CN102863523A - Vegetable yellow dwarf resistant key protein TiRB and coding gene and application thereof - Google Patents
Vegetable yellow dwarf resistant key protein TiRB and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a vegetable yellow dwarf resistant key protein TiRB and a coding gene and application thereof. The protein refers to (a) or (b): (a) the protein consists of amino acid sequences shown in sequence one in a sequence table; and (b) the protein which is relevant to vegetable yellow dwarf resistance is derived from sequence one by subjecting the amino acid sequences in sequence one to substitution and/or deletion and/or addition of one or more amino acid residues. The TiRB gene is introduced into wheat, resistance of transgenic wheat subjecting to overexpression of the gene to yellow dwarf is obviously enhanced, TiRB gene expression suppression in disease-resistant wheat enables a plant to lose resistance to the yellow dwarf, and all that explains the TiRB gene is the key protein for the vegetable yellow dwarf resistance. The key protein TiRB for the vegetable yellow dwarf resistance and the coding gene of the key protein TiRB can be used for improving the vegetable yellow dwarf resistance and is of great value to plant breeding. The vegetable yellow dwarf resistant key protein TiRB and the coding gene and application thereof are of great theoretical and practical significance and play an important role in vegetable genetic improvement.
Description
Technical field
The present invention relates to a kind of Genes For Plant Tolerance yellow dwart key protein TiRB and encoding gene and application.
Background technology
Yellow stunt of wheat is by barly yellow dwarf virus (barley yellow dwarf virus, BYDV) the important disease of the wheat that causes, wheat in case infect namely pasts medical help, and causes wheat yield and quality to descend, so yellow dwart is also referred to as " the yellow pestilence of wheat " and reaches " wheat cancer ".Each Mai Qu all has generation to yellow dwart in the world.Because of yellow stunt of wheat harm, U.S.'s wheat yield in 1978 German Yield Reduction of Winter Wheat 40% in 60%～80%, 1988, annual Australia loses about 3,000 ten thousand dollars because of yellow dwart.All there is the generation of yellow dwart in more than 40 countries such as New Zealand, Argentina, Turkey, Tunisia, Hungary.The northwest of China, some areas, North China and northeast are once in 1966,1970,1973,1978,1980,1987,1999 big area generation yellow dwart, only the Mai Qu such as Shaanxi in 1999, Shanxi just causes wheat yield 20%～30% because of yellow dwart, indivedual serious wheat district underproduction surpass 50%, and the wheat yield loss reaches several hundred million kilograms.In recent years, because climate changes such as warm winters, the wheat aphid of propagating barly yellow dwarf virus is day by day serious in China, and yellow stunt of wheat has spreaded all over a plurality of wheat belts such as Shaanxi, Shanxi, Gansu, Sichuan, Ningxia, the Inner Mongol, Hebei and Jiangsu.Therefore, the control of yellow dwart is for guaranteeing that improving yield of wheat stable yields and agricultural sustainable development are extremely important, and seed selection wheat with resistance to yellow dwarf new variety are the most economical effective approach of this disease of control.
Barly yellow dwarf virus is by aphid mediation and propagation.According to the kind of virus serotype and vector aphid thereof, barly yellow dwarf virus is divided into BYDV-PAV strain, BYDV-MAV strain, BYDV-GAV strain, BYDV-GPA strain, CYDV-RPV strain, SGV strain and RMV strain.The BYDV-GAV strain is China's main flow strain.
Good available disease-resistant gene is the prerequisite of breeding for disease resistance.Up to now, not yet find real effectively resistant gene in the wheat primary gene storehouse.A plurality of strains of Thinopyrum intermedium (wheat kindred plant) high resistance or immune barly yellow dwarf virus have obtained using and further investigation as anti-source.Carry 1 resistance to yellow dwarf gene Bdv2 on Thinopyrum intermedium 7Ai# 1 karyomit(e) is long-armed.Scientist utilizes the pairing of China spring ph mutation induction homeologous chromosome and two approach of tissue culture both at home and abroad, and the long-armed end of the 7Ai# 1 karyomit(e) fragment of carrying resistance to yellow dwarf gene Bdv2 is translocated to the long-armed end of chromosome of wheat 7D, successfully seed selection, identify the Wheat-Thinopyrum intermedium T7D7Ai#1L translocation line of a collection of resistance to yellow dwarf, comprise YW642 (H960642), YW443, YW243 and TC5-TC10, (Banks, P., the Larkin such as TC14, P., Bariana, H., Lagudah, E., Appels, R., Waterhouse, P., Brettell, R., Chen, X., Xu, H., Xin, Z., Qian, Y., Zhou, M., Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405; Zhang Zengyan, horse is strong-willed, and hot will is brave etc., and 1998, applying gene group hybridization in situ technique is identified wheat with resistance to yellow dwarf new germ plasm, Scientia Agricultura Sinica, 31 (3): 1-4; Zhang Zengyan, Xin Zhiyong, horse is strong-willed etc., Mapping of a BYDV resistance gene from Thintermedium intermedium in wheat background by molecular markers, Science in China (Series C), 1999,42 (6): 663-668; Zhang Zengyan, Xin Zhiyong, old filial piety etc., the molecular cytogenetics of wheat with resistance to yellow dwarf new lines YW443 is identified, Acta Genetica Sinica, 2000,27 (7): 614～620; Xie Hao, old filial piety, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lipu, horse is strong-willed, Xu Huijun, the seed selection of wheat with resistance to yellow dwarf new lines YW243 and cellular elements biological assay, Acta Agronomica Sinica, 2000,26(6): 687-691; Xin, Z., Zhang, Z., Chen, X., Lin, Z., Ma, Y., Xu, H., Banks, P., and Larkin, P.Development and characterization of common wheat-Thinopyrum ntermedium translocation lines with resistance to barley yellow dwarf virus.Euphytica.2001,119:161 – 165), Thinopyrum intermedium karyomit(e) 7Ai# 1 long-armed (7Ai#1L) the end small segment that resistance to yellow dwarf gene Bdv2 is carried in discovery translocates to the long-armed end of chromosome of wheat 7D, and (horse is strong-willed etc. for Zhang Zengyan, Xin Zhiyong, Mapping of a BYDV resistance gene from Thintermedium intermedium in wheat background by molecular markers, Science in China (Series C), 1999,42 (6): 663-68; Banks, P., Larkin, P., Bariana, H., Lagudah, E., Appels, R., Waterhouse, P., Brettell, R., Chen, X., Xu, H., Xin, Z., Qian, Y., Zhou, M., Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405).Research is found, carry Wheat-Thinopyrum intermedium translocation line YW642, YW443, YW243, the contour Chinese People's Anti-Japanese Military and Political College of the TC14 wheat yellow dwarf virus of resistance to yellow dwarf gene Bdv2, in theory, these translocation lines should be as the resistance germplasm that is easy to utilize in the wheat with resistance to yellow dwarf breeding.Yet how many wheat with resistance to yellow dwarf new variety these translocation lines successfully do not breed, may to exist disadvantageous Linkage drag relevant with the karyomit(e) 7Ai-#1L fragment of carrying resistance to yellow dwarf gene Bdv2.Therefore, in the urgent need to from the materials such as Wheat-Thinopyrum intermedium translocation line YW642 of resistance to yellow dwarf, cloneing and isolateing the important gene of the upper resistance to yellow dwarf of 7Ai-1L, study its resistance effect molecular mechanism, and be applied to genetic engineering breeding, to cultivate efficiently the new variety of wheat of resistance to yellow dwarf, high yield, high-quality.
Summary of the invention
The purpose of this invention is to provide a kind of Genes For Plant Tolerance yellow dwart key protein TiRB and encoding gene and application.
The invention provides a kind of protein, from the Wheat-Thinopyrum intermedium translocation line YW642 of resistance to yellow dwarf, come in fact from Thinopyrum intermedium from root, called after TiRB albumen is following (a) or (b):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant yellow dwarf resistance protein of being derived by sequence 1.
In order to make the protein in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in the sequence 1 forms in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, 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.
The gene of encoding said proteins (TiRB gene) also belongs to protection scope of the present invention.
Described TiRB gene specifically can be following 1) to 5) in arbitrary described dna molecular:
1) sequence 2 is held the dna molecular shown in the 87th to the 2987th Nucleotide from 5 ' in the sequence table;
2) sequence 2 is held the dna molecular shown in the 87th to the 2990th Nucleotide from 5 ' in the sequence table;
3) dna molecular shown in the sequence 2 in the sequence table;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits and the dna molecular of coded plant yellow dwarf resistance-associated protein;
5) with 1) or 2) or 3) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant yellow dwarf resistance-associated protein.
Described stringent condition is in the solution of 0.1 * SSPE(or 0.1 * SSC), 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described TiRB gene all belong to protection scope of the present invention.
Described recombinant plasmid specifically can be and insert the recombinant plasmid (recombinant plasmid first) that described TiRB gene obtains between the multiple clone site of pAHC25 carrier.
The present invention also protects a kind of method of cultivating transgenic plant, is described TiRB gene is imported in the purpose plant, obtains the transgenic plant that yellow dwarf resistance is higher than described purpose plant.Described TiRB gene specifically can import described purpose plant by described recombinant plasmid first.Described purpose plant is monocotyledons or dicotyledons.Described monocotyledons is preferably wheat, such as wheat line " in 8601 ".Described yellow dwart specifically can be the yellow dwart that is caused by yellow dwart virus BYDV-GAV strain.
The present invention also protects a kind of interference carrier (recombinant plasmid second) be used to suppressing described TiRB genetic expression, is TiRB-RIF fragment and TiRB-RIR fragment are inserted the recombinant plasmid that the carrier that sets out obtains; The sequence 3 that described TiRB-RIF fragment is sequence table is from the double chain DNA fragment shown in 5 ' terminal the 1st to 628 Nucleotide, and the sequence 3 that described TiRB-RIR fragment is sequence table is from the double chain DNA fragment shown in 5 ' terminal the 783rd to 1170 Nucleotide.Described interference carrier specifically can be the double chain DNA molecule shown in the sequence 3 of sequence table is inserted the recombinant plasmid that the multiple clone site of pAHC25 carrier obtains.
The present invention also protects a kind of method of cultivating transgenic plant, is that the material that will suppress described TiRB genetic expression imports in the purpose plant, obtains the transgenic plant that yellow dwarf resistance is lower than described purpose plant; Described purpose plant is the plant that contains described TiRB gene.The material of the described TiRB genetic expression of described inhibition specifically can be described interference carrier (recombinant plasmid second).Described purpose plant is monocotyledons or dicotyledons.Described plant specifically can be the Wheat-Thinopyrum intermedium translocation line YW642 of resistance to yellow dwarf.Described yellow dwart specifically can be the yellow dwart that is caused by yellow dwart virus BYDV-GAV strain.
Described TiRB gene is imported wheat, the transgenic wheat of this gene overexpression obviously improves the resistance of yellow dwart, the genetic expression of TiRB described in the anti-disease wheat suppresses then to make this plant forfeiture to the resistance of yellow dwart, illustrates that described TiRB gene is plant yellow dwarf resistance key protein.Plant yellow dwarf resistance key protein TiRB and encoding gene thereof can be used for improving the yellow dwarf resistance of plant, and plant breeding is had great value.The present invention has important theory and practical significance, will play a significant role in the genetic improvement of plant.
Description of drawings
Fig. 1 is the pcr amplification electrophorogram among the embodiment 1; 1:Ti(Thinopyrum intermedium Z1146); 2:YW642; 3: " in 8601 "; M:1Kb plus DNA ladder.
Fig. 2 is the result that evaluation-PCR identifies of TiRB gene overexpression wheat; 1-8: for the identification of T
1For plant; P1: recombinant plasmid pA25-TiRB-O; WT1: acceptor " in 8601 "; The VC:pAHC25 carrier.
Fig. 3 is the result that evaluation-quantitative RT-PCR is analyzed of TiRB gene overexpression wheat.
Fig. 4 is the Disease Resistance Identification-photo of TiRB gene overexpression wheat.
Fig. 5 is inoculation yellow dwart virus relative expression quantity of CP gene in the plant after 35 days among the embodiment 2.
Fig. 6 is the result that evaluation-PCR identifies that the TiRB gene inhibition is expressed plant; 1-11: for the identification of T
1For plant; P2: recombinant plasmid pA25-TiRB-RNAi; WT2: acceptor YW642; H: water.
Fig. 7 is the result that evaluation-quantitative RT-PCR is analyzed that the TiRB gene inhibition is expressed plant.
Fig. 8 is Disease Resistance Identification-photo that the TiRB gene inhibition is expressed plant.
Fig. 9 is inoculation yellow dwart virus relative expression quantity of CP gene in the plant after 35 days among the embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Wheat-Thinopyrum intermedium translocation line YW642(is called for short YW642 or H960642): the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Zhang Zengyan, horse is strong-willed, and hot will is brave etc., and 1998, applying gene group hybridization in situ technique is identified wheat with resistance to yellow dwarf new germ plasm, Scientia Agricultura Sinica, 31 (3): 1-4; Zhang Z, Xin Z, Ma Y, Chen X, Xu Q, Lin Z.1999, Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers.Sci China C Life Sci.42 (6): 663-668.; This translocation line is that the hot will of Institute of Crop Science, Chinese Academy of Agricultural Science in 1991 bravely waits initiative, and 1996 yearbooks such as Zhang Zengyan are fixed.
The pAHC25 carrier (is called again monocotyledon expression vector pAHC25; PAHC25 is transformed by pUC8 and forms, contain 2 expression cassettes, the 1st expression cassette has corn Ubiquitin promotor, Exon, Intron, GUS, Nos terminator, the GUS two ends have restriction enzyme site, and the 2nd expression cassette has corn Ubiquitin promotor, Exon, Intron, Bar, Nos terminator): the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Christensen and Quail, 1996; Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Research, 5,213 – 218.
The pHMW-Adh-Nos carrier: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Reference: Gao Dongyao, the Chillon qin, horse is strong-willed, Xu Zhaoshi, Xu Huijun, Du Lipu, Nie Lina, Li Yanfang, former inferior duckweed, Li Liancheng, Chen Ming, Sun Jinhai, structure and the genetic transformation of wheat VP-1 gene RNA interference expression vector, plant genetic resources journal 2009,10 (1): 9-15.
Thinopyrum intermedium (Ti): available from Institute of Crop Science, Chinese Academy of Agricultural Science's resource Germplasm Bank, be numbered Z1146.Wheat line " in 8601 " (wheat line of sense yellow dwart): available from Institute of Crop Science, Chinese Academy of Agricultural Science.Yellow Dwarf Virus BYDV BYDV-GAV strain: available from Plant Protection institute, Chinese Academy of Agricultral Sciences.Carry the aphid of Yellow Dwarf Virus BYDV BYDV-GAV strain: available from Plant Protection institute, Chinese Academy of Agricultral Sciences.
The discovery of embodiment 1, TiRB gene and clone
The present inventor is in previous work, and the clone has obtained the TiDPK1 gene, by Molecular Detection and the disease-resistant functional analysis of transgenic wheat, has proved that the TiDPK1 gene is 1 important gene in the resistance to yellow dwarf reaction.
With the aminoacid sequence of TiDPK1 albumen as probe, at Phytozome(http: //www.phytozome.net) search false bromegrass (Brachypodium distachyon) Proteome database, with the Bradi3g05520.2 similarity up to 83.1%, be positioned at Bd3:3,901,063-3,904,141 intervals.Hold the 70kb that stretches out in 5 ' of this interval, obtained Bd3:3,831,063-3, the Nucleotide of 974,141 sections.This interval Nucleotide is carried out predictive genes, there is the gene of two coding NBS-ARC structure products in discovery in its 5 ' outer end, 789 amino acid of NBS-ARC1 coding (functional gene of the more general the type of length is short, tentatively is judged to pseudogene) wherein, NBS-ARC2 921 amino acid of encoding.
With false bromegrass NBS-ARC2 sequence (Bradi3g05480.1) blast comparison on the NCBI website, found the Chinese sorghum sequence Sb05g002950 of 1 homology.According to the conserved regions (LRR district) of this two sequences design primer (NBS1018F:5 '-CCGCCTTACACTCCAGAA-3 ' and NBS1018R:5 '-GATGAAGTCTTTGGACACCT-3 '), as template, pcr amplification obtains a specific fragment with the cDNA of resistance to yellow dwarf translocation line YW642.Recovery PCR product also is cloned on the pMDl8T carrier, through bacterium colony PCR, selects positive colony and carries out sequencing analysis, obtains one section sequence (886bp) of TiRB gene.According to the sequences Design 3 ' in the conservative territory of this section TiRB that obtains-RACE and 5 '-RACE primer, adopt 5 ' RACE System for Rapid Amplification of cDNA Ends of the TaKaRa 3 '-Full RACE Core Set Ver.2.0 of company and Invitrogen company, Version 2.0 test kits are from TI(Z1146), separate TiRB 3 ' end and 5 ' terminal sequence the YW642cDNA.On the basis of homology conserved sequence, take turns 3 ' RACE pcr amplification by 1, sequencing analysis finds that the sequence that obtains extended 694bp, comprised complete ployA structure to 3 ' end.On this splicing basis, take turns 5 ' RACE, sequencing analysis by 4, sequence has been extended 1740bp to 5 ' end, the TiRB full length cDNA sequence (3319bp, the sequence 2 of seeing sequence table) that splicing obtains.
According to TiRB full length cDNA sequence design 2 pairs of Auele Specific Primers (43U/3241L and 57U/3183L).Adopt nested PCR method, induce respectively resistance to yellow dwarf translocation line YW642 and the Ti(Z1146 of processing take yellow dwart) cDNA carry out pcr amplification as template, all successfully obtained the PCR product of expection size.And do not have amplification to obtain expecting the PCR product (see figure 1) of size among the cDNA of susceptible material " in 8601 ".
The pcr amplification product of the expection size that resistance to yellow dwarf translocation line YW642 and Ti are obtained reclaims respectively and checks order, sequencing result such as the sequence 2 of sequence table from (3189bp) shown in the of 5 ' the terminal the 56th to 3244.
Protein called after TiRB albumen with shown in the sequence 1 of sequence table is comprised of 967 amino-acid residues, has CC, NB-ARC, LRR structure, and its molecular weight is that 109.9KDa, iso-electric point are 7.51.The similarity of TiRB albumen and Bradi3g05480.1 and Sb05g002950 proteins encoded is respectively 53.39% and 49.54%.
Be the TiRB gene with the unnamed gene of coding TiRB albumen, its open reading frame such as the sequence 2 of sequence table from (2904bp) shown in 5 ' terminal the 87th to the 2990th Nucleotide.The similarity of TiRB gene and Bradi3g05480.1 and Sb05g002950 nucleotide sequence is respectively 58.59% and 64.61%.
The analysis of the acquisition of embodiment 2, TiRB gene overexpression wheat and TiRB gene resistance to yellow dwarf function
One, the structure of recombinant expression vector
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, take the double chain DNA molecule of step 1 as template, the primer that forms with pO-TiRBF and pO-TiRBR obtains pcr amplification product to carrying out pcr amplification.
PO-TiRBF:5 '-at
CCCGGGThe restriction endonuclease recognition sequence of atggaaggcagcgacccac-3'(underscore mark Restriction enzyme Sma I);
pO-TiRBR：5’-ctaggataggatatcgat-3’。
3, cut the pcr amplification product of step 2 with restriction endonuclease sma I enzyme, reclaim enzyme and cut product.
4, produce sticking end with restriction endonuclease sma I() and restriction enzyme EcoICR I (flat terminal) double digestion pAHC25 carrier, carrier framework (about 7812bp) reclaimed.
5, the enzyme of step 3 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pA25-TiRB-O.According to sequencing result, it is as follows that recombinant plasmid pA25-TiRB-O is carried out structrual description: inserted the sequence 2 of sequence table from the double chain DNA molecule shown in 5 ' terminal the 87th to the 2990th Nucleotide between the Sma of pAHC25 carrier I and EcoICR I restriction enzyme site.Among the recombinant plasmid pA25-TiRB-O, TiRB genetic expression is subjected to Ubi promotor (composition promotor) control.Also have 1 Bar expression casette that is subjected to the control of Ubi promotor among the recombinant plasmid pA25-TiRB-O, be used for follow-up two propylamine phosphines (Bialaphos) screening.
Two, the acquisition of TiRB gene overexpression wheat
1, with the rataria callus of wheat line " in the 8601 " acceptor as the particle gun bombardment, with particle gun recombinant plasmid pA25-TiRB-O is bombarded callus, concrete steps are as follows:
(1) the rataria callus of 1600 wheat lines " in 8601 " is permeated substratum (SD2 substratum+0.2mol/L N.F,USP MANNITOL+0.2mol/L sorbyl alcohol) at height and carry out osmotic treated 4-6h(25 ℃, the dark cultivation); In the above-mentioned processing, callus is placed in the about 2.5cm scope of culture dish mid-diameter, and every culture dish is placed 40 callus.
(2) with recombinant plasmid pA25-TiRB-O parcel diameter 110 μ m bronzes, adopt PDS-1000/He particle gun Bio-Red company to produce), the split film of selection 1100Psi, load sample is bombarded apart from target material 6cm.
(3) callus after the bombardment continues to cultivate 16-18h(25 ℃ at former substratum, the dark cultivation).
2, the callus of completing steps 1 is transferred to the SD2 substratum (MS substratum+1mg/L VB1+150mg/L asparagus fern door acid amides+2mg/L 2,4-D) on, 2 weeks of renewal cultivation (25 ℃, dark cultivate).
3, the callus of completing steps 2 is transferred on the differentiation screening culture medium (the two propylamine phosphines of 1/2MS substratum+1mg/L a-naphthylacetic acid+1mg/L kinetin+4mg/L) 24-26 ℃ of illumination cultivation 14-21d.
4, the Calli Differentiation seedling that step 3 is obtained is transferred in the growth screening culture medium (1/2MS substratum+2mg/L two propylamine phosphines), 24-26 ℃ illumination cultivation 14-28 days, obtained the regeneration plant that 100 strains are taken root.
5, the regeneration plant that step 4 is obtained is transferred on the strong seedling culture base (1/2MS substratum+0.5mg/L a-naphthylacetic acid), about 21 days of 24-26 ℃ of illumination cultivation.
6, with the plantlet of transplant of height of seedling 7-8cm and well developed root system in the step 5 to flowerpot, be positioned in the greenhouse and cultivated for 3 weeks, have 95 strain plant to survive (T
0Generation).
7, with T
0For the plant selfing and gather in the crops seed (T
1For seed), with T
1Be T for cultivating seeds
1For plant.
8, PCR identifies
The T that respectively step 7 is obtained
0For plant and T
1Carry out following evaluation for plant: in 4 leaf phases, every strain plant is got 1 blade, extracts genomic dna, and the primer that forms with UBIPF and TiRB-TR take genomic dna as template is identified carrying out PCR; With recombinant plasmid pAHC25-TiRB-O as positive control (representing with P1), with acceptor " in 8601 " (representing with WT1) and T
0In generation, turn empty carrier plant (preparation method sees step 3, represents with VC) as negative control.
UBIPF:5’-gctctgccttcatacgcta-3’；
TiRB-TR:5’-aggctccgtatgtcttcgtt-3’。
The results are shown in Figure 2, the purpose band of the about 579bp of arrow mark.
For a certain T
0For plant, if this plant and T thereof
1Identify all positively for plant PCR, this plant is transgenosis " in the 8601 " plant of the TiRB gene overexpression that isozygotys, and this plant and self progeny thereof are a TiRB gene overexpression strain.Obtain altogether 6 TiRB gene overexpression strains (respectively called after 92-3 strain, 196-3 strain, 200-1 strain, 201-1 strain, 203-3 strain and 205-4 strain).
9, quantitative RT-PCR analysis
TiRB-QF:5’-cgcctacagaacgacgagc-3’；
TiRB-QR:5’-ctggtcaacggcgtcctc-3。
Actin-QF:5′-cactggaatggtcaaggctg-3′；
Actin-QR:5′-ctccatgtcatcccagttg-3′。
The relative expression quantity of TiRB gene is seen Fig. 3 in each plant.Among Fig. 3,92-3-3 is that a strain, the 196-3-3 in the 92-3 strain is that a strain, 200-1-5 in the 196-3 strain is that a strain, 201-1-4 in the 200-1 strain is that a strain, 203-3-8 in the 201-1 strain is that a strain, 205-4-4 in the 203-3 strain is the strain in the 205-4 strain.Compare with acceptor " in 8601 ", the relative expression quantity of TiRB gene is significantly increased in 6 TiRB gene overexpression strains, and the relative expression quantity of TiRB gene is higher than Wheat-Thinopyrum intermedium translocation line YW642 in the part strain (such as 200-1).
Three, turn the acquisition of empty carrier wheat
Replace recombinant plasmid pAHC25-TiRB-O to carry out step 2 with the pAHC25 carrier, obtain turning the empty carrier plant.
Four, the Disease Resistance Identification of TiRB gene overexpression wheat
Respectively with the T of each TiRB gene overexpression strain (92-3 strain, 196-3 strain, 200-1 strain, 201-1 strain, 203-3 strain and 205-4 strain)
1For plant, the disease-resistant positive control of Wheat-Thinopyrum intermedium translocation line YW642(), acceptor " in 8601 " (negative control), turn the T of empty carrier plant
1Carry out following Disease Resistance Identification for plant, each strain 20 strain: in Seedling Inoculation yellow dwart virus BYDV-GAV strain, be that the aphid that yellow dwart virus BYDV-GAV strain is carried in tweezer places on the wheat plant, 10 aphids of every strain, continue to observe the morbidity sign of plant, inoculate and take pictures after 30 days, inoculate after 35 days according to the plant phenotype and carry out the disease resistance classification and detect the relative content of BYDV in the plant by Q-RT-PCR.
The domestic standard of yellow stunt of wheat severity is adopted in the disease resistance classification, i.e. the standard of IT (seeing Table 2), and reference: " Li Guangbo, Ceng Shimai, Li Zhenqi chief editor. the sick Chinese caterpillar fungus plague of rats comprehensive regulation [M] of wheat. Beijing: Chinese agriculture science and technology press, 1990 ".
Table 2 yellow stunt of wheat Seriousness gradation standard
| Rank (level value) | Domestic standard (11 grades) |
| 0 | |
| 1 | The yellow of |
| 2 | The lower 1 leaf yellow of |
| 3 | The lower 2 leaf yellows of |
| 4 | Boot leaf yellow 1/4, the lower 1 leaf yellow of |
| 5 | Boot leaf yellow 1/4, the lower 2 leaf yellows of |
| 6 | The boot leaf yellow |
| 7 | The boot leaf yellow, the lower 1 leaf yellow of |
| 8 | The lower 2 leaf yellows of boot leaf and boot leaf |
| 9 | Plant is downgraded, but can |
| 10 | Plant is downgraded significantly, does not ear |
Inoculation yellow dwart virus BYDV-GAV strain is after 25 days, acceptor " in 8601 " and turn the empty carrier plant and begin to manifest morbidity and characterize, and sequela characterized obviously in 35 days.Each TiRB gene overexpression strain and Wheat-Thinopyrum intermedium translocation line YW642 all show the high resistance performance to yellow dwart virus BYDV-GAV strain.The photo of part wheat strain inoculation yellow dwart virus BYDV-GAV strain after 30 days seen Fig. 4.The plant phenotype of other transgenic line is with the 196-3 strain.Turn the phenotype isoacceptor " in 8601 " of empty carrier plant.
Inoculation yellow dwart virus BYDV-GAV strain is after 35 days: the average sick level of 92-3 strain is 0.25; The average sick level of 196-3 strain is 0; The average sick level of 200-1 strain is 0; The average sick level of 201-1 strain is 0; The average sick level of 203-3 strain is 0.5; The average sick level of 205-4 strain is 0; The average sick level of Wheat-Thinopyrum intermedium translocation line YW642 is 0; The average sick level of acceptor " in 8601 " is 7.5; The average sick level that turns the empty carrier plant is 7.2.
Inoculation yellow dwart virus BYDV-GAV strain is after 35 days, total RNA and the reverse transcription of extracting the wheat plant blade are cDNA, with cDNA as template, use the Auele Specific Primer for the coat protein gene (CP gene) of yellow dwart virus BYDV-GAV strain that (being comprised of GAV-CP-U and GAV-CP-L) carried out pcr amplification, analyze the relative content of BYDV-GAV.(adopt primer that 18S rRNA-QF and 18S rRNA-QR form pair with 18S rRNA gene as confidential reference items; The ratio of CP gene and 18S rRNA gene expression amount as standard value, as 1, is calculated the relative expression quantity of CP gene in other plant with the standard value of Wheat-Thinopyrum intermedium translocation line YW642).
GAV-CP-U:5′-CAGGCAGGACTGAGGTATT-3′；
GAV-CP-L:5′-：GTTGCTGATTTTGAGAGGG-3′。
18S rRNA-QF：5'-GTGACGGGTGACGGAGAATT-3'；
18S rRNA-QR：5’-GACACTAATGCGCCCGGTAT-3’。
The relative expression quantity of CP gene the results are shown in Figure 5.Among Fig. 5,92-3-3 is that a strain, the 196-3-3 in the 92-3 strain is that a strain, 200-1-5 in the 196-3 strain is that a strain, 201-1-4 in the 200-1 strain is that a strain, 203-3-8 in the 201-1 strain is that a strain, 205-4-4 in the 203-3 strain is the strain in the 205-4 strain.Compare with acceptor " in 8601 ", the CP gene content significantly reduces (namely the content of yellow dwart virus BYDV-GAV strain significantly reduces) in each TiRB gene overexpression strain, some strain is (such as the 196-3 strain, the 200-1 strain, 201-1 strain etc.) basically identical with Wheat-Thinopyrum intermedium translocation line YW642 to the reaction (wherein viral level and resistance level) of BYDV, illustrate that the TiRB gene is the resistance to yellow dwarf key gene, it is expressed in susceptible wheat and can suppress infecting of yellow dwart virus BYDV-GAV strain, breeding and motion give transgenic wheat very high disease resistance.
One, the structure of rna interference vector
As the RNA interference fragment, forward and reverse insertion pHMW-Adh-Nos carrier forms hairpin structure with TiRB gene 3 ' end (comprising the part of ORF 3 ' end and the part of 3 ' UTR).
ZF1:5 '-ga
Gaattc CccgggTtggaggagacaaaggtga-3 ' (underscore mark EcoR I restriction endonuclease recognition sequence and Sma I restriction enzyme site recognition sequence);
ZF2:5 '-gt
Aagctt GagctcTgtgccgtcattcatggt-3 ' (underscore mark Hind III restriction endonuclease recognition sequence and Sac I restriction endonuclease recognition sequence);
ZR:5 '-gc
AgatctTccattttcatttaacgtcct-3 ' (underscore mark Bgl II restriction endonuclease recognition sequence).
1, take the cDNA of Wheat-Thinopyrum intermedium translocation line YW642 as template, adopt primer that ZF2 and ZR form to carrying out pcr amplification, obtain pcr amplification product (TiRB-RIR fragment).
2, with the pcr amplification product (TiRB-RIR fragment) of restriction enzyme BglII and HindIII double digestion step 1, reclaim enzyme and cut product.
3, with restriction enzyme Bgl II and Hind III double digestion pHMW-Adh-Nos carrier, reclaim carrier framework (about 4388bp).
4, the enzyme of step 2 is cut the carrier framework connection that product is connected with step, obtained middle interstitial granules pHMW-Adh-Nos-TiRB-RIR.
5, take the cDNA of Wheat-Thinopyrum intermedium translocation line YW642 as template, adopt primer that ZF1 and ZR form to carrying out pcr amplification, obtain pcr amplification product (TiRB-RIF fragment).
6, with the pcr amplification product (TiRB-RIF fragment) of restriction enzyme EcoR I and Bgl II double digestion step 5, reclaim enzyme and cut product.
7, the middle interstitial granules pHMW-Adh-Nos-TiRB-RIR that obtains with restriction enzyme EcoRI and BamH I double digestion step 4 reclaims carrier framework (about 4436bp has removed the Nos fragment in the middle interstitial granules).
8, the enzyme of step 6 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pTiRB-RIF-HMW-Adh-TiRB-RIR.According to sequencing result, it is as follows that recombinant plasmid pTiRB-RIF-HMW-Adh-TiRB-RIR is carried out structrual description: inserted TiRB-RIF fragment (628bp between the EcoRI of pHMW-Adh-Nos carrier and BamH I restriction enzyme site, such as the sequence 3 of sequence table from shown in 5 ' terminal the 1st to 628 Nucleotide), inserted TiRB-RIR fragment (388bp, such as the sequence 3 of sequence table from shown in 5 ' terminal the 783rd to 1170 Nucleotide) between Bgl II and the Hind III restriction enzyme site.
9, with restriction endonuclease sma I and Sac I double digestion recombinant plasmid pTiRB-RIF-HMW-Adh-TiRB-RIR, reclaim the enzyme of about 1176bp (containing restriction enzyme site) and cut product.
10, with restriction endonuclease sma I and Sac I double digestion pAHC25 carrier, reclaim carrier framework (about 7812bp).
11, the enzyme of step 9 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pA25-TiRB-RNAi(RNA interference carrier).According to sequencing result, it is as follows that rna interference vector is carried out structrual description: inserted the double chain DNA molecule shown in the sequence 3 of sequence table between the Sma of pAHC25 carrier I and Sac I restriction enzyme site.
Two, acquisition and the Molecular Identification thereof of TiRB gene inhibition express transgenic plant
The recombinant plasmid pA25-TiRB-RNAi that makes up with step 1 replaces recombinant plasmid pA25-TiRB-O, and as transformation receptor, other obtain T with the step 2 of embodiment 2 to replace wheat line " in 8601 " with Wheat-Thinopyrum intermedium translocation line YW642 simultaneously
0For plant and T
1For plant.
1, PCR identifies
Respectively with T
0For plant and T
1Carry out following evaluation for plant: in 4 leaf phases, every strain plant is got 1 blade, extracts genomic dna, take genomic dna as template, identifies carrying out PCR with the primer that UBI 1910F and RIR1 form; With recombinant plasmid pA25-TiRB-RNAi as positive control (representing with P2), with acceptor Wheat-Thinopyrum intermedium translocation line YW642(WT2) as negative control, with water as blank (representing with H).
UBI1910F:5’-gctctgccttcatacgcta-3’；
RIR1:5’-gtatctaatcagccatcccatt-3’。
The results are shown in Figure 6, the purpose band is about 869bp.
For a certain T
0For plant, if this plant and T thereof
1All positive for plant PCR evaluation, this plant is a transgenosis YW642 plant that the TiRB gene inhibition of isozygotying is expressed, and this plant and self progeny thereof are that a TiRB gene inhibition is expressed strain.
Obtain altogether 7 TiRB gene inhibition and express strain (respectively called after 29-19 strain, 30-1 strain, 33-1 strain, 57-2 strain, 60-2 strain, 61-2 strain and 68-2 strain).
2, quantitative RT-PCR analysis
TiRB-QF:5’-cgcctacagaacgacgagc-3’；
TiRB-QR:5’-ctggtcaacggcgtcctc-3。
Actin-QF:5′-cactggaatggtcaaggctg-3′；
Actin-QR:5′-ctccatgtcatcccagttg-3′。
The relative expression quantity of TiRB gene is seen Fig. 7 in each plant.Among Fig. 7,29-19-3 is that a strain, the 31-1-4 in the 29-19 strain is that a strain, 33-1-6 in the 31-1 strain is that a strain, 57-2-2 in the 33-1 strain is that a strain, 60-2-1 in the 57-2 strain is that a strain, 61-2-20 in the 60-2 strain is that a strain, 68-2-8 in the 61-2 strain is the strain in the 68-2 strain.(Wheat-Thinopyrum intermedium translocation line YW642) compares with acceptor, the relative expression quantity that 7 TiRB gene inhibition are expressed TiRB gene in the strain all significantly reduces, and the relative expression quantity of TiRB gene approaches the wheat line " in 8601 " of sense yellow dwart in the part strain.
Three, turn the acquisition of empty carrier wheat
Replace rna interference vector to carry out step 2 with the pAHC25 carrier, obtain turning the empty carrier plant.
Four, the TiRB gene inhibition is expressed the Disease Resistance Identification of plant
Respectively each TiRB gene inhibition is expressed the T of strain (29-19 strain, 30-1 strain, 33-1 strain, 57-2 strain, 60-2 strain, 61-2 strain and 68-2 strain)
1For plant, the disease-resistant positive control of resistance to yellow dwarf translocation line YW642(), wheat line " in 8601 " (negative control), turn the T of empty carrier plant
1Carry out following Disease Resistance Identification for plant, each strain 20 strain: in Seedling Inoculation yellow dwart virus BYDV-GAV strain, be that the aphid that yellow dwart virus BYDV-GAV strain is carried in tweezer places on the wheat plant, 10 aphids of every strain, continue to observe the morbidity sign of plant, inoculate and take pictures after 30 days, carry out the disease resistance classification, detect the relative content of BYDV in the plant by Q-RT-PCR according to the plant phenotype after 35 days.
Concrete experimental technique is with the step 4 of embodiment 2.
Each TiRB gene inhibition is expressed strain and is all showed typical yellow dwart and characterize, and severity and wheat line " in 8601 " are close, turn the high resistance to yellow dwarf of empty carrier plant and acceptor (Wheat-Thinopyrum intermedium translocation line YW642).The photo of part wheat strain inoculation yellow dwart virus BYDV-GAV strain after 30 days seen Fig. 8.The plant phenotype of other transgenic line is with the 29-19 strain.Turn the phenotype isoacceptor (Wheat-Thinopyrum intermedium translocation line YW642) of empty carrier plant.
Inoculation yellow dwart virus BYDV-GAV strain is after 35 days: the average sick level of 29-19 strain is 7.1; The average sick level of 30-1 strain is 6.3; The average sick level of 33-1 strain is 6.8; The average sick level of 57-2 strain is 7.3; The average sick level of 60-2 strain is 7.1; The average sick level of 61-2 strain is 6.5; The average sick level of 68-2 strain is 7.2; The average sick level of acceptor (Wheat-Thinopyrum intermedium translocation line YW642) is 0; The average sick level of wheat line " in 8601 " is 7.6; The average sick level that turns the empty carrier plant is 0.
The relative expression quantity of CP gene the results are shown in Figure 9.Among Fig. 9,29-19-3 is that a strain, the 31-1-4 in the 29-19 strain is that a strain, 33-1-6 in the 31-1 strain is that a strain, 57-2-2 in the 33-1 strain is that a strain, 60-2-1 in the 57-2 strain is that a strain, 61-2-20 in the 60-2 strain is that a strain, 68-2-8 in the 61-2 strain is the strain in the 68-2 strain.(Wheat-Thinopyrum intermedium translocation line YW642) compares with acceptor, each TiRB gene inhibition expresses that the CP gene content significantly raises in strain (namely the content of yellow dwart virus BYDV-GAV strain significantly raises), some strain is (such as the 29-19 strain, the 57-2 strain, the 60-2 strain, 68-2 strain etc.) basically identical to the wheat line of the reaction of BYDV and sense yellow dwart " in 8601 ", illustrate by importing TiRB gene interference fragment, the expression of TiRB gene is effectively suppressed in the acceptor (Wheat-Thinopyrum intermedium translocation line YW642), thereby lost BYDV-GAV is infected, breeding, the control of motion, cause its body inner virus content to increase, performance is susceptible to yellow dwart virus BYDV-GAV strain.
The presentation of results TiRB gene of embodiment 2 and embodiment 3 is the key gene of anti-yellow stunt of wheat.
Claims (10)
1. protein is following (a) or (b):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant yellow dwarf resistance protein of being derived by sequence 1.
2. the gene of coding claim 1 described albumen.
3. gene as claimed in claim 2, it is characterized in that: described gene is following 1) to 5) in arbitrary described dna molecular:
1) sequence 2 is held the dna molecular shown in the 87th to the 2987th Nucleotide from 5 ' in the sequence table;
2) sequence 2 is held the dna molecular shown in the 87th to the 2990th Nucleotide from 5 ' in the sequence table;
3) dna molecular shown in the sequence 2 in the sequence table;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits and the dna molecular of coded plant yellow dwarf resistance-associated protein;
5) with 1) or 2) or 3) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant yellow dwarf resistance-associated protein.
4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described genes.
5. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that yellow dwarf resistance is higher than described purpose plant.
6. method as claimed in claim 5, it is characterized in that: described purpose plant is monocotyledons or dicotyledons; Described monocotyledons is preferably wheat.
7. a method of cultivating transgenic plant is that the material that will suppress claim 2 or 3 described genetic expressions imports in the purpose plant, obtains the transgenic plant that yellow dwarf resistance is lower than described purpose plant; Described purpose plant is the plant that contains claim 2 or 3 described genes.
8. such as arbitrary described method in the claim 5 to 7, it is characterized in that: described yellow dwart is caused by yellow dwart virus BYDV-GAV strain.
9. being used for suppressing the interference carrier of genetic expression shown in the claim 2 or 3, is that TiRB-RIF fragment and TiRB-RIR fragment are inserted the recombinant plasmid that the carrier that sets out obtains; The sequence 3 that described TiRB-RIF fragment is sequence table is from the double chain DNA fragment shown in 5 ' terminal the 1st to 628 Nucleotide, and the sequence 3 that described TiRB-RIR fragment is sequence table is from the double chain DNA fragment shown in 5 ' terminal the 783rd to 1170 Nucleotide.
10. interference carrier as claimed in claim 9 is characterized in that: the recombinant plasmid that described interference carrier obtains for the multiple clone site of the double chain DNA molecule shown in the sequence 3 of sequence table being inserted the pAHC25 carrier.
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| CN1570102A (en) * | 2003-07-25 | 2005-01-26 | 中国农业科学院作物育种栽培研究所 | Wheat yellow dwarf disease candidate resistance gene and its coded protein and uses |
| US7569389B2 (en) * | 2004-09-30 | 2009-08-04 | Ceres, Inc. | Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics |
| CN101704882A (en) * | 2009-11-16 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein, coding gene and application thereof |
| CN101704883A (en) * | 2009-11-26 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof |
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| CN1570102A (en) * | 2003-07-25 | 2005-01-26 | 中国农业科学院作物育种栽培研究所 | Wheat yellow dwarf disease candidate resistance gene and its coded protein and uses |
| US7569389B2 (en) * | 2004-09-30 | 2009-08-04 | Ceres, Inc. | Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics |
| CN101704882A (en) * | 2009-11-16 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein, coding gene and application thereof |
| CN101704883A (en) * | 2009-11-26 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof |
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