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Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof

A yellow dwarf disease and gene technology, applied in plant gene improvement, application, plant peptides, etc., can solve problems such as difficult exchange and separation, positioning Bdv2 and molecular marker genetic distance, distant relationship, etc.

Inactive Publication Date: 2010-05-12
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the very distant genetic relationship between Tripyrum intermedia and wheat, it is difficult to exchange and segregate between the Chromosome (7XL, 7Ai#1L) fragment carrying Bdv2 and some homologous chromosomes (7D) of wheat, and it is difficult to segregate through the normal F2 The population finely maps the genetic distance between Bdv2 and molecular markers. Therefore, the map-based cloning method is not suitable for isolating important genes such as Bdv2 on the fragment of clone 7Ai#1L that determine the resistance to yellow dwarf disease

Method used

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  • Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof
  • Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof
  • Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, discovery of new gene

[0052] 1. Screening and sequence analysis of TiDPK1 gene-specific EST functional markers

[0053]Since Chromosome 7XL (7Ai#1L) of T. intermedia can translocate to chromosome 7DL of wheat, it indicates that Chromosome 7XL of T. intermedia (7Ai#1L) has a partial homology relationship with chromosome 7DL of wheat. Although the functional genome research on Tripyrum intermedia has not yet been carried out, many ESTs have been mapped to the 21 chromosomes of wheat due to the implementation of the wheat functional genome, especially the EST project. Therefore, we used 70 EST sequences on wheat chromosome 7DL to design PCR primers, and optimized the conditions for PCR amplification and gel electrophoresis analysis. DNA of arm (7Ai#1L) double end addition line DT7XL, wheat-Thinopyrum intermedium translocation line YW642) and Bdv2-free yellow dwarf wheat material (Zhong 8601, CS, YW641S) was amplified by PCR and SDS-PAGE Gel electrophoresi...

Embodiment 2

[0056] Embodiment 2, the cloning of gene and its analysis

[0057] 1. Gene cloning

[0058] According to the full-length cDNA (nucleotide sequence shown in the sequence 1 of 1842bp) spliced ​​according to the gene fragment (BQ238952 sequence and 3' RACE obtained sequence) of embodiment 1, the PCR amplification primer (QC-U of full-length sequence) of design : 5'-GCAGCACGCCAATCCGC-3', QC-L: 5'-GCAGCCGCTATCAACACAAGAC-3'), using the cDNA of Thinopyrum intermedia Z1146 (purchased from the resource germplasm bank of the Institute of Crop Science, Chinese Academy of Agricultural Sciences) as a template, PCR amplification The full-length cDNA sequence of TiDPK1 gene (specific conditions: 94°C for 3min; 94°C for 30s, 62°C for 35s, 72°C for 1min30s, 5 cycles; 94°C for 30s, 60°C for 35s, 72°C for 1min30s, 5 cycles; 94°C for 30s , 58°C for 35s, 72°C for 1min30s, 25 cycles; 72°C for 10min;), obtain the fragment shown in the 32-1827th position from the 5' end of the sequence 1 in the sequ...

Embodiment 3

[0070] Example 3, Anti-yellow dwarf functional analysis of TiDPK1 gene

[0071] 1. Silencing the TiDPK1 gene in YW642 (virus-mediated gene silencing technology)

[0072] The anti-yellow dwarf function of TiDPK1 was verified by BMSV virus-mediated gene silencing technology. Primer TiDPK1-FV: 5'-AA ACATGCCGTATTACCACAAG-3' italicized sequence is the NotI restriction site, TiDPK1-RV: 5'-CC GCCAATGCGTCCTCCTTG-3' sequence in italics is the PacI restriction site, the high-fidelity TAQ enzyme amplifies the specific fragment of the TiDPK1 gene (from the 289th to the 548th position of the 5' end of Sequence 1), and obtains PacI and NotI restriction enzymes on both sides respectively A specific fragment of the TiDPK1 gene at the locus. Recover the TiDPK1 gene-specific fragment digested by PacI and NotI, and connect to the γ vector of BSMV virus digested by PacI and NotI (the γ vector of BSMV virus, refer to Burch-Smith TM, Anderson J C, Martin G B, Dinesh-Kumar S P .Applications an...

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Abstract

The invention discloses a plant yellow dwarf resistance-associated protein TiDPK1, a coding gene and application thereof. The protein is a protein 1) or 2) as follows: 1) the protein consisting of an amino acid sequence shown by a sequence 2 in a sequence table; and 2) the protein which is derived from 1) by performing substitution and / or deletion and / or addition on the amino acid sequence of the sequence 2 in the sequence table and is related to stress resistance of plants. The coding gene TiDPK1 of the protein, transgenic lines and a method also belong to the protection of the invention. Experiment proves that the gene is specifically expressed in yellow dwarf resistant wheat (such as YW642) and is regulated up by the induction of BYDV; TiDPK1 is a membrane protein; BYDV concentration is raised and susceptibility index rises in silent original yellow dwarf resistant wheat YW642 of the TiDPK1; over-expressed transgenic wheat improves resistance to yellow dwarf; and the TiDPK1 gene is shown to be an important gene resistant to yellow dwarf.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a plant anti-yellow dwarf related protein extraction TiDPK1 and its coding gene and application. Background technique [0002] Barley yellow dwarf virus can infect small-grained cereals and cause yellow dwarf disease of small-grained cereals such as wheat, barley, oats and pastures, resulting in serious loss of yield. Barley yellow dwarf virus (BYDV) is transmitted by aphids. According to the transmission characteristics of different aphids, barley yellow dwarf virus was divided into the following BYDV-PAV, BYDV-MAV, BYDV-GAV, BYDV-GPA, CYDV-RPV and SGV strains and RMV strains. Among them, BYDV-GPV (a unique strain type in my country) and BYDV-GAV are the mainstream strains in my country. [0003] Yellow dwarf is an important viral disease of wheat. Once wheat is infected with yellow dwarf disease, there is no cure, which will reduce the yield and quality of wheat. Therefore, yell...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N15/82C12N1/15C12N1/19C12N1/21C12N5/10C12N15/11A01H5/00
Inventor 张增艳陈亮辛志勇高兰英徐惠君刘艳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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