CN101265294B - Disease-resistant correlated wheat MYB albumen and coding gene - Google Patents
Disease-resistant correlated wheat MYB albumen and coding gene Download PDFInfo
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Abstract
The invention discloses wheat MYB protein correlated to plant disease resistance, encoding gene and application thereof. The protein is protein (a) and protein (b): protein (a) is composed of amino acid sequence of sequence 2 in a sequence table; and protein (b) is made by performing substitution and/or deletion and/or addition of one or more amino acid residues on amino acid residue sequence of the sequence 2 in the sequence table, is correlated to flowering time, and is derived from protein (a). The wheat MYB protein correlated to plant disease resistance is transferred into plants to improve disease resistance of the plants. The method for culturing transgenic plant having improved disease resistance has important theoretical and practical significance and exerts important effect on genetic improvement of the plants.
Description
Technical field
The present invention relates to a kind of disease-resistant relevant wheat MYB albumen and encoding gene and application.
Background technology
Along with the change of factors such as cropping system, fertilizer and water condition and weather condition, the wheat disease and pest is on the rise to the threat of China's main product wheat, and gas borne fungus diseases such as wheat powdery mildew, rust have become the main disease of China's wheat.At present, Powdery Mildew has spreaded all over more than 20 provinces and cities of China, and soil-borne diseases such as banded sclerotial blight, root rot and gaeumannomyces graminis disease are also serious day by day in the wheat main producing region, and head blight is except that the middle and lower reach of Yangtze River is popular, the trend that the trend of going up north in addition, virus diseases such as yellow dwart are expansion, rise.And China most of wheat planting area has been subjected to the threat of multiple diseases.Seed selection and promote that disease-resistant wheat breed is that control disease popular is most economical, safety and valid approach.
Utilize resistant gene to carry out a large amount of mildew-resistances, rust breeding work both at home and abroad, the resistant variety of breeding plays an important role on producing.The mode of action between present employed disease-resistant gene and the pathogenic bacteria nontoxic gene (avr) is followed " gene pairs gene " pattern of Flor mostly.These pathogenic bacteria physiological strain colonies are big, variation is fast, as adopting term single gene, can cause wheat breed resistance frequent " forfeiture ", in addition exist the wheat resistant variety breed, its resistance passive state of " forfeiture " when intending wideling popularize.And the research of genetics of resistance such as wheat hypochnus, root rot and gaeumannomyces graminis disease is weak, and resistance breeding is made slow progress.Therefore, strengthen the further investigation of disease-resistant wheat reaction mechanism and then to open up the breeding new way very necessary and important.
Study lessly to the disease-resistant wheat reaction mechanism at present, to studies show that in a large number of Arabidopis thaliana isotype plant disease-resistant reaction mechanism, the disease resistance that pathogenic micro-organism induces the host to produce is very complicated network system, comprise the mutual identification and the effect of plant disease resistance genes and pathogenic bacteria nontoxic gene, activate conduction of host's a series of signal and gene regulating thus, activate, suppress a series of downstream defence expression of gene in phase as some transcription factors (MYB, WRKY, MYB, bZIP), and then produce resistance of wide spectrum.
Disease-resistant relevant transcription factor can be as the latent gene source of broad spectrum antidisease gene engineering.Proved Arabidopis thaliana ERF1 (AtERF1, At3g23240) ERF-AP2 can interact with GCC box cis-acting elements in the target gene promotor in conjunction with the territory and regulate a plurality of defence expression of gene in downstream, and then make plant show resistance (Solano R to downright bad nutritious fungus, soil-borne pathogen, Stepanova A, Chao Q, Ecker J is events in ethylene signaling:a transcriptional cascade mediated byETHYLENE-INSENESITIVE 3 R.1998.Nuclear, Gen Dev 12:3703-3714; Berrocal-Lobo M, Molina A.2004, ETHYLENE RESPONSE FACTOR 1 mediates Arabidopsis resistance tosoilborne Fungus Fusarium oxysporum.Mol Plant-Microbe Interact, 17:763-770; Berrocal-Lobo M, Molina A, Solano R.2002, Constitutive expression of ETHYLENE-RESPONSE-FACTOR 1 in Arabidopsis confers resistance to several necrotrophic fungi.Plant J, 29:23-32).The transgenic arabidopsis plant of overexpression ERF1, to the resistance of soil-borne pathogens such as downright bad nutritious fungus such as Botrytis cinerea, Plectosphaerella cucumerina and F.oxysporum (the Berrocal-Lobo M that is improved, Molina A.2004, ETHYLENE RESPONSE FACTOR 1mediates Arabidopsis resistance to soilborne Fungus Fusarium oxysporum.MolPlant-Microbe Interact, 17:763-770).Arabidopis thaliana MYB108 (BOS1) can suppress growth (the Mengsite T of botrytis cinerea by mediated responses oxygen intermediate (ROI), Chen X, Salmeron J, Dietrich R.2004, The botrytis susceotible1 gene encodes an R2R3 MYB transcription factor protein that isrequired for biotic and abiotic stress responses in Arabidopsis.The plant cell.15:2551-2565).Therefore, with the separating, identify of plant disease resistance-related protein gene, to illustrate disease-resistant mechanism, to carry out molecular breeding research effectively very necessary, become the focus of domestic and international plant science research.Yet, do not appear in the newspapers as yet about clone, expression characterization and the functional study of the relevant MYB protein gene of disease-resistant wheat.
Summary of the invention
The purpose of this invention is to provide a kind of albumen relevant and encoding gene thereof with plant disease-resistant.
The albumen relevant provided by the present invention with plant disease-resistant, name is called TaPIMP1, derives from wheat (Triticumaestivum L.), is following (a) or protein (b):
(a) protein of forming by the amino acid residue sequence shown in the sequence in the sequence table 2;
(b) with the amino acid residue sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant disease-resistant by (a) deutero-protein.
For the ease of the purifying of TaPIMP1, label as shown in table 1 on proteinic N-terminal that can the amino acid residue sequence of sequence 2 is formed in by sequence table or C-terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the TaPIMP1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of TaPIMP1 in above-mentioned (b) can be by the codon that lacks one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 48-1019 bit base with sequence in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Above-mentioned and cDNA gene plant disease resistance-related protein also belongs to protection scope of the present invention.
Specifically can be following 1 with the cDNA gene of plant disease resistance-related protein)-4) in arbitrary described gene:
1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 48-1019 position deoxyribonucleotide;
2) its nucleotide sequence is the sequence 1 in the sequence table;
3) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 1 and encode above-mentioned and dna molecular plant disease resistance-related protein.
4) with 1) or 2) gene have homology 90% or more, and encode above-mentioned and dna molecular plant disease resistance-related protein.
Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
Increase above-mentioned TaPIMP1 full length gene or its arbitrary segmental primer to also belonging to protection scope of the present invention.
Contain above-mentioned and recombinant vectors, transgenic cell line and reorganization bacterium the plant disease resistance-related protein encoding gene and also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of TaPIMP1 gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries of the present invention and plant disease resistance-related protein encoding gene TaPIMP1 can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.By the plant transformed host both can be monocotyledonss such as wheat, paddy rice, corn, also can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, cucumber, tomato, cotton, willow, lucerne place.
When using the gene constructed recombinant expression vector of TaPIMP1, can before its transcription initiation Nucleotide, add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (gentamicin marker, kantlex marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.
Described recombinant expression vector specifically can be and insert the above-mentioned recombinant expression vector that obtains with encoding gene plant disease resistance-related protein between the multiple clone site of pBI121, as pBI121-TaPIMP1.
Another object of the present invention provides a kind of method of cultivating the transgenic plant of disease resistance raising.
The method of the transgenic plant that cultivation disease resistance provided by the present invention improves is that above-mentioned encoding gene TaPIMP1 with plant disease resistance-related protein is imported in the plant, obtains the transgenic plant that disease resistance improves.
Described is to import in the plant by described recombinant expression vector with encoding gene TaPIMP1 plant disease resistance-related protein.
Described plant both can be monocotyledonss such as wheat, paddy rice, corn, also can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, cucumber, tomato, cotton, willow, lucerne place.
Described plant specifically can be wheat or tobacco.
Described disease resistance specifically can be the disease resistance to bacterial wilt, wheat hypochnus or gibberella saubinetii.
Transcription factor gene is subjected to the expression pattern of pathogenic bacterium inducing, is Rapid identification and disease-resistant correlation candidate transcription factor gene, further carries out the foundation of functional analysis.Of the present invention and expression analysis result plant disease resistance-related protein shows that sheath blight fungus, gibberellic hypha infect can induce the TaPIMP1 expression of gene to raise.
The transgenic experiments that TaPIMP1 is imported tobacco proves, the tobacco disease resistance that changes TaPIMP1 over to obviously improves, illustrate that TaPIMP1 is the albumen relevant with plant disease-resistant, can be used for improving the disease resistance of plant with plant disease resistance-related protein TaPIMP1 and encoding gene thereof.
Description of drawings
Fig. 1 is the gel electrophoresis figure of RT-PCR amplification TaPIMP1 full length gene cDNA sequence
Fig. 2 is R2, the R3 conserved regions homology comparison result of TaPIMP1 and other plant MYB
Fig. 3 is the full length amino acid sequence homology comparison result of TaPIMP1 and other plant MYB
Expression Fig. 4 is induced by wheat hypochnus for RT-PCR analyzes TaPIMP1 after
Expression Fig. 5 is induced by gibberella saubinetii for RT-PCR analyzes TaPIMP1 after
Fig. 6 is the schema of vector construction and transformation of tobacco
Fig. 7 is T
1Pcr amplification result for TaPIMP1 gene in the transgene tobacco
Fig. 8 is T
1Resistance to bacterial wilt phenotypic evaluation result for transgene tobacco
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
One, the amplification of 5 ' terminal sequence
The wheat breed Soviet Union wheat No. 3 (available from the Jiangsu Province Agriculture Science Institute germplasm resource banks) that grows to the anti-gibberella in two leaf periods is handled with gibberella, concrete treatment process is: No. 3 wheat leaf blades of Soviet Union wheat that grew to for two leaf periods with gibberella saubinetii (Fusarium graminearum) (academy of agricultural sciences, Jiangsu biotechnology institute) mycelia piece frictional inoculation, and gibberella mycelia piece is put in leaf sheath portion, the above-mentioned wheat leaf blade of handling with the gibberella mycelia of clip behind the 48h, liquid nitrogen is handled, method according to Invitrogen TRIZOL Reagent total RNA extraction reagent specification sheets is operated, and extracts total RNA of above-mentioned wheat leaf blade.According to the program of the Invitrogen company first chain cDNA synthetic agent box, with the synthetic first chain cDNA of the RNA sample reverse transcription of extracting.
With Arabidopis thaliana BOS1 (MYB108, NP-187301) sequence B last searches GenBank, the EST public database, the result finds and AtMYB108 homologous wheat est sequence CN011324, this est sequence according to wheat, design and synthesize a pair of primer: MYB-F15 '-ACTCGCGTACGTCTTCCTGA-3 ' and MYB-R1:5 '-TCCAGACTTGCCTGACGAC-3 ', the cDNA that handles the Soviet Union wheat No. 3 of 48h with the gibberella of above-mentioned acquisition is a template, carry out pcr amplification, the pcr amplification system is: 10 * GC damping fluid, 1 μ L, cDNA 2 μ L (20ng), 2.5mM dNTPs 2 μ l, each 1 μ L of 5 μ mol/L upstream and downstream primers, 5U/ μ l LA-Taq enzyme 0.2 μ l, ddH
2O mends to 25 μ L.The pcr amplification program is: 94 ℃ of pre-sex change of elder generation 3 minutes; Then 94 ℃ 45 seconds, 61 ℃ 45 seconds, 72 ℃ 45 seconds, totally 5 circulations; Again 94 ℃ 45 seconds, 59 ℃ 45 seconds, 72 ℃ 60 seconds, totally 30 circulations; Last 72 ℃ were extended 10 minutes.The PCR product is carried out agarose gel electrophoresis, and result's amplification obtains the fragment of a 553bp, is connected on the pMD18-T carrier this PCR product and order-checking.Sequencing result shows, the nucleotide sequence of the amplified production of acquisition be in the sequence table sequence 1 from 5 ' terminal 1-553 position nucleotide sequence, be the conservative territory section of the TaPIMP1 relevant with plant disease-resistant.
Two, the amplification of 3 ' RACE
Adopt 3 ' the RACE test kit clone TaPIMP1 of GIBCOBRL company gene, 3 ' terminal sequence.No. 3 blade RNA of Soviet Union wheat that the gibberella of above-mentioned acquisition handled 48h with joint primer AP:5 '-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3 ' carry out reverse transcription earlier, obtain the first chain cDNA, and be template with it, use upstream primer MYB-3-RA1:5 '-ACGGACAACGAGGTCAAGAA-3 ' and downstream primer AUAP:5 '-GGCCACGCGTCGACTAGTAC-3 ' to carry out first round amplification, the pcr amplification system is: 10 * GC damping fluid, 1 μ L, cDNA 2 μ L (20ng), 2.5mM dNTPs 2 μ l, each 1 μ L of 5 μ mol/L upstream and downstream primers, 5U/ μ l LA-Taq enzyme 0.2 μ l, ddH
2O mends to 25 μ L.The pcr amplification program is: 94 ℃ of pre-sex change of elder generation 3 minutes; Then 94 ℃ 50 seconds, 60 ℃ 50 seconds, 72 ℃ 2 minutes, totally 5 circulations; Again 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ were extended 10 minutes.
Be template to dilute 50 times first round PCR product then, use nested primer MYB-3-RA2:5 '-CAGAAGCACGCCAAGCAG-3 ' and downstream primer AUAP:5 '-GGCCACGCGTCGACTAGTAC-3 ' to carry out second and take turns pcr amplification, the pcr amplification system is: 10 * GC damping fluid, 1 μ L, cDNA 2 μ L (20ng), 2.5mM dNTPs2 μ l, each 1 μ L of 5 μ mol/L upstream and downstream primers, 5U/ μ l LA-Taq enzyme 0.2 μ l, ddH
2O mends to 25 μ L.The program of pcr amplification is: 94 ℃ of pre-sex change of elder generation 3 minutes; Then 94 ℃ 55 seconds, 58 ℃ 45 seconds, 72 ℃ 2 minutes, totally 3 circulations; Again 94 ℃ 55 seconds, 56 ℃ 45 seconds, 72 ℃ 2 minutes, totally 33 circulations; Last 72 ℃ were extended 10 minutes.Get second and take turns the PCR reaction product and carry out agarose gel electrophoresis and detect, result's amplification obtains the fragment of a 637bp, is connected on the pMD18-T carrier this PCR product and order-checking.Sequencing result shows, the nucleotide sequence of the amplified production of acquisition be in the sequence table sequence 1 from 3 ' terminal 481-1117 position nucleotide sequence.
Three, the amplification of total length TaPIMP1 gene cDNA sequence
5 ' terminal sequence of the TaPIMP1 gene of above-mentioned acquisition and 3 ' terminal sequence in external splicing, are obtained the full length cDNA sequence of the TaPIMP1 gene of 1117bp.For obtaining effable and gene clone plant disease resistance-related protein, according to the full length cDNA sequence that splices the TaPIMP1 gene that obtains, design a pair of Auele Specific Primer myq-QU:5 '-ACTCGCGTACGTCTTCCTGA-3 ' and MYB-QL:5 '-GCGCTCTAGTTAAGTTCATCGTC-3 ', the total RNA that handles No. 3 blades of Soviet Union wheat of 48h with the gibberella of above-mentioned acquisition is a template, carry out the RT-PCR amplification, the pcr amplification system is: 10 * GC damping fluid, 1 μ L, cDNA 2 μ L (20ng), 2.5mM dNTPs 2 μ l, each 1 μ L of 5 μ mol/L upstream and downstream primers, 5U/ μ l LA-Taq enzyme 0.2 μ l, ddH
2O mends to 25 μ L.The program of pcr amplification is: 94 ℃ of pre-sex change of elder generation 3 minutes; Then 94 ℃ 50 seconds, 62 ℃ 50 seconds, 72 ℃ 2 minutes, totally 5 circulations; Again 94 ℃ 50 seconds, 60 ℃ 50 seconds, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ were extended 10 minutes.
The PCR product is carried out agarose gel electrophoresis, detected result as shown in Figure 1, wherein, swimming lane M is the dna molecular amount standard of 100bp, swimming lane C is the RT-PCR amplified production.The result shows the band that obtains 1038bp, reclaims this band and is connected on the pMD18-T carrier, will obtain recombinant expression vector called after pT-TaPIMP1.This sequence is checked order, and sequencing result shows that this sequence and external splicing sequence (1117bp) can be mated fully, illustrates that the splicing sequence is a complete nucleotide sequence of TaPIMP1 gene cDNA.The nucleotide sequence of TaPIMP1 is shown in sequence in the sequence table 1, and its amino acid sequence coded is shown in sequence in the sequence table 2.
The aminoacid sequence of TaPIMP1 is carried out the BLASTP compare of analysis in NCBI, the result shows, TaPIMP1 and the relevant myb transcription factor of disease-resistant, drought-enduring function, as Arabidopis thaliana MYB78 (NP-199773), BOS1 (MYB108, NP-187301), Cpm5 (AAB58313) and OsMYB (Os05g0132700), the JAMYB (AAK08983) of Cpm10 (AC051631) and paddy rice, the P-type R2R3-MYB (AF474132-1) of Chinese sorghum etc. have homology.With DNAMAN software aminoacid sequence and the proteic aminoacid sequence of above-mentioned MYB of TaPIMP1 carried out homology analysis, the result shows that these proteic aminoacid sequences are about 86.2% in the homology of R2, R3 conserved regions, and comparison result as shown in Figure 2.Wherein, wheat MYB albumen TaPIMP1 relevant with plant disease-resistant and above-mentioned MYB albumen R2 conserved regions homology comparison result are shown in Fig. 2 A, and wheat MYB albumen TaPIMP1 relevant with plant disease-resistant and above-mentioned MYB albumen R3 conserved regions homology comparison result are shown in Fig. 2 B.But the homology of the full length amino acid sequence of the wheat MYB albumen TaPIMP1 relevant with plant disease-resistant and the proteic full length amino acid sequence of above-mentioned MYB is very low, even and also having only 57.27% consistence between the highest OsMYB (Os05g0132700) of homology, comparison result is as shown in Figure 3.The result shows that the wheat MYB albumen TaPIMP1 relevant with plant disease-resistant of the present invention is the newcomer in the plant myb transcription factor family.
The abduction delivering analysis of embodiment 2, TaPIMP1 gene
Cultivate No. 3 wheat leaf blades of Soviet Union wheat of 10d with gibberella saubinetii mycelia piece frictional inoculation flowerpot, and head blight mycelia piece is put in the leaf sheath portion of wheat; Cultivate the red wheat in mountain (China national germplasm resource bank) seedling of the anti-banded sclerotial blight of 15d (two leaf phases) with rhizoctonia cerealis (Rhizoctonia cerealis) (academy of agricultural sciences, Jiangsu biotechnology institute) mycelia piece frictional inoculation flowerpot, and banded sclerotial blight mycelia piece is put in leaf sheath portion.Get the wheat leaf blade of above-mentioned different treatment behind the 48h respectively, it is standby to be stored in-80 ℃ of Ultralow Temperature Freezers behind the liquid nitrogen flash freezer.
Extract the RNA (each sample is got the total RNA of about 5 μ g) of the wheat leaf blade of above-mentioned different treatment respectively, according to the program of the Invitrogen company first chain cDNA synthetic agent box, reverse transcription becomes cDNA.Method (the Okubara P A that utilizes Okubara etc. to propose, Blechl A E, McCormick S P, Alexander N J, Dill-Macky R, Hohn T is M.2002, Engineering deoxynivalenol metabolism in wheat through theexpression of a fungal trichothecene acetyltransferase gene.Theor Appl Genet, 106:74-83), carry out sxemiquantitative RT-PCR and analyze, do internal reference with actin.With the homogenization of sample cDNA concentration, use special primer the ACT-F:5 '-cactggaatggtcaaggctg-3 ' of TaPIMP1 gene and ACT-R:5 '-ctccatgtcatcccagttg-3 ' to carry out pcr amplification then, with 2
-Δ Δ CTMethod (Livak KJ, SchmittgenTD.2001.Analysis of relative gene expression data using real-time quantitative PCR andthe 2
-Δ Δ CTMethod.Methods.25:402-408) expression of analyzing gene TaPIMP1 under rhizoctonia cerealis or fusarium graminearum processing, simultaneously with the Soviet Union wheat No. 3 that do not inoculate gibberella and the red wheat in mountain that do not inoculate sheath blight fungus in contrast, every group of sample repeats 3 times at least.
The result shows that the TaPIMP1 gene transcription is expressed and is subjected to inducing of sheath blight fungus.Do not inoculate in the red wheat in mountain of sheath blight fungus, detect less than the TaPIMP1 expression of gene; Sheath blight fungus infects after 3 hours the TaPIMP1 gene expression amount both to have begun to increase, and infected 12 hours TaPIMP1 expression of gene amounts and reached the peak the 1st time, infected 48 hours TaPIMP1 expression of gene amounts and reached the peak the 2nd time.Concrete analytical results as shown in Figure 4.The TaPIMP1 expression of gene also is subjected to the rapid induction of gibberella, and once peak expression infects and peaked in 12 hours, infects 24 hours and 48 hours expression amounts reduce gradually, but still is higher than expression amount in the Soviet Union wheat No. 3 that does not inoculate gibberella.Concrete analytical results as shown in Figure 5.The result shows, the TaPIMP1 gene all responds at above-mentioned two kinds of pathogenic bacterium inducings early stage (3 hours), transcriptional level significantly strengthens, but be subjected to the expression pattern difference of above-mentioned two kinds of pathogenic bacterium inducings, illustrate that TaPIMP1 may participate in the defensive raction of host to rhizoctonia cerealis and gibberella, but control methods are different.
The disease resistance analysis of embodiment 3, commentaries on classics TaPIMP1 genetic tobacco
1, changes the acquisition of TaPIMP1 genetic tobacco
With the plasmid pT-TaPIMP1 that contains TaPIMP1 full length gene ORF is template, to have the MBD-F:5-CA of XbaI and BmaHI restriction enzyme site
TCTAGAATGGACATGGACAAGG-3 and MBD-R:5-GT
GGATCCTTCATCGTCAGCAGTAA-3 is a primer, the ORF of pcr amplification TaPIMP1.Cut the PCR product of this 982bp with XbaI and BmaHI enzyme, be connected to then with XbaI and BmaHI enzyme and cut on the big fragment that carrier pBI121 (Beijing Baeyer enlightening company) obtains, connect product transformed into escherichia coli TOP10 competent cell, from bacterium colony, filter out recon to kalamycin resistance, carry out sequencing analysis, identifying the recombinant plasmid called after pBI121-TaPIMP1 that contains TaPIMP1 gene ORF through order-checking, this recombinant plasmid is controlled by 35S promoter.Utilize electric shocking method that pBI121-TaPIMP1 is transformed in Agrobacterium LAB4404 (Beijing Baeyer enlightening company) cell, screen with the MS substratum that contains the 100ug/ml kantlex, the agrobacterium strains LAB-pBI121-TaPIMP1 of 35S promoter and TaPIMP1 gene is carried in acquisition, LBA-pBI121-TaPIMP1 is adopted leaf dish method transformation of tobacco (W38, Tobacco Institute, Chinese Academy of Agricultural Science), above-mentioned transgene tobacco is moved to hot-house culture, the seed of selfing, collection transgene tobacco.Concrete schema as shown in Figure 6.
With the planting seed of above-mentioned transgene tobacco on the MS substratum that contains the 100ug/ml kantlex, the T that will on above-mentioned substratum, survive
1Seedling for transgene tobacco moves to hot-house culture, with above-mentioned T
1Genomic dna for transgene tobacco is a template, is primer with MBD-F and MBD-R, carries out the PCR reaction.
Above-mentioned PCR reaction product is carried out agarose gel electrophoresis to be detected.With plasmid pBI121-TaPIMP1 is over against photograph, to change the T of plasmid pBI121 over to
1Be negative contrast for transgene tobacco, the result as shown in Figure 7.Wherein, 1 is the pcr amplification result of plasmid pBI121-TaPIMP1, and 2 for changing the T of plasmid pBI121 over to
1For the pcr amplification result of transgene tobacco, 3-5 is the T that changes plasmid pBI121-TaPIMP1 over to
1Pcr amplification result for transgene tobacco.The result shows at the T that changes plasmid pBI121-TaPIMP1 over to
1For have in the transgene tobacco with over against take a picture with size be about the goal gene band of 982bp, and in negative contrast band does not appear.The result obtains the positive T1 that changes plasmid pBI121-TaPIMP1 over to of the PCR assay of 12 strains system altogether for transgene tobacco.
2, detect the disease resistance of changeing the TaPIMP1 genetic tobacco
Getting 4 strains arbitrarily from the transgene tobacco that above-mentioned 12 strains are is M21, M8, M27 and M33, according to excised leaf injection (Zhang HB, Zhang DB, Chan J, Yang YH, Huang ZJ, Huang DF, Wang XC, Huang R is stress-responsive factor TSRF1 interacts withethylene responsive element GCC box and regulates pathogen resistance to Ralatoniasolanacearum.Plant Mol.Biol.55:825-834 F.2004.Tomato), with ralstonia solanacearum (Ralatonia solanacearum) (Tobacco Institute, Chinese Academy of Agricultural Science) spore suspension (1 * 10
7Spore/ml) infect its blade, simultaneously to inject the T of equal volume distilled water
1In generation, changeed TaPIMP1 genetic tobacco blade in contrast.Each strain is every kind and handles 3 blades.Each blade is injected 50 μ L ralstonia solanacearums or distilled water in the both sides of blade, postvaccinal blade is put into enamel tray, preserve moisture with thieving paper, preservative film seals, place 25 ℃ of incubators of the dark alternative of 16h illumination and 8h, observe the incidence of blade behind the 4-6.Simultaneously to change the T of plasmid pBI121 over to
1For transgene tobacco and not genetically modified tobacco plant in contrast.
The result shows that in the time of 4 days, the transgene tobacco of injection distilled water is not morbidity all in the inoculation of ralstonia solanacearum spore suspension; The T of not genetically modified tobacco, Pignus pignoris grain pBI121
1All fall ill for the transgene tobacco blade, a large amount of necrotic plaques occur; And the T of commentaries on classics TaPIMP1 gene
1All show disease-resistant for transgene tobacco.Wherein, strain is that 3 blades of M21 do not have peculiar symptom, and strain is that 3 blade inoculations of M8, M27 and M33 are put flavescence slightly.Inoculate 5 days halfs, the T of not genetically modified tobacco, Pignus pignoris grain pBI121
1For all seriously morbidities of transgene tobacco blade, downright bad putrefactive phenomenon appears; And the T of commentaries on classics TaPIMP1 gene
1All show disease-resistant for transgene tobacco.Wherein, strain is that the blade inoculation of M21 is put flavescence slightly, shows as high resisting, and strain is that the blade inoculation point of M8, M27 and M33 forms scab on every side, but indiffusion.Concrete phenotypic evaluation result as shown in Figure 8.Wherein, WT-W38 is the T that changes plasmid pBI121 over to for not genetically modified tobacco, pBI121
1For transgene tobacco, M21, M8, M27 and M33 are the T that changes the TaPIMP1 gene over to
1For transgene tobacco.The result shows that commentaries on classics TaPIMP1 genetic tobacco obviously improves the resistance of Ralstonia solanacearum, shows that TaPIMP1 is the albumen relevant with plant disease-resistant.
Sequence table
Claims (12)
1. albumen, the protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2.
2. the described proteic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described proteic encoding gene is following 1) or 2) described gene:
1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 48-1019 position deoxyribonucleotide;
2) its nucleotide sequence is the sequence 1 in the sequence table.
4. the recombinant expression vector that contains claim 2 or 3 described genes.
5. the transgenic cell line that contains claim 2 or 3 described genes.
6. the reorganization bacterium that contains claim 2 or 3 described genes.
7. recombinant expression vector according to claim 4 is characterized in that: described recombinant expression vector is for inserting the recombinant expression vector that claim 2 or 3 described genes obtain between the multiple clone site of pBI121.
8. the primer of nucleotide fragments shown in the sequence 1 is right in the extension increasing sequence table, it is characterized in that: described primer is to being made up of primer 5 '-ACTCGCGTACGTCTTCCTGA-3 ' and primer 5 '-GCGCTCTAGTTAAGTTCATCGTC-3 '.
9. a method of cultivating the transgenic plant of disease resistance raising is that claim 2 or 3 described encoding genes are changed in the plant, obtains the transgenic plant that disease resistance improves.
10. method according to claim 9 is characterized in that: claim 2 or 3 described encoding genes are to import in the plant by the described recombinant expression vector of claim 4.
11. according to claim 9 or 10 described methods, it is characterized in that: described plant is wheat or tobacco.
12. method according to claim 11 is characterized in that: described disease resistance is the disease resistance to bacterial wilt, wheat hypochnus or gibberella saubinetii.
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| CN102464710B (en) * | 2010-11-09 | 2014-01-29 | 中国科学院遗传与发育生物学研究所 | Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof |
| CN102161698B (en) * | 2011-02-28 | 2013-04-10 | 吉林大学 | Soybean MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor as well as coding gene and application thereof |
| CN102146128B (en) * | 2011-04-12 | 2013-04-10 | 中国农业大学 | Protein MtMYB1 related to stress tolerance of plants and encoding gene and application thereof |
| CN102234653B (en) * | 2011-06-29 | 2012-12-12 | 济南大学 | Salt-tolerant and drought-resistant gene TaMYB33 of wheat and coding protein as well as application thereof |
| CN103588868B (en) * | 2012-08-17 | 2015-07-22 | 中国科学院遗传与发育生物学研究所 | Wheat protein TaMYB1, and coding gene and application thereof |
| CN106146634B (en) * | 2015-04-15 | 2019-08-16 | 中国农业科学院作物科学研究所 | Plant disease-resistant protein B jMYB9 and its encoding gene and application |
| CN108440659A (en) * | 2018-05-28 | 2018-08-24 | 青岛大学 | A kind of wheat powdery mildew resistance-associated protein TaMYB15 and its encoding gene and application |
| CN110117322A (en) * | 2019-06-11 | 2019-08-13 | 中国农业科学院作物科学研究所 | The MYB class transcription factor and its encoding gene that are separated from purple plague purpura Trifolium repense and application |
| CN111574606B (en) * | 2020-06-29 | 2021-11-16 | 中国农业科学院作物科学研究所 | Wheat disease-resistant and heading regulation gene TaCOK and related biological material and application thereof |
| CN112111499B (en) * | 2020-10-29 | 2022-04-12 | 南京农业大学 | Transcription factor PbMYB7 sensitive to abiotic stress expression, protein, expression vector and application thereof |
| CN114276429B (en) * | 2021-12-28 | 2022-10-11 | 中国农业科学院作物科学研究所 | Method for cultivating TaLRK-R gene-transferred wheat with resistance to sheath blight and stem base rot and related biological material thereof |
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