PCR response procedures is: the first round: 94 ℃ of sex change 5min; Second takes turns: 94 ℃ of sex change 45sec, and 43 ℃ of renaturation 50sec, 72 ℃ are extended 1min, 30 circulations; Third round: 72 ℃ are extended 10min.After reaction finishes, 1.0% agarose gel electrophoresis detected result.The result is as shown in Figure 4, is MtMYB5 gene PCR amplified production electrophorogram.WT is a unconverted Arabidopis thaliana PCR product (negative control), and 1-15 is a transgenic arabidopsis PCR product.From figure, find out that (WT) compares with the wild-type Arabidopis thaliana, be numbered the T of 1-15 ₁In generation, changeed the PCR product that the MtMYB5 Arabidopis thaliana obtains 1293bp, proves that it contains the MtMYB5 gene.Wild-type Arabidopis thaliana and T ₁Generation is changeed empty carrier Arabidopis thaliana result does not have significant difference.

Whether RT-PCR detects: transcribed in order to detect in the PCR positive plant MtMYB5 gene, further carry out RT-PCR and detect.Extract PCR with the Trizol method and identify that male is numbered the T of 5-1,5-2,5-10,5-12 ₃In generation, changeed total RNA of MtMYB5, is template with good in integrity, free of contamination RNA, and reverse transcription RNA is cDNA with M-MLV enzyme (available from Promage company), with wild-type Arabidopis thaliana (WT) and T ₃In generation, changeed the empty carrier Arabidopis thaliana.The primer of internal control gene Actin2 is 5 '-GGTAACATTGTGCTCAGTGGTGG and 3 '-AACGACCTTAATCTTCATGCTGC.

The reverse transcription reaction system:

RNA 2μg

Oligd?T(18) 2.0μl

RNase Free H ₂O adds to 15.0 μ l

Behind the said mixture mixing, of short duration centrifugal it is collected in managed at the end, and 72 ℃ of incubation 5min place 5min on ice more immediately, add following composition again:

With the said mixture mixing, of short durationly centrifugal it is collected in the pipe end, at 42 ℃ of incubation 60min, 70 ℃ of reaction 15min take out and to place on ice, are stored in-20 ℃ after centrifugal.

With 0.5 μ l reverse transcription product is template, carries out pcr amplification, PCR reaction system and reaction conditions such as preceding.

The result is as shown in Figure 5, and WT is unconverted Arabidopis thaliana RT-PCR product (negative control, a wild-type Arabidopis thaliana), and 5-1,5-2,5-10,5-12 are T ₃In generation, changeed MtMYB5 Arabidopis thaliana RT-PCR product, and Actin2 is a reference.(WT) compares with the wild-type Arabidopis thaliana, is numbered the T of 5-1,5-2,5-10,5-12 ₃The MtMYB51 gene obtains expressing in the generation commentaries on classics MtMYB5 Arabidopis thaliana.Wild-type Arabidopis thaliana and T ₃Generation is changeed empty carrier Arabidopis thaliana result does not have significant difference.

5, change the resistance of reverse research of MtMYB5 Arabidopis thaliana

Will be through the above-mentioned T that is numbered 5-1,5-2,5-12 ₃In generation, changes the MtMYB5 Arabidopis thaliana and carries out Function detection, and detection method is following:

1) germination period salt stress experiment:

Be numbered the T of 5-1,5-2,5-12 ₃In generation, changeed MtMYB5 Arabidopis thaliana and contrast (wild-type Arabidopis thaliana and T ₃In generation, changeed the empty carrier Arabidopis thaliana) seed plants respectively on the MS substratum that contains 150mM NaCl, 4 ℃ of vernalization 72 hours, illumination (16h light/8h is dark) is cultivated, and 22 ℃ of temperature were added up germination rate since the 2nd day every day, until the 7th day, carried out germination rate calculating.Each each 40 strain of strain system.

The calculation formula of germination rate is germination rate=sprouting number/sum.

Germination rate calculation result is as shown in Figure 6, the T of the 7th day 5-1,5-2,5-12 ₃The germination rate that generation is changeed MtMYB5 Arabidopis thaliana and wild-type Arabidopis thaliana is respectively 91.11%, 82.07%, 93.92%, 36.15%;

Wild-type Arabidopis thaliana and T ₃Generation is changeed empty carrier Arabidopis thaliana result does not have significant difference.

2) arid experiment:

Be numbered the T of 5-1,5-2,5-12 ₃In generation, changeed MtMYB5 Arabidopis thaliana and contrast (wild-type Arabidopis thaliana and T ₃Generation commentaries on classics empty carrier Arabidopis thaliana) seed is planted respectively on the MS substratum; 4 ℃ of vernalization 72 hours; Illumination cultivation (16h light/8h is dark) 6 days; 22 ℃ of temperature, (concrete grammar is with reference to Paul E.Verslues et al., (2006) Methods and concepts in quantifying resistance to drought to transfer to the MS substratum that PEG solution (the MS liquid nutrient medium that contains 45% (quality percentage composition) PEG8000) balance crosses; Salt and freezing; Abiotic stresses that affect plant water status.The Plant Journal, Protocol S1 in the Supplementary Material) on, the statistics root is long after 10 days.Each each 15 strain of strain system.Statistics is as shown in Figure 7,

The T of 5-1,5-2,5-12 ₃The root length that generation is changeed MtMYB5 Arabidopis thaliana and wild-type Arabidopis thaliana is respectively 1.6567,1.4567,1.6900,0.9300.

Claims

1. cut the application of type clover MtMYB5 albumen in cultivating anti-contrary Arabidopis thaliana, the said section proteic aminoacid sequence of type clover MtMYB5 is the sequence 2 in the sequence table,

Said resistance of reverse is drought tolerance and/or salt tolerance.

2. application according to claim 1 is characterized in that: said being applied as imports in the Arabidopis thaliana cutting the proteic encoding sox of type clover MtMYB5, obtains the transgenic arabidopsis that resistance of reverse is higher than said Arabidopis thaliana.

3. according to claim 1 or claim 2 application, it is characterized in that: the nucleotides sequence of the said section proteic encoding sox of type clover MtMYB5 is classified the sequence 1 in the sequence table as.

4. application as claimed in claim 3 is characterized in that: the said section proteic encoding sox of type clover MtMYB5 imports in the said Arabidopis thaliana through expression vector.

5. application as claimed in claim 4 is characterized in that: said expression vector is the carrier that the MCS that the said section proteic encoding sox of type clover MtMYB5 inserts pCAMBIA1302 is obtained.