CN102643830A - Application of cotton gene GbMYB 5 related to drought resistance - Google Patents

Abstract

The invention discloses a cotton gene GbMYB 5 related to plant drought resistance and further relates to application of the gene GbMYB 5 in improving drought resistance capability of tobaccos. The cotton drought-resistant gene GbMYB5 has a nucleotide sequence shown by SEQ ID NO.1. A transcription factor is one of the following amino acid residue sequences: 1) SEQ ID NO.2 in a sequence table; and 2) protein which is obtained through carrying out substitution, deletion or addition of one or multiple amino acid residues on the amino acid residue sequence of SEQ ID NO.2 in the sequence table, and has a plant drought resistance regulation function. The protein and coding gene thereof have great theoretical and practical significance on researching the plant drought resistance mechanism and improving plant drought resistance, can play an important role in drought resistance gene engineering improvement of plants, and have broad application prospect.

Description

One grow cotton gene related to drought tolerance GbMYB5 application

Technical field

The invention belongs to gene engineering technology field, be specifically related to the gene related to drought tolerance GbMYB5 that grows cotton, relate to the application of GbMYB5 gene in improving the tobacco drought-resistant ability simultaneously.

Technical background

MYB class transcription factor belongs to the transcription factor family of band tryptophane bunch; The one type of transcription factor that contains one section conservative DNA land (DBD)----MYB structural domain; The DBD district generally comprises 1-3 not exclusively Tumor-necrosis factor glycoproteins (R); Each repeated fragment contains 51-53 amino acid, comprises the amino-acid residue and the intervening sequence of a series of high conservatives.The amino-acid residue of these high conservatives makes the MYB district combine with the DNA major groove with the form that becomes helix turn helix (HTH), and these 3 residues are 18-19 amino acid at interval, plays the hydrophobic core effect, and are significant to the HTH configuration of keeping MYB.According to the number of repeated fragment R, can MYB class transcription factor be divided into single MYB domain protein (R1/R2), 2R albumen (R2R3) and 3R albumen (R1R2R3).Also there is the 4RMYB gene in the discovery Arabidopis thalianas such as Stracke.

Research to MYB class transcription factor in recent years shows, MYB class transcription factor wide participation development of plants and metabolic all respects.Combinatorial regulation is the important way of eukaryotic gene expression regulation and control, mainly realizes the precision regulation and control to target gene through the interaction between the multiple transcription factor.MYB class transcription factor and other transcription factor family member regulate morphogenesis, the secondary metabolism of plant through the mode of combinatorial regulation, and environmental stimulus, hormone induction and disease and pest etc. are made responsing reaction to external world.Reply that biology is coerced and during abiotic stress, the expression of myb gene plays an important role plant.

Myb gene is proved that involved in plant is drought-resistantly coerced, high-salt stress, high temperature stress, low temperature stress, UV radiation etc.; Can coerce generation replying to a certain degree to PEG (arid), high salt, low temperature etc. like the BcMYB1 of boea crassifolia; The AtMYB2 of Arabidopis thaliana and the drought resisting of AtMYB60 involved in plant, rice Os myb4 a large amount is expressed can improve plant to arid, high salt, UV radiating tolerance.In the Arabidopis thaliana, the MYB5 gene is the member of R2R3MYB gene family.R2R3MYB albumen is a huge family in the plant, and its function is also very various.Only in the arabidopsis gene group, just having 135 genes at least is the R2R3MYB transcription factors of encoding, and its function major part is relevant with regulating cell death, phenylpropyl alcohol alkanes pathways metabolism, tryptophane route of synthesis and drought resisting etc.Therefore, this genoid has comparison extensive studies and application prospect.

After plant receives pathogen infection, trigger cell programmed cell death, i.e. anaphylaxis (Hypersensitive response; HR), Whitfield's ointment (SA) is played an important role therein, and isolated AtMyb30 is the member of R2R3MYB gene family in the Arabidopis thaliana; It can be at a myb transcription factor of the early stage transient expression of HR; The overexpression of this gene and inhibition experiment showed, that AtMYB30 can participate in the synthetic of SA, thereby regulating cell is dead.

Show that according to statistics tillable land area accounts for about 10% of the land total area in the world wide, most of land area then is under the adverse circumstances such as arid, saline and alkaline, marsh.Along with the aggravation of aging population and the continuous deterioration of environment, cultivate that to be adapted under the adverse circumstance environment growing crop imperative.The relevant gene of the adjustable a plurality of and similar proterties of transcription factor can be through regulating the comprehensive change that key factor reaches stress resistance of plant, and the resistance of therefore using transcription factor raising plant has become research focus in recent years.Cotton is as one of most important farm crop in the world and cash crop; From cotton, clone and isolate myb transcription factor and help further understanding and resolve the regulatory mechanism of transcription factor of people, also help further carrying out of cotton quality improvement and tolerance studies work for abiotic stress etc.

Summary of the invention

The purpose of this invention is to provide a MYB type transcription factor relevant that derives from cotton with drought resisting.

The present invention utilizes a BAC clone who is obtained in the upland cotton MaxxaBAC library to carry out in the sequencing analysis process; Found a new MYB class transcription factor; And the design special primer clones GbMYB5 from sea island cotton kind H7124, its nucleotide sequence shown in SEQ ID NO.1, this gene 277 amino acid of encoding altogether; Its aminoacid sequence belongs to myb transcription factor shown in SEQ ID NO.2.

Those skilled in the art can not influence under its active prerequisite according to aminoacid sequence disclosed by the invention, replace, lack or add one or several amino acid, obtain said proteic mutant nucleotide sequence.Therefore, albumen of the present invention comprises that also aminoacid sequence shown in the SEQ ID NO.2 through replacing, lack or adding one or several amino acid, has equal active by the said protein derived protein that obtains.Gene of the present invention comprises the nucleic acids encoding said proteins sequence.

In addition, consider the degeneracy of codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.

Gene of the present invention can be cloned or separate from sea island cotton with protein and obtained, and perhaps obtains through DNA or peptide synthetic method.

To invent gene is connected with expression vector; Obtain to express the proteic recombinant vectors of the present invention; And then can institute's statement expression vector be imported host cell, the transformant that obtains changeing the GbMYB5 gene through such as transgenic methods such as agrobacterium-mediated transformation, pollen tube passage methods.

The present invention through with external source GbMYB5 gene integration to the tobacco gene group, the drought-resistant ability of transfer-gen plant strengthens, it is grown and is not received the remarkably influenced of drought stress.Show that GbMYB5 can be used for improving the resistance of tobacco to drought stress.

Of the present invention said cotton gene GbMYB5 is provided the application in the tobacco bred seed selection.

Said cotton gene GbMYB5 is provided the application in improving the tobacco drought-resistant ability.

Description of drawings

Fig. 1: the RT-PCR that the GbMYB5 gene is expressed at the cotton different sites analyzes.

Fig. 2: the GbMYB5 gene is expression analysis under abiotic stress.

Fig. 3: the genetic transformation A. callus culture of tobacco; B. screening and culturing; C. root culture; D. regeneration plant.

Fig. 4: the PCR of reconstituted tobacco detects 1:Marker DS ^TM2000 2-21: reconstituted tobacco PCR result.

Fig. 5: the RT-PCR of transgene tobacco detects 1:Marker DS ^TM2000 2-15: transgene tobacco RT-PCR result.

Fig. 6: kantlex screening transgene tobacco.

Fig. 7: 25%PEG6000 simulating drought lower blade upgrowth situation.

Fig. 8: plant growth condition under the 2%PEG6000 substratum simulating drought.

Fig. 9: before the 2%PEG6000 substratum simulating drought, the upgrowth situation of plant.

Figure 10: after 2 weeks of 2%PEG6000 substratum simulating drought, the upgrowth situation of plant.

Figure 11: after 3 weeks of 2%PEG6000 substratum simulating drought, the upgrowth situation of plant.

Figure 12: after 3 weeks of 2%PEG6000 substratum simulating drought, the growing state of plant (removal substratum).

Figure 13: after 4 weeks of 2%PEG6000 substratum simulating drought, the growing state of plant.

Figure 14: after 4 weeks of 2%PEG6000 substratum simulating drought, the growing state of plant (removal substratum).

Embodiment

Method therefor is ordinary method like no specified otherwise among the following embodiment.

1. the used biomaterial of the present invention

Sea island cotton (Gossypium barbadense) 7124, tobacco (Nicotiana tabacum) K326 (Charles S.Johnson; Jeremy A.Pattison; Elizabeth M.Clevinger et al.Clarifying the source of black shank resistance in flue-cured Tobacco.Plant Health Progress.Doi:10.1094/PHP-2008-0618-02-RS) and Arabidopis thaliana (Arabidopsis thaliana) Colombia's type (ZHANG Jian; XU Jin-Xiang; KONG Ying-Zhen et al.Generation of chemical-inducible activation tagging insertion lines of Arabidopsis thaliana.Acta Genetica Sinica.2005; 32 (10): 1082-1088), plant expression vector pCAMBIA2301 (Peter Hajdukiewicz; Zora Svab; Pal Maliga.The small; Versatile pPZP family of Agrobacterium binary vectors for plant transformation.Plant Molecular Biology.1994,25:989-994) and agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404 available from precious biotechnology (Dalian) ltd.The BAC clone is available from Clemson University Genomics Institute.

2.GbMYB5 the clone of gene

According to the sequences Design special primer BamMYB19:5 ' ACTGGATCCACATGGGTCGAGCTCCTTGCTGT3 ' (SEQ ID NO.3) that contains the MYB structural domain among the BAC clone who obtains, KpnMYB1122:5 ' TCCAGGTACCTTGTCTCTTGCTACATAGGGTAACC3 ' (SEQ ID NO.4).Primer adds BamH I and Kpn I restriction enzyme site respectively.

The CTAB method is extracted sea island cotton RNA, with TransScript First-Strand cDNA Synthesis SuperMix (Beijing Quanshijin Biotechnology Co., Ltd's product) preparation cDNA first chain.With cDNA first chain is template, primer BamMYB19 and KpnMYB1122, pcr amplification GbMYB5 gene; The pcr amplification program is: 94 ℃ of preparatory sex change 3 minutes, 94 ℃ of sex change 30 seconds, 59 ℃ of annealing 50 seconds; 72 ℃ were extended 1 minute and 20 seconds, totally 36 circulations, and last 72 ℃ were extended 10 minutes.Electrophoresis detection obtains the PCR product fragment of 1.1Kb size, and this PCR product purification rear clone to pEASY-T3 carrier (Beijing Quanshijin Biotechnology Co., Ltd), is made up intermediate carrier T-GbMYB5, and examining order is accomplished by the biological ltd of Shanghai English fine horse.Sequencing result shows that this exogenous segment total length is 1105bp, has one longly to be the open reading frame of 832bp, 277 amino acid of encoding, and called after GbMYB5 in the accession number of GenBank is: JF820389.Warp carries out the BLAST analysis with the gene that NCBI goes up the registration login, and (accession number: AAC83600) (accession number: homologous gene similarity ABH02912) is new gene about 51% with big shield beans MYB71 for GbMYB5 gene and model plant Arabidopis thaliana.

3.GbMYB5 the expression of gene in different tissues

According to sea island cotton GbMYB5 full length gene sequence; Utilize a pair of special primer MYBFrt:5 ' AGGCTTCTAATGGAAACCCTAACC3 ' of Primer5 software design (SEQ ID NO.5), MYBRrt:5 ' TCCATTGGTTGAAGAACAGAAGT3 ' (SEQ ID NO.6).Extract total RNA of stem, leaf, flower bud, wadding and the immature seed of sea island cotton 7124 respectively, the cDNA that obtains with reverse transcription increases as pcr template, and preliminary experiment is confirmed the RT-PCR condition.With sea island cotton cDNA is template, and MYBFrt, MYBRrt are that primer carries out the RT-PCR amplification, and the result shows; The GbMYB5 gene all has expression at these organs; But at the expression amount of vegetative organ expression amount apparently higher than reproductive organ, the highest with the expression in leaf especially, secondly higher expression is also arranged in stem; And in wadding and flower bud, slight expression (see Fig. 1, wherein Histone is for contrasting) is only arranged.

4.GbMYB5 gene is coerced the expression analysis under handling in difference

Sea island cotton lint seed directly is seeded in nutrition soil, when treating that seedling grows to 2～3 true leaves, respectively seedling is carried out heavy metal, arid, dormin and gibberic acid and induces processing.Heavy metal is handled the CuSO with the 10uM of 5ml ₄Solution directly sprays cotton plant; Arid processing use 5ml concentration is 20% PEG6000 pouring cotton plant; The ABA solution that dormin (ABA) is handled with the 15mg/L of 5ml directly sprays plant; The gibberic acid solution that gibberic acid (GA) is handled with the 10ppm of 5ml directly sprays cotton plant.More than all in treatment times consistent, get respectively and handle back 24h, 48h and inductive plant leaf (CK) not, place liquid nitrogen rapidly, deposit for-70 ℃ and extract total RNA fully and use.At heavy metal (CuSO ₄) after the processing, the GbMYB5 gene expression amount is compared obvious decline with contrast, but after handling 48h, the expression amount comparison is according to slightly rising.The PEG simulating drought is handled down, and GbMYB5 genetic expression has raising slightly when 24h, when 48h, increase rapidly.ABA and GA hormone induction are handled can significantly improve the GbMYB5 expression of gene in 24h; But pass in time; GbMYB5 is different to both responses, after ABA handles 48h, and the high level when the GbMYB5 gene expression amount also maintains 24h; And behind the GA processing 48h, the GbMYB5 gene expression amount descends rapidly.This shows that GbMYB5 gene pairs heavy metal, arid, ABA and GA induce response is all arranged, maybe relevant with plant stress-resistance (see Fig. 2, wherein Histone is contrast).

5. the structure of recombinant vectors and tobacco transform

With a BamH I and Kpn I enzyme carrier T-GbMYB5 that hits; Obtain the purpose fragment of 1.1.Kb size; Be connected with the pCAMBIA2301 plasmid after handling through same double digestion; Obtain recombinant expression vector pCAMBIA2301-GbMYB5, adopt freeze-thaw method that pCAMBIA2301-GbMYB5 is transformed Agrobacterium LBA4404, transformant is preserved subsequent use after the PCR checking.

Leaf dish culture method transformation of tobacco is altogether adopted in this experiment.Infect: the explant tobacco leaf that disinfects is cut into 1cm ²Size places the Agrobacterium LBA4404/pCAMBIA2301-Gb (OD that contains recombinant plasmid ₆₀₀=0.3-0.4) soak 5-6min in the dip-dyeing solution, blot with the filter paper after the sterilization then.Cultivate altogether: be total to reason 48h in dark place on the culture medium in MS.Screening and culturing: behind the 48h blade of contaminating is transferred on the screening culture medium that contains kantlex and Rifampin.Succeeding transfer culture: change a subculture about every separated 10d.Root culture: filter out the seedling of taking root, the seedling that takes root that filters out is transferred on the root media.Transplant: the long 2-3 of the seedling of taking root transplanted to soil behind the refining seedling during week.Used substratum sees the following form.

The table tobacco transforms used substratum

6. the Molecular Detection of positive plant

Molecular Detection is divided into dna level and transcriptional level detects.Dna level detects: the tobacco regeneration plant extracts the blade genome DNA with the CTAB method, carries out pcr amplification GbMYB5 gene with primer BamMYB19 and KpnMYB1122, is contrast with non-transformation of tobacco.Transcriptional level detects: extract the total RNA of the positive tobacco plant of PCR, RT-PCR detects GbMYB5 expression of gene level.

Insert and select on the screening culture medium to cultivate cultivating tobacco leaf disc behind the 48h altogether, the leaf dish begins to occur resistant buds about 10-15d, and unconverted then chlorisis gradually is dead to albefaction.Containing on the screening culture medium of Kan, can grow up through most of indefinite bud of conversion processing, and can be on root media normal root of hair (Fig. 3).

Extract reconstituted tobacco plant DNA and carry out pcr amplification, 14 strain regeneration plants amplify the specific band (Fig. 4) of treaty 1.1.Kb size, but not transformed plant does not then have specific band, explains that the GbMYB5 gene has been incorporated in the tobacco gene group.Further extract this 14 strain transgene tobacco RNA and carry out the RT-PCR detection, obtain the transfer-gen plant (Fig. 5) that the GbMYB5 gene is expressed in 9 strains, finally keep 2 strains systems.

7. the cultivation of tobacco seedling

T1 earlier with behind the 75% alcohol surface sterilization 1min, uses 15%H for the transgene tobacco seed again ₂O ₂Soaking disinfection 10～15min, sterilized water washing 5 times.Wild type seeds is seeded in 1/2MS (PH=5.8) solid medium, and transgenic seed is seeded in 1/2MS+25mg/L Kan+ (PH=5.8) solid medium, the diel rhythm that 16h illumination/8h is dark, and intensity of illumination is 1600～1800lx.

8. the screening of transgene tobacco and evaluation

T1 after germination for some time, if plant is a transfer-gen plant, then can contain normal growth on the substratum of kantlex for the tobacco transgenic seed, otherwise then the chlorisis albefaction is dead gradually.Select the separation ratio and try material as confession near the transfer-gen plant and the consistent wild-type plant of growing way of 3: 1 strain system; Be transplanted to continued growth in the soil; After treating that growing way is stable; Extracting all with the CTAB method and shift out total DNA of plant, is template with DNA, with primer BamMYB19 and KpnMYB1122PCR amplification GbMYB5 gene.Electrophoresis detection is if can obtain the band of 1.1kb size, and explanation is a transfer-gen plant, otherwise, then be not.After molecular biology method was identified, for fear of false positive occurring, we went out the young leaves of the tobacco plant of specific band with the cotton balls point tobacco of the kantlex that dips in 800ppm to pcr amplification, and 7-10d observes the variation at blade point kantlex position.If the blade position flavescence (Fig. 6) of some kantlex explains that this plant is a false positive, the plant that should blade not changed is as further experiment material, further definite positive seedling.

9. degeneration-resistantly coerce processing

The present invention is primarily aimed at the environment stress of drought resisting and studies.

PEG (polyoxyethylene glycol) simulating drought is coerced processing:

(1) 25%PEG6000 simulating drought: get wild-type plant (WT) and all transgenic lines (TP) plant leaf; Use the punch tool punching of diameter as 2.5cm; The roundlet blade of laying is placed the petridish that contains 25%PEG6000, and each strain system does 3 and repeats and respectively with H ₂O observes the variation (remarks: plant sample number average>=15) of blade as control group.

As shown in Figure 7, handle 8h at the 25%PEG6000 simulating drought, the obvious flavescence of wild-type blade, dehydration is more, and genetically modified two strains are that (TP-1 and TP-2) dehydration is less, blade is flavescence slightly.

(2) 20%PEG6000 waters simulating drought: wild-type (WT) plant and transfer-gen plant (TP-1 and TP-2) use 20%PEG6000 solution are watered, and every strain pouring 5ml rewaters behind 35d, observes the plant variation after handling and after watering.

As shown in Figure 8, before the processing, wild-type and transfer-gen plant (TP-1 and TP-2) be well-grown all, handles the leaf rolling of all plant of back, and plant is wilted condition.After treating to water in the 35th day, continue a few days and observe, find that the wild-type plant fails to restore normal growth, and transfer-gen plant (TP-1 and TP-2) recovers all to grow, show change this gene over to after, tobacco has certain drought-resistant ability.

(3) 2%PEG6000 substratum simulating drought: transgenic aseptic seedling that normal growth on 1/2MS+25mg/L Kan+ solid medium is filtered out and the wild-type aseptic seedling of on 1/2MS, growing; The excision root; Be put on the solid 1/2MS substratum that contains 2%PEG6000 and grow, observe the variation of plant.

Handle preceding wild-type and transfer-gen plant on form, no significant difference (Fig. 9).The back observation of two weeks, the transfer-gen plant upgrowth situation is better than wild-type, mainly shows: blade is big, take root many (Figure 10).The back observation of three weeks; The upgrowth situation of transfer-gen plant will obviously be superior to wild-type, and wild-type mainly shows as poor growth, stagnation, and transfer-gen plant growing state good (Figure 11); Its root also has significant difference; TP-1 plant root growth situation is influenced by PEG hardly, and TP-2 plant root growth situation also obviously is better than WT (Figure 12, wherein WT (PEG) representing not add 2%PEG6000 in this control medium).Explain that the plant that obtains through transgenic has more the drought resisting performance than common plant.The back is observed all around, and the result is consistent with the 3rd week, and the TP-1 plant growth condition is not received the influence of PEG basically, and the upgrowth situation of TP-2 plant also obviously is better than WT plant (Figure 13,14).

PEG through using different concns coerces processing to the tobacco simulating drought, and we find that the plant that changes the GbMYB5 gene over to has stronger drought-resistant ability, has the resistance to arid.

SEQUENCE LISTING

< 110>Nanjing Normal University

< 120>one grow cotton gene related to drought tolerance GbMYB5 application

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Claims (2)

1.SEQ the application of cotton gene GbMYB5 in the tobacco bred seed selection shown in the ID NO.1.

2.SEQ the application of the cotton gene GbMYB5 shown in the ID NO.1 in improving the tobacco drought-resistant ability.

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