WO2012006866A1

WO2012006866A1 - BOTANICAL YELLOW DWARF DISEASE RESISTANT PROTEIN TiSTKI, CODING GENE AND APPLICATION THEREOF

Info

Publication number: WO2012006866A1
Application number: PCT/CN2011/001120
Authority: WO
Inventors: 张增艳; 陈亮; 汪信东; 徐惠君; 辛志勇; 杜丽璞
Original assignee: 中国农业科学院作物科学研究所
Priority date: 2010-07-14
Filing date: 2011-07-06
Publication date: 2012-01-19
Also published as: CN101892315A; CN101892315B

Abstract

Provided are a botanical yellow dwarf disease resistant protein TiSTKI, derived from a yellow dwarf disease resistant material, and a coding gene thereof, for use in enhancing resistance against yellow dwarf disease in plants such as wheat. Also provided are an interfering RNA to inhibit TiSTKI gene expression and an application of such RNA in transgenic plant cultivation; a primer pair capable of expanding TiSTKI gene or a cDNA characteristic segment thereof, and an application of the primer pair in aiding to identify a yellow dwarf disease resistant plant.

Description

Plant yellow dwarf resistance protein TISTK1 and its coding gene and application

Technical field

The invention relates to a plant yellow dwarf resistance key protein TiSTK1 and a coding gene thereof and application thereof.

Background technique

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. In 1999, only Shaanxi, Shanxi and other wheat areas were yellow dwarf. The disease caused wheat to reduce production by 20% to 30%, individual severe wheat areas reduced production by more than 50%, and wheat yield losses amounted to hundreds of millions of kilograms. In recent years, wheat bran, which spreads in winter and spreads barley yellow dwarf virus, is widespread and growing in China. Wheat yellow dwarf disease has spread to many wheat producing areas such as Shaanxi, Shanxi, Gansu, Sichuan, Ningxia, Inner Mongolia, Hebei and Jiangsu. Therefore, the prevention and treatment of yellow dwarf disease is very important to ensure high yield and stable yield of wheat and sustainable development of agriculture. Breeding new varieties of wheat resistant to yellow dwarf disease is the most economical and effective way to control the disease.

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. According to the sequence of different aphids and barley yellow dwarf viruses, 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.

Excellent available disease resistance genes are prerequisites for disease resistance breeding. To date, no truly effective resistance genes have been found in the wheat primary gene pool. However, multiple strains of intermediate buckwheat (wheat relatives), highly resistant or immune to barley yellow dwarf virus have been applied and studied intensively. The intermediate long-armed arm of the 7Ai#l chromosome carries an anti-yellow dwarf gene 3⁄4 2 . Domestic and foreign scientists use the Chinese spring/^ mutant to induce partial homologous chromosome pairing and tissue culture to translocate the long arm end fragment of the 7Ai#l chromosome carrying the anti-yellow dwarf gene 3⁄4 2 to the long arm of the wheat chromosome 7D. At the end, a batch of wheat-intermediate buckwheat T7D* 7Ai#lL translocation lines resistant to yellow dwarf disease, including YW642 (HW642), YW443, YW243, and TC5_TC10, TC14, etc. (Banks, P.) were successfully selected and identified. , Larkin, P. , Bariana, H. , Lagudah, E. , Appels, R. , Waterhouse, P. , Brettel l, R. , Chen, X. , Xu, H. , Xin, Z. , Qian, Y . Zhou, M. , Cheng, Z. , and Zhou, G. The use of cel l culture For sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat. Genome. 1995, 38: 395-405; Zhang Zengyan, Xin Zhiyong, Chen Xiao et al. Molecular cytogenetic identification of a new wheat line YW443 resistant to yellow dwarf disease, Acta Genetica Sinica, 2000, 27 (7) : 614-620; Xie Yi, Chen Xiao, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lizhen, Ma Youzhi, Xu Huijun, Breeding and Cellular Molecular Bioassay of YW243, a New Wheat Line with Resistance to Yellow Dwarf Disease , Crop Journal, 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 intermedium translocation lines with resistance to barley yellow dwarf virus. Euphytica. 2001, 119: 161 - 165) , found that carrying the anti-yellow dwarf gene ^ intermediate buckwheat chromosome 7Ai# l Long arm (7Ai#lL) end small fragment translocation to the long arm end of wheat chromosome 7D (Zhang Zengyan, Xin Zhiyong, Ma Youzhi, etc., Mapping of a BYDV resistance gene fro m Thin termedium intermedium in wheat background by molecular markers, Science in China ( Series C) , 1999, 42 (6) : 663-668; 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). The study found that the wheat-intermediate buckwheat translocation lines YW642, YW443, YW243, TC14, etc., which are resistant to the yellow dwarf disease gene, are highly resistant to barley yellow dwarf virus. In theory, these translocation lines should be used as anti-yellow Resistant germplasm that is easy to use in dwarf wheat breeding. However, these 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.

Invention disclosure

The invention provides a plant yellow dwarf resistance important protein TiSTK1 and a coding gene thereof and application thereof. The protein provided by the present invention, named TiSTKl, is derived from the intermediate ryegrass Thinopyrum intermedium) and is as follows (a) or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing;

(b) a sequence 1 derived protein which has the amino acid sequence of SEQ ID NO: 1 substituted and/or deleted and/or added to one or several amino acid residues and which is associated with plant yellow dwarf resistance.

In order to facilitate the purification of the protein in (a), 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.

Table 1 Sequence of labels

Tag residue sequence Poly-Arg 5-6 (usually 5) RRRRR

Poly-His 2-10 (usually 6) HHHHHH

FLAG 8 DYKDDDDK

Strep-tag II 8 WSHPQFEK

C-myc 10 EQKLISEEDL

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):

1) the DNA molecule of sequence 2 in the sequence listing from nucleotides 171-1448 at the 5' end;

2) the molecule shown in sequence 2 in the sequence listing;

3) the molecule shown in SEQ ID NO: 3 in the sequence listing;

4) a DNA molecule that hybridizes under stringent conditions to a DNA sequence defined by 1) or 2) or 3) and encodes a plant yellow dwarf resistance-associated protein;

5) A DNA molecule that has more than 90% homology to the defined DNA sequence of 1) or 2) or 3) and encodes a plant yellow dwarf resistance-associated protein.

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. When the recombinant plant expression vector is constructed using the gene, 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; Furthermore, when constructing a plant expression vector using the gene of the present invention, 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. In order to facilitate the identification and screening of transgenic plant cells or plants, 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. Specifically, 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.

In any one of the above methods, 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. Anti-yellow dwarf translocation line YW243, anti-yellow dwarf translocation line TC14, wheat medium 8601, wheat Chinese spring, anti-yellow dwarf wheat-intermediate buckwheat cultivar Zl, wheat-intermediate buckwheat translocation line P961341 , wheat Pm97034, wheat Y15 or wheat Y26. 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.

DRAWINGS

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 best way to implement the invention

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.

Barley yellow dwarf virus (BYDV-GAV strain or BYDV-PAV strain): All purchased from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Aphids carrying barley yellow dwarf virus (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.

Middle ryegrass (Ti): purchased from the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences, numbered Z 1146.

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.

Wheat Chinese Spring (CS; Yellow Dwarf Wheat): purchased from the Institute of Crop Sciences, 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. 42 (6) : 663-668.; The translocation system was created by Xin Zhiyong of the Crop Science Research Institute of the Chinese Academy of Agricultural Sciences in 1991, Zhang Zengyan and others were identified in 1996; the Crop Science Research Institute of the Chinese Academy of Agricultural Sciences guarantees to the public provide.

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. The use of cel l cul ture for sub-chromosomal introgress Isolation of barley yel low dwarf virus res i stance from Thinopyrum in termedium to wheat. Genome. 1995, 38 : 395-405; The translocation system was 1991-1994 Australian scientist Banks, Larkin and Chinese scientists Xin Zhiyong, Chen Xiao, Xu Huijun Etc., Banks, Larkin, etc. were identified in 1995; the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences introduced and preserved; the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences promised to provide it to the public.

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. The use of cel l culture for Sub-chromosomal introgress ions of barl ey yel low dwarf virus res i stance from Thinopyrum in termedium to wheat. Genome. 1995, 38 : 395-405; The translocation system was 1991-1994 Australian scientist Banks, Larkin and Chinese scientist Xin Zhiyong , Chen Xiao, Xu Huijun and other creations, Banks, Larkin et al. identified in 1995; Institute of Crop Sciences, Chinese Academy of Agricultural Sciences introduced, preserved; Institute of Crop Sciences, Chinese Academy of Agricultural Sciences guaranteed to provide to the public.

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. intermedium , Journal of Genetic and Genomics ( Yi Chuan Xue Bao), 932: 942-947; This department is the susceptible sister of YW642, which was China 1996-1998 Zhang Zengyan, Institute of Crop Sciences, Academy of Agricultural Sciences, etc., selected and identified; Research Group of Wheat Molecular Research Group, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (Researcher Zhang Zengyan); Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, guaranteed to provide to the public.

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

Breeding, 2005, 124: 203-205; The department was founded in 1997-2001 by scientists such as Chen Xiao and Xin Zhiyong of the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences, Li Hui, etc.; the Wheat Molecular Research Group of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences Preservation; The Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences guarantees to the public.

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. The use Of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium Xo wheat. Genome. 1995, 38: 395-405; Zhang, ZY, Xin, ZY, and Larkin, P, J. 2001. Molecular character i Zat ion of a Thinopyrum intermedium group 2 chromosome (2Ai-2) conferring resistance to barley yellow dwarf virus. Genome, 44: 1129 -- 1135; Zl is a pair of intermediate buckwheat chromosome 2Ai-2 added to the wheat genome. Carrying anti-yellow dwarf disease gene 3⁄4 i, by Australian scientist Larkin and Chinese scientists Xin Zhiyong, Chen Xiao, Xu Huijun equal to 1992-1995 creation, identification, crop science research of Chinese Academy of Agricultural Sciences; Crop Science Research, Chinese Academy of Agricultural Sciences Guarantee available to the public.

Wheat-intermediate buckwheat translocation line P961341 (referred to as P961341; carrying anti-yellow dwarf gene 3⁄4/ 3): created and presented by Professor Ohm of Purdue University, USA, Crop Science Research, Chinese Academy of Agricultural Sciences, China Agriculture The Institute of Crop Sciences of the Academy of Sciences is guaranteed to be available to the public; references: Ohm, IL W., Anderson, JM, Sharma, Η, C., Ayala, L., Thompson, N., and Uphaus, j. J. 2005. Registration Of yellow dwarf viruses resistant wheat germplasm line P961341. Crop Sci, 45:805 - 806.

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).

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

I. Discovery of ^ 3⁄4 gene fragments

Because the middle buckwheat chromosome 7Ai#lL can translocate to the wheat chromosome 7DL, it indicates that the intermediate buckwheat 7Ai#lL has a partial homology with the wheat chromosome 7DL. 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. It was found that the amplified fragment of BQ238952 (a part of the cDNA sequence of the ^73⁄4 gene) was specifically found in the middle of the buckwheat grass 7Ai#lL (carrying

Among the anti-yellow dwarf materials (Ti, DT7Ai#lL and YW642), the yellow-streaked wheat material does not have this specific band. Subsequently, a TiSTK1-specific amplification band (260 bp fragment) derived from the anti-yellow dwarf material was isolated from the gel, and the specific primer (F: 5'-GACATGCCGTATTACCACAAG-3', R: 5' - GCCAATGCGTCCTCCTTG-3') was used. Secondary PCR amplification, recovery, cloning, colony-PCR screening of positive clones, 5 positive clones containing the anti-yellow dwarf material (Ti, DT7Ai#lL and YW642) were sequenced and analyzed. The sequencing results were identical, and the nucleotide sequence of the ^73⁄4 gene fragment was obtained (for example, the sequence 2 of the sequence listing is shown from nucleotides 264-523 of the 5' end).

Second, the full length cDNA of the ^73⁄4 gene and the discovery of the genome

Design a 3' RACE primer (3RA1: 5' - TGACGTCCCCACACCAAACTATTC-3', 3RA2: 5' - TCATCTATCCCAACAAGGC-3') based on the nucleotide sequence of the 73⁄4 gene fragment obtained in step 1. Using a nested PCR strategy with Ti or YW642 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).

By sequence splicing, the following primers were designed:

QC-U: 5' -GCAGCACGCCAATCCGC-3';

QC-L: 5' -GCAGCCGCTATCAACACAAGAC-3'.

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).

Third, the sequence analysis

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.

There are only 54 single nucleotide polymorphism (SNP) differences between the TiSTK1 gene shared by Ti and YW642 and the TaSTK1 sequence derived from the middle 8601 gene, and the 5' non-coding region (5' UTR) has 19 At the SNPs.

WS-F: 5' -CGCAGCACGCCAATCCGCC-3 ' ;

STK-L: 5' -GCAGCCGCTATCAACACAAGAC-3'. Example 2. Acquisition of S3⁄4 overexpressing wheat and analysis of resistance to yellow dwarf disease

I. Construction of overexpression vector

1. Total RNA from the intermediate buckwheat grass was extracted and reverse transcribed into cDNA.

2. Using the cDNA of step 1 as a template, 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);

TiSTK-0-SACI: 5, - TAGAGCTCTCAGTCGGTGGTCATGGGCT-3, (introduced 53⁄4cl restriction site).

3. The product was amplified by restriction enzyme digestion with Sim restriction enzyme Sim and 53⁄4cl to obtain an enzyme-cut product.

4. The pAHC25 vector was digested with restriction endonuclease Siml and double-transgested, and the vector backbone (about 7818 bp) was recovered.

5. The restriction enzyme product of step 3 is ligated with the vector backbone of step 4 to obtain a recombinant plasmid pAHC25-Ji'OTio. According to the sequencing results, 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. G) ; ^ 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.

Second, ^ 3⁄4 overexpression of wheat

1. 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.

2. The callus after bombardment with the gene gun was treated on osmotic medium for 16 h.

3. 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).

4. Transfer the callus after restoration to the differentiation screening medium (1/2 MS medium + naphthaleneacetic acid 1 mg/L + kinetin 1 mg/L + bialaphos 2-5 mg/L), 24_26 ° C light After cultured for 14 days, the callus was differentiated into seedlings and transferred to growth screening medium (1/2 8 medium + bialaphos 2-3 ₁ ^ few), cultured at 24-26 ° C; 135 regenerated Plant.

5. Transfer the regenerated plants to the strong seedling medium (1/2MS medium + 0.5 mg/L naphthaleneacetic acid), transplant the transformed seedlings with 7-8 cm seedling height and roots to the pots, and transplant them to the greenhouse for 3 weeks. In the future, the living plant is T. Generation plants. Will T. The plants were selfed, and the plants were obtained. The plants were self-crossed and the plants were obtained.

6, PCR identification and Southern hybridization detection

In the 4-leaf stage, one leaf of each surviving 1\ generation plant (or T. 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).

UBI-F: 5' -GCTCTGCCTTCATACGCTA- 3, ;

TiSTKl-Rl:: 5' -TATCTCCGTcGGATGAGTTGG-3'.

Some results are shown in Figure 2. Positive control, part 1\ generation plant and part T. The expected fragment of about 750 bp was amplified from the plants, but the corresponding fragment was not amplified in the negative control. The plants amplified with the expected fragment were ^73⁄4 overexpressing plants (^73⁄4 overexpressing transgenic wheat).

To further confirm the results of PCR identification, determine whether the target gene has been integrated into the wheat genome, and the number of copies inserted, extract the genomic DNA of each of the over-expressed plants, using restriction endonuclease ral (or 3⁄4 ₀ 7?V) After digestion, the transgene-specific amplified fragment was used as a probe (probe as shown in SEQ ID NO: 4 of the Sequence Listing) for Southern hybridization; the genomic DNA of Medium 8601 was used as a negative control.

Some results are shown in Figure 3. The ^73⁄4 overexpressing plants showed a positive hybridization signal, and the middle 8601 did not show a hybridization signal. The number of hybridization bands can represent the copy number of the gene of interest into the genome, and the ^3⁄4 gene is successfully transferred into the genome of the plant in 1-3 copies.

According to T. A total of 7 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).

7. Quantitative RT-PCR analysis of the expression of ^3⁄4 gene in ^73⁄4 overexpressing plants The total RNA of the 1\ generation plants of each TiSTK1 overexpressing line was extracted and reverse transcribed into cDNA, diluted 10 times and used as a template, and Q-RT-PCR amplification was performed using primer pairs consisting of TiSTK1-SNPF and TiSTK1-SNPR. The expression of the 73⁄4 gene was analyzed; the 18S rRNA gene was used as the internal reference (primer pair consisting of 18S rRNA-QF and 18S rRNA-QR; the ratio of the expression of ^73⁄4 gene and 18S rRNA gene was used as the ^73⁄4 gene in the plant) Relative expression). The anti-yellow dwarf translocation line TC14 and the anti-yellow dwarf translocation line YW642 were used as positive controls, and the middle 8601 was used as a negative control.

TiSTKl-SNPF : 5 ' _gctcccctccttcccctt_3 ' ;

TiSTKl-SNPR: 5, -cgaccttgtggtaatacggca-3 '.

18S rRNA-QF: 5' - GTGACGGGTGACGGAGAATT- 3';

18S rRNA-QR: 5 ' - GACACTAATGCGCCCGGTAT- 3, .

Some results are shown in Figure 4. Compared with Zhong 8601, the relative expression levels of TiSTK1 gene in seven TiSTK1 overexpressing lines were significantly increased.

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.

Identification of disease resistance of transgenic plants

1\ generation plants of each of the ^73⁄4 overexpressing lines, YW642 (positive control), medium 8601 (negative control), and transgenic vector control plants of the control vector were subjected to the following disease resistance. Identification, 20 strains per 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. .

1. Disease resistance classification

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.

Table 2 Classification criteria for the severity of wheat yellow dwarf disease

Domestic Standard (Level 11) Act

0 health plant

1 part of the tip yellowing

2 under the flag leaf, one leaf yellowing

3 under the flag leaf 2 leaves yellowing

4 flag leaf yellowing 1/4, under the flag leaf, one leaf yellowing

5 flag leaf yellowing 1/4, under the flag leaf 2 leaves yellowing

6 flag leaf yellowing

7 flag leaf yellowing, under the flag leaf, one leaf yellowing

8 2 leaves under the flag leaf and flag leaf 9 plants dwarf, but can head

10 Plant dwarfing is significant, no heading

2, the relative content of BYDV

The specific primers 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 '.

The results are shown in Figure 6 (the result is the average of the plants of this line).

3, ELISA test

Detection by double antibody sandwich (DAS) in enzyme-linked immunosorbent assay. PBS buffer (currently available) was used as a blank control. Take 1-2 leaves under the flag leaf as test samples. The BYDV-GAV and BYDV-PAV ELISA test kits were purchased from Agida AG of AGDIA USA, containing BYDV-GAV antiserum and BYDV-PAV antiserum. Follow the instructions in the kit as follows:

(1) Add the virus antiserum (diluted 1:1000 in the coating buffer) to the well of the enzyme-linked plate, 200μ per well, place the enzyme plate in the white porcelain plate, cover the moisturizer, 37° C was incubated for 4 hours.

(2) Weigh 0.2 g of the test sample, add PBS buffer (50X) lml, squeeze the juice on the juicer, and collect the supernatant.

(3) Drain the solution on the enzyme plate of step (1) and wash the plate 3 times with washing buffer (PBS containing 0.5% Tween20) for 5 minutes each time.

(4) In the enzyme-linked plate of step (3), add 200 μl of each step to the supernatant of step (2) and incubate at 4 °C overnight.

(5) 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.

(6) In the enzyme-linked plate of step (5), add 200 μl per well and dilute 1000-fold alkaline phosphatase-labeled IgG with ligation buffer (PBST containing 2% PVP), 200 μl per well, capped Moisturize, incubate for 5-6 hours at 37 °C.

(7) Drain the solution on the enzyme plate of step (6) and wash the plate 3 times with washing buffer (PBS containing 0.5% Tween20) for 5 minutes each time.

(8) Add the newly prepared substrate solution (0.6 g PNP per ml) to the enzyme-linked plate of step (7), 200ulo per well.

(9) After the color reaction was observed, the 0D value at 405 nm was measured on a microplate reader within 1 hour of the reaction.

The results are shown in Table 3. Table 3 Results of disease resistance identification (results are the average of the plants)

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.

The above-mentioned molecular detection and disease resistance identification of the T ₂ generation plants of the ^ 3⁄4 gene overexpressing lines were basically consistent with the results of the 1\ generation plants, indicating that the transferred ^73⁄4 gene can be stably inherited and transcribed, which confers transgenes. The broad-spectrum resistance of wheat can be inherited.

The same result was obtained when the above experiment was carried out by changing the nucleotide number 1271 from G to A. Example 3: RNA interference expression vector and its anti-yellow dwarf function analysis of transgenic wheat I. Construction of RNA interference vector

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. The repeat sequence of the RNA interference vector pAHC25_Adh-TiSTKl.

1, primer design

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). In order to enable the inverted repeat sequence to be inserted into the multiple cloning site of the vector PAHC25, 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, .

2. Construction of RNA interference vector

(1) The total RNA of the intermediate buckwheat grass was extracted and reverse transcribed into cDNA.

(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).

(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).

(4) The reverse fragment was digested with restriction endonucleases oRI and Bglll, and the digested product was recovered.

(5) The pHMW-Adh_Nos vector was digested with restriction endonuclease coRI and BwiRl {Bglll homologous enzyme), and the vector backbone (about 4554 bp) was recovered.

(6) The digested product of the step (4) is ligated to the vector backbone of the step (5) to obtain an intermediate plasmid pHMW-STKR-Adh.

(7) The forward fragment was digested with restriction endonucleases Bglll and Ncol, and the digested product was recovered.

(8) The intermediate plasmid pHMW-STKR-Adh was digested with restriction endonucleases Bgll I and Ncol, and the vector backbone (about 5062 bp) was recovered.

(9) The digested product of the step (7) is ligated to the vector backbone of the step (8) to obtain an intermediate plasmid pHMW-STKR-Adh-STKF.

(10) The intermediate plasmid pHMW-STKR-Adh_STKF was digested with restriction endonucleases Sma I and Seic I to recover a fragment of about 1150 bp.

(11) Restriction of pAHC25 vector by restriction endonuclease Sma I and Sac I, recovery of vector backbone

(about 7818 bp.

(12) 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). According to the sequencing results, 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).

Second, ^ 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.

Some results are shown in Figure 7. Positive control and partial T. The expected fragment of about 419 bp was amplified from the plant, while the corresponding fragment was not amplified in the negative control, and the T of the expected fragment was amplified. The plant is the S 3⁄4 inhibitory plant. A total of 7 strains of T were obtained. When the S73⁄4 gene was inhibited, seven S73⁄4 inhibitory strains (strain 124, strain 125, strain 127, strain 142, strain 147, strain 153, and strain 157) were obtained.

In order to further confirm the results of PCR identification, determine whether the target gene has been integrated into the wheat genome and the number of copies inserted, 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.

The total 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 above-mentioned molecular detection of the T ₂ generation plants of the ^73⁄4 gene-inhibiting strain was basically consistent with the results of the 1\ generation plants, indicating that the transferred interference fragments of the ^73⁄4 can be inherited and interfered with expression.

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.

Identification of disease resistance of transgenic plants

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:

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) .

Some of the results of the Q-RT-PCR assay are shown in Figure 9. The photo is shown in Figure 10.

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.

The results showed that by introducing the interference functional fragment, the expression of the ^73⁄4 gene in the yellow dwarf translocation line YW642 was effectively inhibited, and the control of BYDV-GAV infection was lost, resulting in a viral content in the body, showing a feeling of BYDV. Disease, indicating that the ^3⁄4 gene is a key gene against yellow dwarf disease.

The above-mentioned disease resistance identification was carried out on the plants of the ^73⁄4 gene-inhibiting strain, which was basically consistent with the results of the 1\-generation plants, indicating that the transferred interference fragments of the ^73⁄4 can be inherited, and the interference of the resistance can be stably inherited. It is proved that the ^73⁄4 gene is a key gene against yellow dwarf disease and a gene essential for expression in response to yellow dwarf disease. Example 4: Identification of disease-resistant plants and susceptible plants based on ^ 3⁄4 gene

Based on the two consecutive SNPs of ^73⁄4 gene 5, UTR, 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'. Using primer pairs consisting of TiSTK1-SNPF and TiSTK1-SNPR, 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^).

Specific conditions of PCR: pre-variation of 3 min at 94 °C; 45 °, 45 °, 63 V, 35 s, 72 °C, lmin, 2 cycles; 94 °C 45 s, 61 V 35s, 72 V l min, 5 cycles; 94 °C 45 s, 59 V 35s, 72 °C lmin, 30 cycles; 72 °C lOmin; 4 °C storage.

As a result, as shown in Fig. 11, 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.

Using the SNP marker specific to the ^3⁄4 gene to detect 100 individuals in the F2 population of the yellow transgenic material wheat translocation line YW642 and the yellow-yellow disease material wheat YW641S hybrid, the results showed that the molecular marker detection and disease resistance The results were consistent, further confirming that the ^3⁄4 gene is a specific gene carrying 3⁄4 2 anti-yellow dwarf wheat. Example 5, Analysis of TiSTK1 protein and its coding gene

I. Subcellular localization of TiSTK1 protein

According to the structural analysis of TiSTK1 protein, the TiSTK1 kinase protein has a membrane binding site. To verify the function of this protein structure, 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. coli, 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 ΒίΐηΆΙ (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. A novel ERF transcript ion act ivator in wheat and its induct ion kinet i cs after pathogen and hormone treatments. Journal of Experimen tal Bo tany 58, 2993 - 3003 ) Put P35S : : TiSTKl-GFP into onion epidermal cells for transient expression, while empty vector _P35S:: GFP in onion epidermal cells When expressed as a control. The results shown in Figure 11, TiSTKl-GFP is only expressed in the cell membrane, and GFP was expressed on the membrane onion epidermal cells in the nucleus, indicating TiSTKl is a membrane protein.

Second, the transcriptional expression characteristics of ^3⁄4 gene

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 results are shown in Fig. 13. After induction by BYDV-GAV, the expression of ^3⁄4 in YW642 leaves was increased to the highest level of 12 hours, indicating that BYDV-GAV infection up-regulated the transcriptional expression of S73⁄4.

Industrial application

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. It is indicated that 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.

Claims

Rights request

1. A protein, as follows (a) or (b):

2. A gene encoding the protein of claim 1.

The gene according to claim 2, wherein the gene is a DNA molecule according to any one of the following 1) to 5):

1) a DNA molecule of sequence 2 from nucleotides 171 to 1448 at the 5' end;

2) the molecule shown in SEQ ID NO: 2 in the sequence listing;

3) the molecule shown in SEQ ID NO: 3 in the sequence listing;

5) A DNA molecule having more than 90% homology with a DNA sequence defined by 1) or 2) or 3) and encoding a plant yellow dwarf resistance-related protein.

A recombinant expression vector, expression cassette, transgenic cell line or recombinant strain comprising the gene of claim 2 or 3.

The recombinant expression vector according to claim 4, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene of claim 2 or 3 into a multiple cloning site of the vector pAHC25.

6. A primer pair that amplifies the full length of any of the genes of claim 2 or 3 or any of its fragments.

7. The primer pair according to claim 6, wherein the primer pair consists 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.

A method for cultivating a transgenic plant, which comprises introducing the gene of claim 2 or 3 into a plant of interest to obtain a transgenic plant having yellow dwarf resistance higher than said plant of interest.

The method according to claim 8, wherein the gene of claim 2 or 3 is introduced into the plant of interest by the recombinant expression vector of claim 4 or 5.

10. Method according to claim 8 or 9, characterized in that the plant of interest is a monocot or a dicot; the monocot is preferably wheat.

The method according to any one of claims 8 to 10, wherein: said yellow dwarf disease is

Caused by B YD V-GA V strain or B YD V-PA V strain.

12. An interfering RNA for inhibiting expression of a protein according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO:9 of the sequence listing.

13. The DNA encoding the interfering RNA according to claim 12, comprising a fragment A and a DNA fragment B; wherein the fragment A is as shown in the sequence 2 of the sequence listing from nucleotides 2 to 493 at the 5' end;

DNA fragment B is inversely complementary to the DNA fragment A.

The DNA according to claim 13, wherein: the sequence of the sequence is 5 Shown.

A recombinant plasmid comprising the specific fragment of claim 13 or 14.

The recombinant plasmid according to claim 15, wherein the recombinant plasmid is a recombinant plasmid obtained by introducing the sequence shown in the sequence 5 of the sequence into the multiple cloning site of the vector pAHC25.

A method for cultivating a transgenic plant, which comprises introducing the recombinant plasmid of claim 15 or 16 into a plant of interest to obtain a transgenic plant having yellow dwarf resistance lower than the plant of interest; 2 or 3 plants of the stated gene.

A method for assisting in the identification of a plant carrying the gene of claim 2 or 3, comprising the steps of: PCR-amplifying the primer pair according to claim 7 using the genomic DNA of the plant to be tested; if 739 bp is obtained A fragment of the plant to be tested is a candidate plant carrying the gene; if a 739 bp fragment is not obtained, the plant to be tested is a candidate plant that does not carry the gene.

A method for assisting in the identification of a plant carrying the gene of claim 2 or 3, comprising the steps of: PCR-amplifying the primer pair according to claim 7 using the cDNA of the plant to be tested; if 235 bp is obtained A DNA fragment, the plant to be tested 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 method according to any one of claims 18 or 19, wherein: the plant to be tested is an intermediate buckwheat grass, a diploid addition line L1 against yellow dwarf disease, and an anti-yellow dwarf translocation line YW642. Anti-yellow dwarf translocation line YW243, anti-yellow dwarf translocation line TC14, wheat medium 8601, wheat Chinese spring, yellow dwarf-resistant wheat - intermediate buckwheat grass Z1, wheat-intermediate buckwheat translocation line P961341, Wheat Pm97034, wheat Y15 or wheat Y26.

21. Use of the method according to any one of claims 18 to 20 for assisting in the identification of plants against yellow dwarf disease; plants carrying said genes are candidate anti-yellow dwarf plants, and plants not carrying said genes are candidates Yellow-light dwarf plants.

22. The protein of claim 1, or the gene of claim 2 or 3, or the recombinant expression vector, expression cassette, transgenic cell line or recombinant strain of claim 4 or 5, or the primer of claim 6 or Or the interfering RNA of claim 12, or the DNA of claim 13 or 14, or the recombinant plasmid of claim 15 or 16, or the method of any one of claims 8 to 11, or claim 17 to The use of any of the methods of 20 in plant breeding.