WO2012006866A1 - Protéine tistki résistante à la maladie du nanisme jaune touchant les végétaux, gène codant pour celle-ci et application de ceux-ci - Google Patents

Protéine tistki résistante à la maladie du nanisme jaune touchant les végétaux, gène codant pour celle-ci et application de ceux-ci Download PDF

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WO2012006866A1
WO2012006866A1 PCT/CN2011/001120 CN2011001120W WO2012006866A1 WO 2012006866 A1 WO2012006866 A1 WO 2012006866A1 CN 2011001120 W CN2011001120 W CN 2011001120W WO 2012006866 A1 WO2012006866 A1 WO 2012006866A1
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plant
gene
wheat
yellow dwarf
sequence
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PCT/CN2011/001120
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Chinese (zh)
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张增艳
陈亮
汪信东
徐惠君
辛志勇
杜丽璞
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中国农业科学院作物科学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

Definitions

  • Plant yellow dwarf resistance protein TISTK1 Plant yellow dwarf resistance protein TISTK1 and its coding gene and application
  • the invention relates to a plant yellow dwarf resistance key protein TiSTK1 and a coding gene thereof and application thereof.
  • Wheat yellow dwarf disease is an important disease of wheat caused by barley yellow dwarf virus (BYDV). Once infected, wheat has no cure, which causes wheat yield reduction and quality decline. Therefore, yellow dwarf disease is also called "wheat cancer". Yellow dwarf disease occurs in all wheat regions of the world. In 1978, due to the outbreak of wheat yellow dwarf disease in the United States, wheat production was reduced by 60% to 80%. In 1988, German winter wheat was reduced by 40% due to the prevalence of yellow dwarf disease. Every year Australia suffers from wheat yellow dwarf disease, and wheat losses are about $30 million. Yellow dwarf disease occurs in more than 40 countries including New Zealand, Argentina, Turkey, Tunisia and Hungary. In the northwestern part of China, parts of north China and northeast China, yellow dwarf disease occurred in 1966, 1970, 1973, 1978, 1980, 1987, and 1999.
  • BYDV barley yellow dwarf virus
  • the barley yellow dwarf virus is a positive single-stranded RNA virus that relies on mediator aphids to spread among host plants and has a wide range of hosts that can infect almost all members of the grass family, including important cereal crops such as wheat, barley, and oats.
  • the barley yellow dwarf virus is divided into BYDV-PAV strain, BYDV-MAV strain, BYDV-GAV strain, CYDV-RPV strain, GPV strain, SGV strain. And RMV strains.
  • Zhou Guanghe et al (Zhou Guanghe, Zhang Shuxiang, Qian Youting, Identification and Application of Four Strains of Wheat Yellow Dwarf Virus, Chinese Agricultural Sciences, 1987, 20: 7 ⁇ 12) identified four wheat yellow dwarf virus strains prevalent in China , GAV, GPV, PAV and RMV, respectively, wherein the GPV strain is a unique strain type of wheat in China, and the BYDV-GAV strain is the mainstream strain of wheat yellow dwarf virus in China in recent years, and the serology of BYDV-MAV Very similar, the main difference is that there is one more mediator than the latter.
  • translocation lines have not successfully bred many new wheat varieties resistant to yellow dwarf disease, which may be related to the unfavorable linkage of the chromosome 7Ai-#lL fragment carrying the anti-yellow dwarf gene. Therefore, it is urgent to isolate and clone the important genes against yellow dwarf disease on 7Ai-lL from the wheat-intermediate buckwheat translocation line YW642, which is resistant to yellow dwarf disease, and study its molecular mechanism of resistance, and apply it to genetic engineering breeding. In order to efficiently cultivate new varieties of wheat resistant to yellow dwarf disease, high yield and high quality.
  • TiSTKl is derived from the intermediate ryegrass Thinopyrum intermedium) and is as follows (a) or (b):
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • the protein in (b) above can be synthesized synthetically, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in (b) above may be deleted by one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and/or one or several base pairs may be missed.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein (S3 ⁇ 4 gene) is also within the scope of the present invention.
  • the gene may be the DNA molecule of any one of the following 1) to 5):
  • the above stringent conditions may be to hybridize and wash the membrane at 65 ° C in a solution of 0.1 X SSPE (or 0.1 X SSC), 0.1% SDS.
  • a recombinant expression vector, expression cassette, transgenic cell line or recombinant strain containing the gene is within the scope of the present invention.
  • a recombinant expression vector containing the gene can be constructed using an existing plant expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment and the like.
  • the plant expression vector may further comprise a 3' untranslated region of the foreign gene, i.e., comprising a polyadenylation signal and any other fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal directs the addition of polyadenylation to the 3' end of the mRNA precursor.
  • any of the enhanced promoters or constitutive promoters may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters;
  • an enhancer including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be an ATG start codon or a contiguous region start codon, etc., but The same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.
  • the source of the translational control signal and the initiation codon is extensive, either natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound that can be expressed in plants, and a resistant antibiotic marker. Or anti-chemical reagents, etc. From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • the recombinant expression vector may specifically be obtained by inserting the gene into the multiple cloning site of the vector PAHC25. Recombinant plasmid.
  • Primer pairs that amplify the full length of the gene or any fragment thereof are also within the scope of the invention.
  • the primer pair may specifically be a primer pair consisting of the DNA shown in SEQ ID NO: 7 of the Sequence Listing and the DNA shown in SEQ ID NO: 8 of the Sequence Listing.
  • the present invention also contemplates a method of cultivating a transgenic plant by introducing the gene into a plant of interest to obtain a transgenic plant having a yellow dwarf resistance higher than the plant of interest.
  • the gene can be specifically introduced into the plant of interest through the recombinant expression vector.
  • An expression vector carrying the gene can be transformed into a plant cell or tissue by using conventional methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, gene gun, and the like, and The transformed plant tissue is grown into plants.
  • the plant of interest may be either a monocot or a dicot.
  • the monocotyledonous plant may specifically be wheat (e.g., wheat variety 8601, stone 4185, yangmai 18, etc.).
  • the yellow dwarf disease may specifically be caused by a BYDV-GAV strain or a BYDV-PAV strain (or MAV, CYDV-RPV, CPV strain, etc.).
  • the present invention also contemplates an interfering RNA for inhibiting expression of said protein, the nucleotide sequence of which is shown in SEQ ID NO:9 of the Sequence Listing.
  • the DNA (specific DNA fragment) encoding the interfering RNA is also within the scope of the present invention.
  • the encoding of the interfering RNA may include a fragment A and a fragment B; the fragment A is as shown in the sequence 2 of the sequence listing from nucleotides 2 to 493 at the 5' end; Reciprocally complementary to the DNA fragment A.
  • the specific encoding of the interfering RNA can be as shown in the sequence 5 of the sequence listing.
  • a recombinant plasmid (inhibiting expression vector) containing the specific fragment is also within the scope of the present invention.
  • the recombinant plasmid may be a recombinant plasmid obtained by introducing the cloning site of the vector pAHC25 as shown in the sequence 5 of the sequence listing.
  • the present invention also protects a method for cultivating a transgenic plant by introducing the inhibitory expression vector into a plant of interest to obtain a transgenic plant having yellow dwarf resistance lower than the plant of interest; the plant of interest is a plant containing the gene .
  • the invention also protects a method for assisting in identifying a plant carrying the gene, comprising the steps of: PCR amplification using the primer pair of the genomic DNA of the plant to be tested; if a 739 bp fragment is obtained, the test is to be tested A plant is a candidate for a plant carrying the gene; if no 739 bp is obtained
  • a DNA fragment, the plant to be tested is a candidate plant that does not carry the gene.
  • the invention also protects another method for assisting in identifying a plant carrying the gene, comprising the steps of: PCR amplification using the primer pair as a template; if a 235 bp fragment is obtained, the test is to be tested The plant is a candidate plant carrying the gene; if a 235 bp DNA fragment is not obtained, the plant to be tested is a candidate plant that does not carry the gene.
  • the plant to be tested may be an intermediate buckwheat grass, a diploid ligation line against yellow dwarf disease, a double-end system 7Ai#lL against yellow dwarf disease, and a translocation line YW642 resistant to yellow dwarf disease.
  • Any of the above methods can be used to assist in the identification of plants against yellow dwarf disease; the plant carrying the gene is a candidate anti-yellow dwarf plant, and the plant not carrying the gene is a candidate yellow-dwarf plant.
  • the protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain, the primer pair, the RNA and the DNA encoding the same, the recombinant plasmid, or the method can be used for the plant Breeding.
  • the gene encoding the gene provided by the present invention is specifically expressed in wheat against yellow dwarf disease (such as YW642), and is up-regulated by BYDV; TiSTK1 is a membrane protein; TiSTKl is silenced (RNA interference) in YW642 which is anti-yellow dwarf disease. Increased BYDV concentration and increased susceptibility index; over-expressed transgenic wheat increased resistance to yellow dwarf disease and decreased BYDV concentration; indicating that ⁇ 3 ⁇ 43 ⁇ 4 gene is a key gene against yellow dwarf disease.
  • Figure 1 shows PCR amplification and SDS-PAGE coagulation of the anti-yellow dwarf material (Ti, DT7AI#1L, YW642) and the yellow-sensitive dwarf wheat material (CS, Zhong8601, YW641S) carrying no ⁇ 2. Gel electrophoresis analysis.
  • Figure 2 shows the PCR identification of 1 ⁇ generation TiSTK1 overexpressing transgenic wheat; M: lOObp DNA ladder; P: positive control; WT: negative control (receptor wheat Zhong8601); 1-15: 3 ⁇ 4 overexpressing different plants of transgenic wheat.
  • Figure 3 is a Southern hybridization test of 1 ⁇ generation ⁇ 73 ⁇ 4 overexpressing transgenic wheat; 1, 4, 31, 40, 41, 44, 45 are 1 ⁇ generation ⁇ 73 ⁇ 4 overexpressing different lines of transgenic wheat; Zhon g 8601 is Receptor wheat (infected).
  • Figure 4 is a quantitative analysis of the expression of the S73 ⁇ 4 gene in transgenic wheat by fluorescence quantitative RT-PCR; 1-1 represents 1 strain in strain 1; 4-3 represents 1 strain in strain 4; -5, 40-9, 40-14, 40-16 represent 4 strains in strain 40; 41-14 represents 1 strain in strain 41; 44_2 represents 1 strain in strain 44; 45-4 represents One strain of strain 45; Zhon g 8601 is the recipient wheat (sense), YW642, and TC14 is the resistant wheat control.
  • Figure 5 shows the resistance of yellow dwarf disease in 8601 (infected) in 1 ⁇ generation ⁇ 3 ⁇ 4 overexpressing transgenic wheat and recipient wheat.
  • Figure 6 is a quantitative quantitative analysis of BYDV-gene expression in 1T generation TiSTK1 overexpressing transgenic wheat by fluorescence quantitative RT-PCR; 1-1 represents 1 strain in strain 1; 4-3 represents 1 strain in strain 4; 40 -5, 40-9, 40-14, 40-16 represent 4 strains in strain 40; 41-14 represents 1 strain in strain 41; 44-2 represents 1 strain in strain 44; 45- 4 represents one strain of strain 45; Zhon g 8601 is a recipient wheat (infected), and YW642 and TC14 are resistant wheat controls.
  • Figure 7 shows T. Generation S73 ⁇ 4 inhibited PCR identification of wheat; P: positive control; 124, 125, 127, 142, 147, 153 and 157 were different S73 ⁇ 4 inhibition wheat lines; YW642 was recipient wheat.
  • Figure 8 is a Southern hybridization assay for 1/generation 73 ⁇ 4 inhibition of wheat; 124, 125, 127, 142, 147, 153, and 157 are different S73 ⁇ 4 inhibition wheat lines; YW642 is recipient wheat (resistant).
  • Figure 9 is a quantitative RT-PCR analysis of TiSTK1 inhibiting the expression of TiSTK1 gene and BYDV_i gene in wheat; 124, 125, 127, 142, 147, 153 and 157 are 7 S73 ⁇ 4 inhibition, respectively.
  • Wheat strain; YW642 is the recipient wheat (resistance), and Zhon g 8601 is the susceptible wheat control.
  • Figure 10 shows the resistance of leaves to yellow dwarf disease in 1 ⁇ generation ⁇ 73 ⁇ 4 inhibition of wheat and YW642 as recipient wheat (resistant to disease) and its susceptible wheat control; 124, 125, 127, 142, 147, 153 and 157, respectively For different ⁇ 73 ⁇ 4 inhibitory strains; YW642 is the recipient wheat (resistance), and Zhon g 8601 is the susceptible wheat control.
  • Figure 11 shows the identification of resistant wheat and susceptible wheat based on the 73 ⁇ 4 gene.
  • Figure 12 shows the subcellular localization of the TiSTK1 protein.
  • Figure 13 shows the transcriptional expression characteristics of the ⁇ 3 ⁇ 4 gene.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • PAHC25 vector also known as monocotyledonous expression vector pAHC25; pAHC25 is transformed from pUC8 and contains two expression cassettes, the first expression cassette has maize Ubiquit in promoter, Exon, Intron, GUS, Nos terminator, GUS two The end has a 53 ⁇ 4al and 5" acl cleavage site, and the second expression cassette has a maize Ubiquit in promoter, Exon, Intron, Bar, Nos terminator): The public can obtain it from the Crop Science Institute of the Chinese Academy of Agricultural Sciences; Chri stensen and Quai l, 1996; Ubi quit in promoter-based vectors for high-level express ion of selectable and/ or screenable marker genes in monocotyledonous plants. Transgenic Research, 5, 213-218.
  • BYDV-GAV strain or BYDV-PAV strain All purchased from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.
  • BYDV-GAV strain or BYDV-PAV strain All purchased from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.
  • pHMW-Adh-Nos vector The public can obtain it from the Crop Science Research Institute of the Chinese Academy of Agricultural Sciences; References: Gao Dongyu, Xia Lanqin, Ma Youzhi, Xu Zhaoshi, Xu Huijun, Du Lizhen, Nie Lina, Li Yanzhen, Yuan Yaping, Li Liancheng, Chen Ming, Sun Jin Hai, Construction and Genetic Transformation of RNA Interference Expression Vector of Wheat VP-1 Gene, Journal of Plant Genetic Resources 2009, 10 (1): 9-15.
  • Wheat 8601 also known as Zhon g 8601; common wheat strain with yellow dwarf disease: purchased from the Crop Science Institute of the Chinese Academy of Agricultural Sciences.
  • Anti-yellow dwarf translocation line YW642 (referred to as YW642 or HW642) : References: Zhang Zengyan, Ma Youzhi, Xin Zhiyong et al., 1998, Identification of new wheat germplasm resistant to yellow dwarf disease by genomic in situ hybridization, China Agriculture Science, 31 (3): 1-4; Zhang Z, Xin Z, Ma Y, Chen X, Xu Q, Lin Z. 1999, Mapping of a BYDV res i stance gene from Thinopyrum intermedium in wheat background by molecular markers. Sci China C Life Sci.
  • YW243 (YW243) for reference to anti-yellow dwarf disease: References: Xie Wei, Chen Xiao, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lizhen, Ma Youzhi, Xu Huijun, Breeding and cell molecular biology of new wheat line YW243 resistant to yellow dwarf disease Identification, Journal of Crop Science, 2000, 26 (6): 687-691; The translocation system was created by Xin Zhiyong and Chen Xiao of the Crop Science Research Institute of the Chinese Academy of Agricultural Sciences from 1991 to 1995. Xie Wei et al. were identified in 1998-2000; The Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences guarantees to the public.
  • Anti-yellow dwarf translocation line TC14 (referred to as TC14): References: Banks, P., Larkin, P., Bariana, H., Lagudah, E., Appel s, R., Waterhouse, P., Brettel l, R. , Chen, X. , Xu, H. , Xin, Z. , Qian, Y. , Zhou, M. , Cheng, Z. , and Zhou, G.
  • L1 (L1) for anti-yellow dwarf disease References: Cauderon, Y., Saigne, B., and Dauge, M. (1973) The res i stance to wheat rusts of Agropyron in termedium and its use In wheat improvement. Proc Int Wheat Genet Symp Wheat Improvement, Vol. 4, ER Sears and LMS Sears eds (Univ of Mi ssouri, Columbia, MD), pp 401 - 407 ; LI was created in 1973 by French scientist Cauderon et al; The Institute of Crop Sciences of the Academy of Agricultural Sciences has introduced and preserved it; the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences guarantees to provide it to the public.
  • the double-ended system of anti-yellow dwarf 7Ai#lL (referred to as DT7Ai#lL): References: Banks, P., Larkin, P., Bariana, H., Lagudah, E., Appel s, R., Waterhouse, P . , Brettel l, R. , Chen, X. , Xu, H. , Xin, Z. , Qian, Y. , Zhou, M. , Cheng, Z. , and Zhou, G.
  • Wheat YW641S (YW641S for short) : References: Xiaodong Liu, ZengYan Zhang (Corresponding author), Zhiyong Xin, 2005, Molecular evidence of barley yel low dwarf Virus replication/movement suppressed by the resistance gene Bdv2 derived from Th.
  • Pm97034 Resistance to powdery mildew wheat germplasm Pm97034 (referred to as Pm97034; carrying powdery mildew resistance gene / 3 ⁇ 4) : References: Li H, Chen X, Xin ZY, Ma YZ, Chen XY, Jia X. Development and identification of wheat-ffaynaldia villosa T6DL .6VS chromosome translocation lines conferring resistance to powdery mildew. Plant
  • Wheat-intermediate ryegrass-added line Z1 against yellow dwarf disease (Z1; carrying anti-yellow dwarf gene Bdv ⁇ References: 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.
  • P961341 carrying anti-yellow dwarf gene 3 ⁇ 4/ 3
  • P961341 carrying anti-yellow dwarf gene 3 ⁇ 4/ 3
  • P961341 carrying anti-yellow dwarf gene 3 ⁇ 4/ 3
  • Y15 Resistance to stripe rust wheat germplasm Y15 (referred to as Y15; carrying stripe rust resistance gene Yrl5: Professor Mcintosh from the University of Sydney, Australia, the Crop Science Research Institute of the Chinese Academy of Agricultural Sciences, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, guaranteed to the public; References: Peng JH , Fahima T, R5der MS, Huang QY. Dahan A, Li YC, Grama A, Nevo E, 2000. High-density molecular Map of chromosome region harbouring stripe-rust resistance genes YrH52 and Yrl5 derived from wild emmer wheat, Triticum dicoccoides. Genetica, 109:199 - 210).
  • Y26 Resistance to stripe rust wheat germplasm Y26 (referred to as Y26; carrying stripe rust resistance gene: a gift from Professor Mcintosh of the University of Sydney, Australia, preservation of crop science research of Chinese Academy of Agricultural Sciences, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, guaranteed to the public; References: Li GQ Li ZF, Yang WY, Zhang Y, He ZH, Xu SC, Singh RP, Qu YY, Xia XC, 2006. Molecular mapping of stripe rust resistance gene YrCH42 in Chinese wheat cultivar Chuanmai 42 and its allelism with Yr24 and Yr26. And Applied Genetics, 112: 1434 - 1440.
  • Example 1 Discovery of the TiSTK1 gene
  • PCR primers were designed using 70 EST sequences on wheat chromosome 7DL to optimize the conditions of PCR amplification and gel electrophoresis analysis for anti-yellow dwarf materials (Ti, DT7Ai#lL, YW642) and BdVZ without 3 ⁇ 4/ 2 DNA of yellow-streaked dwarf wheat (medium 8601, CS, YW641S) was analyzed by PCR amplification and SDS-PAGE gel electrophoresis. see picture 1.
  • the cDNA is used as a template to rapidly amplify the 3' cDNA sequence of TiSTK1 gene (specific conditions: 94 °C 3 min ; 94 °C 30s, 68 °C 3 min, 5 cycles; 94 °C 30s, 60 °C lmin, 68 ° C 3 min, 30 cycles; 72 °C lOmin), obtain a partial sequence of the S 3 ⁇ 4 gene (sequence 2 of the sequence listing is shown from nucleotides 472 to 1823 at the 5' end).
  • Primers consisting of QC-U and QC_L using cDNA or genomic DNA of Ti or YW642 as a template For PCR amplification.
  • PCR amplification conditions 94 ° C 3 min ; 94 ° C 30 s, 62 ° C 45 s, 72 ° C 2 min, 3 cycles; 94 ° C 30 s, 60 ° C 45 s, 72 ° C 2 min, 5 cycles; 94 ° C 30s, 58 °C 40s, 72 °C 2min, 30 cycles; 72 °C 10 min.
  • the PCR amplification products were sequenced, and the sequencing results were identical, and the nucleotide sequence of the cDNA, the nucleotide sequence of the genomic DNA, and the amino acid sequence of the protein were obtained.
  • the protein shown in SEQ ID NO: 1 is named TiSTK1 protein, which is composed of 425 amino acid residues and is a serine/threonine protein kinase.
  • the gene encoding the TiSTK1 protein is named S73 ⁇ 4 gene, that is, the cDNA is shown in SEQ ID NO: 2 of the sequence listing (opening frame from nucleotides 171-1448 at the 5' end), and genomic DNA is shown in sequence 3 of the sequence listing. (containing 2 introns, the first intron is sequence 3 of the sequence listing from nucleotides 92-595 at the 5' end, and the second intron is sequence 3 of the sequence listing from the 5' end Nucleotides 1612-1692).
  • the primers of WS-F and STK-L were used for PCR amplification using the cDNA of SEQ ID NO: 6601 as a template, and PCR amplification products were obtained and sequenced, and the sequencing results were compared with sequence 2 of the sequence listing.
  • SNP single nucleotide polymorphism
  • WS-F 5' -CGCAGCACGCCAATCCGCC-3 ' ;
  • STK-L 5' -GCAGCCGCTATCAACACAAGAC-3'.
  • RNA from the intermediate buckwheat grass was extracted and reverse transcribed into cDNA.
  • a primer pair consisting of TiSTK-0-SMAI and TiSTK-O-SACI was used for PCR amplification to obtain a PCR amplification product.
  • TiSTK-0-SMAI 5, - ATCCCGGGATGATTGAGGGGGCAAGGTTC-3, (introduced Smal cleavage site);
  • the product was amplified by restriction enzyme digestion with Sim restriction enzyme Sim and 53 ⁇ 4cl to obtain an enzyme-cut product.
  • the pAHC25 vector was digested with restriction endonuclease Siml and double-transgested, and the vector backbone (about 7818 bp) was recovered.
  • the restriction enzyme product of step 3 is ligated with the vector backbone of step 4 to obtain a recombinant plasmid pAHC25-Ji'OTio.
  • the recombinant plasmid pAHC25-i ⁇ 73 ⁇ 4 is described as follows:
  • the PAHC25 vector is used as a skeleton vector, Between the 53 ⁇ 4al and the cleavage site of the backbone vector, the sequence 2 of the sequence listing was inserted from the nucleotides at positions 171 to 1448 of the 5' end (0RF; the sequence 2 was from the 5' end of the 1st nucleotide at the 5' end.
  • ⁇ 3 ⁇ 4 gene is under the control of the Ubiquitin promoter;
  • the granule also has a War gene expression cassette controlled by the Ubiquitin promoter, which can provide a resistance marker for screening and regenerating plants using the herbicide bialaphos (Bialaphos) in subsequent work.
  • the callus of the immature embryo of 8601 in 2000 wheat lines was used as a receptor for gene gun bombardment, and the recombinant plasmid pAHC25- ⁇ 73 ⁇ 4 was bombarded with the above-mentioned callus with a gene gun.
  • the callus was then transferred to SD2 medium (VB 1 lmg/L, aspartame 150 mg/L, 2, 4_D 2 mg/L) was added to the inorganic salt component of MS medium, and culture was resumed for 2 weeks ( 26 ° C, dark culture).
  • each leaf of each surviving 1 ⁇ generation plant was used to extract genomic DNA, and the genome was used as a template.
  • a sequence in Ubiquitin was used as the upstream primer (UBI-1F).
  • a sequence of the S 3 ⁇ 4 gene was used as a downstream primer (TiSTKlH for PCR amplification.
  • the recombinant plasmid pAHC25-i ⁇ 73 ⁇ 4 was used as a positive control, and the genomic DNA of the 8601 was a negative control, and the amplified product fragment was expected to be about 758 bp (see the sequence listing). Sequence 4).
  • TiSTKl-Rl 5' -TATCTCCGTcGGATGAGTTGG-3'.
  • TiSTK1 strains resistant to yellow dwarf disease were obtained from the plant and 1 ⁇ generation plants (strain 1, strain 4, strain 31, strain 40, strain 41, strain 44). And strain 45).
  • TiSTKl-SNPR 5, -cgaccttgtggtaatacggca-3 '.
  • 18S rRNA-QF 5' - GTGACGGGTGACGGAGAATT- 3';
  • 18S rRNA-QR 5 ' - GACACTAATGCGCCCGGTAT- 3, .
  • the pAHC25 vector was used to replace the recombinant plasmid pAHC25-S73 ⁇ 4 in transformation 8601, and the method was the same as that of the transgenic plant to obtain the vector of the empty vector control.
  • BYDV-GAV strain or BYDV-PAV strain Aphids inoculated with yellow dwarf virus (BYDV-GAV strain or BYDV-PAV strain) at the seedling stage, that is, aphids carrying the BYDV-GAV strain (or BYDV-PAV strain) are placed on the wheat plants, each Ten aphids were photographed and photographed 40 days after inoculation.
  • the disease resistance was graded according to the plant phenotype, the flag leaf chlorophyll index was detected, the relative content of BYDV in the plants was detected by Q-RT-PCR, and the virus infection in the plants was detected by ELISA. .
  • the disease resistance classification uses the domestic standard of the severity of wheat yellow dwarf disease, that is, the IT standard, see Table 2, References: “Li Guangbo, Zeng Shimai, Li Zhenqi, editor. Integrated management of wheat diseases, pests and diseases [M]. Beijing: China Agricultural Science and Technology Press, 1990". The photo is shown in Figure 5, and the results are shown in Table 3.
  • GAV-CP-U and GAV_CP_L of the coat protein-encoding gene (GAV-gene, also known as BYDV-gene) of the GAV strain of barley yellow dwarf virus (BYDV) were designed, and the cDNA of the plant to be tested was used as a template.
  • Primer pairs consisting of GAV-CP-U and GAV-CP-L were subjected to Q-RT-PCR to detect the accumulation of BYDV in the plants.
  • the 18S rRNA gene was used as an internal reference (primer pair consisting of 18S rRNA-QF and 18S rRNA-QR; the ratio of GYDV- and 18S rRNA gene expression was used as the relative expression of BYDV- ⁇ P gene in the plant).
  • GAV-CP-U 5' -CAGGCAGGACTGAGGTATT-3 ' ;
  • GAV-CP-L 5' -: GTTGCTGATTTTGAGAGGG-3 '.
  • step (3) In the enzyme-linked plate of step (3), add 200 ⁇ l of each step to the supernatant of step (2) and incubate at 4 °C overnight.
  • step (4) Drain the solution on the enzyme plate of step (4) and wash the plate 3 times with washing buffer (PBS containing 0.5% Tween20) for 5 minutes each time.
  • washing buffer PBS containing 0.5% Tween20
  • step (5) In the enzyme-linked plate of step (5), add 200 ⁇ l per well and dilute 1000-fold alkaline phosphatase-labeled IgG with ligation buffer (PBST containing 2% PVP), 200 ⁇ l per well, capped Moisturize, incubate for 5-6 hours at 37 °C.
  • ligation buffer PBST containing 2% PVP
  • the results of disease resistance identification showed that all overexpressing lines of TiSTK1 gene were resistant to BYDV-GAV and BYDV-PAV infection, and the content of BYDV was significantly decreased. Some strains (such as #1, #4, #40, # 44, #45, etc.
  • the resistance level is basically the same as that of the positive control, indicating that the ⁇ 73 ⁇ 4 gene is an important gene against yellow dwarf disease and has a broad spectrum.
  • the TiSTK1 gene overexpressing line is highly resistant to BYDV-GAV, BYDV-PAV during the whole growth period.
  • RNA interference expression vector and its anti-yellow dwarf function analysis of transgenic wheat I Construction of RNA interference vector
  • RNA interference fragment A specific sequence at the 5' end of the ⁇ 73 ⁇ 4 gene was used as an RNA interference fragment (sequence 2 of the sequence listing from nucleotides 2 to 493 at the 5' end), constructed on the basis of pHMW-Adh-Nos vector and pAHC25 vector.
  • Primers were designed based on the restriction fragment and the restriction endonuclease recognition sequence at both ends of the pH MW-Adh-Nos vector Adh intron (about 147 bp).
  • a restriction site is introduced into the forward primer of the reverse fragment, and a restriction site is introduced in the reverse primer of the forward fragment, that is, in the opposite There are Siml and feci restriction sites at both ends of the repeat sequence, respectively.
  • the primer pair that amplifies the inverted fragment consists of TISTKl-RI-BagI I and TISTK-RI- EcoRI.
  • the primer pair that amplifies the forward fragment consists of TISTK1-RI- ⁇ 3 ⁇ 41 I and TISTKl-RI-AfcoI.
  • TISTKl-RI-BagI I 5 ' - AAAGATCTGCAGCACGCCAATCCGC- 3, ;
  • TISTK-RI-EcoRI 5 ' - GAGAATTC CO3 ⁇ 43 ⁇ 4GCAGCCTTGTTGGGATAGA- 3, .
  • TISTKl-RI-AfcoI 5' - ATCCATGG63 ⁇ 4 OnCAGCCTTGTTGGGATAGA- 3, .
  • RNA of the intermediate buckwheat grass was extracted and reverse transcribed into cDNA.
  • step (2) Using the cDNA of step (1) as a template, a primer pair consisting of TISTK-RI-EcoRI and TISTK1-RI-Bagll was subjected to PCR amplification to obtain a PCR amplification product (reverse fragment STKR).
  • step (3) Using the cDNA of step (1) as a template, a primer pair consisting of TISTKl-RI-BagII and TISTK1-RI-Afcol was subjected to PCR amplification to obtain a PCR amplification product (forward fragment STKF).
  • pHMW-Adh_Nos vector was digested with restriction endonuclease coRI and BwiRl ⁇ Bglll homologous enzyme), and the vector backbone (about 4554 bp) was recovered.
  • the intermediate plasmid pHMW-STKR-Adh was digested with restriction endonucleases Bgll I and Ncol, and the vector backbone (about 5062 bp) was recovered.
  • the fragment recovered in the step (10) is ligated to the vector skeleton recovered in the step (11) to obtain a recombinant plasmid pAHC25_STKR-Adh-STKF (RNA interference vector).
  • a recombinant plasmid pAHC25_STKR-Adh-STKF RNA interference vector
  • the recombinant plasmid pAHC25_STKR-Adh-STKF was structurally described as follows:
  • the pAHC25 vector was used as the backbone vector, and the DNA shown in SEQ ID NO: 5 of the sequence listing was inserted between the SiO1 and the restriction sites of the backbone vector (sequence In 5, the nucleotides 7 to 498 from the 5' end are reverse fragments, and the nucleotides 653 to 1144 are forward fragments).
  • ⁇ 3 ⁇ 4 inhibits plant acquisition
  • the anti-yellow dwarf translocation line YW642 was transformed with the RNA interference vector (pAHC25_STKR-Adh-STKF) in the same manner as in steps 1 to 5 of Example 2 except that the RNA interference vector was used instead of the recombinant plasmid pAHC25-J OT.
  • the anti-yellow dwarf translocation line YW642 replaces the middle 8601. Get T. Generation plants. Will T. The plants were selfed, and the plants were obtained. The plants were self-crossed to obtain the plants.
  • the specific primers AI-L and RI-U of the interference fragment were designed based on the Adh intron sequence and the S 3 ⁇ 4 gene inverted fragment on the pAHC25_STKR-Adh-STKF vector.
  • AI-L 5' -CCAAGGTATCTAATCAGCCATC-3 ';
  • RI-U 5' - CGACCTTGTGGTAATACGGCAT- 3, .
  • the genomic DNA of the transgenic plants was subjected to PCR amplification detection, and the amplified product fragment was expected to be about 419 bp.
  • the RNA interference vector was used as a positive control, and the anti-yellow dwarf translocation line YW642 was used as a negative control.
  • the genomic DNA of 7 ⁇ 3 ⁇ 4 inhibitory strains 1 ⁇ generation plants were extracted and digested with restriction endonuclease Dral. Thereafter, Southern hybridization was carried out using the probe shown in SEQ ID NO: 6 of the Sequence Listing; the genomic spirit of the anti-yellow dwarf translocation line YW642 was used as a negative control. Some results are shown in Figure 8.
  • the TiSTK1 gene-inhibited plants all showed a positive hybridization signal, and the yellow dwarf resistance translocation line YW642 did not show a hybrid signal.
  • the interference functional fragment was integrated into the genome of 7 ⁇ 73 ⁇ 4 inhibitory strain plants with 1-2 copies, which can be stably inherited.
  • RNA of the 1 ⁇ generation plants of each TiSTK1 inhibitory strain was extracted and reverse transcribed into cDNA, diluted 10 times and used as a template.
  • Q-RT-PCR amplification was performed using primer pairs consisting of TiSTK1-SNPF and TiSTK1-SNPR.
  • Expression of the S73 ⁇ 4 gene; using the 18S rRNA gene as an internal reference using a primer pair consisting of 18S rRNA-QF and 18S rRNA-QR; the ratio of the expression of the ⁇ 73 ⁇ 4 gene to the 18S rRNA gene was used as the relative of the ⁇ 73 ⁇ 4 gene in the plant
  • the expression level was YW642, which was the anti-yellow dwarf translocation line, and the middle one was the negative control.
  • the partial results are shown in Figure 9. Compared with the YW642 transgenic line of the yellow dwarf disease, the S73 ⁇ 4 gene in the seven S73 ⁇ 4 inhibitory lines The relative expression levels were significantly reduced.
  • the anti-yellow dwarf translocation line YW642 was transformed with pAHC25 vector.
  • the method was the same as above, and the control vector was obtained.
  • the 1 ⁇ generation plants of each control line, the anti-yellow dwarf translocation line YW642 (positive control), the middle 8601 (negative control), and the 1 ⁇ generation plants of the empty vector control plants were identified as follows. , 20 strains per strain:
  • BYDV-GAV strain Aphids inoculated with yellow dwarf virus (BYDV-GAV strain) at seedling stage, that is, aphids carrying BYDV-GAV strains were placed on wheat plants, 10 aphids per plant, photographed 30 days after inoculation and according to plant phenotype
  • the disease resistance grading was carried out, and the relative content of BYDV in the plants was detected by Q-RT-PCR (using the cDNA of the plant to be tested as a template, and the primer pair consisting of GAV-CP-U and GAV-CP-L was used for Q-RT- PCR, detection of BYDV accumulation in plants; 18S rRNA gene as internal reference, primer pair consisting of 18S rRNA-QF and 18S rRNA-QR; ratio of gene and 18S rRNA gene expression as relative expression of genes in the plant the amount) .
  • the method for identifying the disease resistance classification is the same as that of the third step of the second embodiment.
  • Each 3 3 gene suppression strain The IT is 7, the IT resistance of the yellow dwarf translocation line YW642 is 0, and the IT of the 8601 is 6_7.
  • the detection method of the relative content of BYDV is the same as that of the third step of the second embodiment, and the results are shown in Fig. 8.
  • a ⁇ 73 ⁇ 4 gene-specific SNP primer pair was designed, consisting of TiSTKl-SNPF and TiSTKl-SNPR.
  • TiSTKl-SNPF sequence of sequence listing 7: 5, -gctcccctccttcccctt-3';
  • TiSTKl-SNPR (sequence 8 of the sequence listing): 5, -cgaccttgtggtaatacggca-3'.
  • PCR was carried out using the genomic DNA of the leaves of the following materials as a template: Plant material carrying anti-yellow dwarf disease (Ti, Ll, YW642, YW243 and TC14); ⁇ 2 yellow-sensitive dwarf wheat material (medium 8601, CS); anti-yellow dwarf plant material: Z1 (carrying Plant material resistant to powdery mildew: Pm97034 (carrying PwV); wheat material resistant to stripe rust: Y15 (carrying YrlS) and Y26 (carrying ri ⁇ ).
  • the ⁇ 3 ⁇ 4 gene-specific primer can amplify a 739 bp fragment only in the anti-yellow dwarf material (Ti, Ll, YW642, YW243, and TC14) carrying 3 ⁇ 4 2 , but in the absence of 2 Yellow-streaked wheat material (medium 8601 and CS), wheat germplasm carrying other yellow dwarf resistance genes (Z1 and P961341), wheat germplasm resistant to powdery mildew (Pm97034), wheat germplasm resistant to stripe rust (Y15 and Y26) The medium is not amplified, indicating that the 73 ⁇ 4 gene is a specific gene carrying 3 ⁇ 4/2 anti-yellow dwarf wheat.
  • the TiSTK1 kinase protein has a membrane binding site.
  • the P35S : TiSTKl-GFP fusion protein vector was constructed (specific steps: The ORF-F ( 5 , - GCCU ATGATTGAGGGGGCAAGGTTCC-3 , He B GFP-R ( 5, - TA ⁇ 47m TCGGTGGTCATGGGCTCGG-3, ) was used to amplify the ORF region of the TiSTK1 gene from which the stop codon was removed, and was ligated to PMD18- On the T vector; the ligation product was transformed into E.
  • the ORF-F 5 , - GCCU ATGATTGAGGGGGCAAGGTTCC-3 , He B GFP-R ( 5, - TA ⁇ 47m TCGGTGGTCATGGGCTCGG-3, ) was used to amplify the ORF region of the TiSTK1 gene from which the stop codon was removed, and was ligated to PMD18- On the T vector;
  • the positive clone was selected for sequencing, the positive cloned strain was sequenced and the plasmid was isolated; the plasmid was digested with /i 7dI II and the target fragment was digested with #i ?dI
  • the 163hGFP vector digested with II and ⁇ (provided by Wang Daowen, Institute of Genetics, Chinese Academy of Sciences) was ligated and transformed into E. coli; the positive clone was selected for bacterial preservation and plasmid was extracted, and the gene gun was used to mediate (see Zhang ZengYan, Yao WuLan, Dong Na). , Liang HongXia, Liu HongXia, Huang RongFeng. 2007.
  • the aphids carrying the BYDV-GAV strain were placed in the three-leaf stage of the anti-yellow dwarf translocation line YW642 wheat plant, and each plant was inoculated with about 10 or so aphids.
  • the BYDV-GAV strain was directly inoculated with the trifoliate anti-yellow dwarf translocation line YW642.
  • the aphids that did not carry the virus were directly placed in the leaf-leaf of the yellow-wing dwarf translocation line YW642, and each plant was inoculated with about 10 or so aphids. After 3 days, the cockroach was sterilized.
  • the conserved 18SrRNA housekeeping gene ( 18SrR_F : 5'-GTGACGGGTGACGGAGAATT-3', 18SrR-R : 5'-GACACTAATGCGCCCGGTAT-3' ) was used as an internal standard gene to maintain consistent total cDNA concentrations between samples, using TiSTKl-SNPF and TiSTKl- Primer pairs consisting of SNPR and Takara's SYBR Premix Ex TaqTM kit were subjected to Q-RT-PCR to analyze the expression of S73 ⁇ 4 gene in the sample, with at least 3 replicates per sample.
  • PCR amplification procedure denaturation at 95 °C for 1 min, entering 95 °C for 10 s, 60 °C for 31 s for 41 cycles.
  • the gene encoding plant resistance-related protein S73 ⁇ 4 was introduced into wheat, and the transgenic wheat overexpressing the gene significantly increased the resistance to yellow dwarf disease.
  • the inhibition of 73 ⁇ 4 expression in resistant wheat caused the plant to lose resistance to yellow dwarf disease.
  • TiSTKl is a key protein of plant yellow dwarf resistance.
  • the plant yellow dwarf resistance key protein TiSTK1 and its coding gene can be used to improve the resistance of plants to yellow dwarf disease, which is of great value for plant breeding.
  • the primer pair provided by the present invention can assist in identifying whether the plant to be tested has the gene based on the gene, and further determining whether the plant to be tested is a plant against yellow dwarf disease or a sensitive plant.
  • the invention has important theoretical and practical significance and will play an important role in the genetic improvement of plants.

Abstract

La présente invention concerne une protéine TiSTKI résistante à la maladie du nanisme jaune touchant les végétaux, dérivée d'une substance résistante à la maladie du nanisme jaune, et un gène codant pour celle-ci, destinés à être utilisés pour améliorer la résistance de plantes telles que le blé contre la maladie du nanisme jaune. L'invention concerne également un ARN interférent destiné à inhiber l'expression génique de TiSTKI et l'application d'un tel ARN dans une culture de plantes transgéniques; une paire d'amorces capables de réaliser l'expansion du gène TiSTKI ou un segment d'ADNc caractéristique de celui-ci, et une application de la paire d'amorces pour aider à l'identification d'une plante résistante à la maladie du nanisme jaune.
PCT/CN2011/001120 2010-07-14 2011-07-06 Protéine tistki résistante à la maladie du nanisme jaune touchant les végétaux, gène codant pour celle-ci et application de ceux-ci WO2012006866A1 (fr)

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