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Anti- wheat yellow dwarf disease candidate gene cloning , characterization and separation of the pathogen inducible promoter

Author: LiNing
Tutor: FanShouJin
School: Shandong Normal University
Course: Botany
Keywords: Wheat (Triticum aestivum) Barley yellow dwarf virus (BYDV) Resistance-related gene NBS-LRR VIGS Promoter
CLC: S512.1
Type: Master's thesis
Year: 2008
Downloads: 120
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Wheat yellow dwarf disease (WYD) is one of the most serious diseases of cereals worldwide.It is caused by barley yellow dwarf virus, which was spreaded by aphids and often causes serious yield loss. Thinopyrum intermedium, a wheatgrass, shows a high level of resistance to BYDV. The wheatgrass possesses three resistance genes which locate on the 7St. 7E and 2Ai-2 chromosomes in Thirropyrum intermedium. A resistance gene from 7St chromosome was designated as Bdv2. Researchers crossed the wheatgrass to wheat to obtain a series of virus-resistant wheat translocation lines which carries Bdv2, for example, HW642. However, the resistance gene-mediated molecular mechanism reaction against BYDV which will be further studied Research shows that the protein encoded by plant disease resistance gene determines the host plant could open resistance systems or open the system level, and the real role of the resistance is the product from the defense gene expression.It is necessary to isolate BYDV resistance genes and some important resistance signal regulation components for unraveling BYDV resistance mechanism and solving above-mentioned problems. Identification of host genes involved in defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in plants and it is very useful for developing new methods to breeding for disease resistance.In this study,based on screening the fragments of specific expression induced BYDV through cDNA-AFLP technology . Gene specific primers were designed and the full-length cDNA sequence of two resistance-related wheat yellow dwarf disease candidate genes through the 5’ RACE,screening BYDV-induced cDNA library and 3’ RACE methods were obtained. The resistance-related gene were named as P38M16 and P6M3 temporarily. The length of P38M16 was 3218 by which included a 1938 bp-by complete open reading frame encoding P38M16 protein of 646 amino acids. The cDNA sequences of P6M3 containing full-length open reading- frame was isolated from wheat and Thinopyrum intermedium.cDNA with full length ORF was isolated from wheat and Thinopyrum intermedium, temporary named TaP6M3 and TiP6M3. The length of TaP6M3 was 4106 by which included a 3180 bp-by complete open reading frame encoding TiP6M3 protein of 1060 amino acids. The length of P38M16 was 4072 by which included a 3096 bp-by complete open reading frame encoding P38M16 protein of 1032 amino acids.The deduced amino acids the two protein consisted of nucleotide binding site (NBS) domain, a leucine-rich repeats(LRR) domain, and a hydrophobic domain, which were the conserved domains of plant resistance genes. Semi-quantitative RT-PCR analysis showed that treatment with signaling molecules,including salicylic acid and methyl jasmonate ,did not activate transcription of the two candidate genes ,indicating that the two candidate genes expression is not regulated by defence signalling pathways triggered by these molecules.The two candidate genes were not induced by treatment with BYDV. The P38M16-related sequences were presented as 3 copies in resistant wheat and Thinopyrum intermedium genome.according to the result of southern blotting.however, there are 5 copies P38M16 in susceptible lines of wheat genome.The virus-induced gene silencing (VIGS) vectors were constructed,it will be used to set up virus-induced gene silencing (VIGS) system in wheat. It will be single out to analyze function of gene. It will be a powerful tool for determining gene function in wheat and its relative.The expression and regulation of plant gene has been the hotspot study in molecularbiology. Promoter was an important cis-acting element. Cloning promoter was important to study gene regulation, vectors construction and target protein expression. Glu,ERF1b belong to the pathogeny promoter. In order to isolate wheat Glu,ERF1b promoter, wheat genome was digested with HindIII. A special adaptor was ligated to the ends of the digested DNA fragment, and the ligated DNA fragment was used as a template for adaptor PCR amplication. The adaptor ligated HindIII digestion DNA was cloned and sequenced. Cloning these promoters were important to the study of plant resistence. The cloning of resistance-related promoter has been explored using Link Adaptor PCR method. But only part of the promoter sequence is amplified due to large hexaploid wheat genome and too many repeat sequences. Sequence extension often happen deviate rely solely on one end to the sequence 5ˊ-extended. We will explore other cloning methods of promoter in future. Our ultimate goal is to get full-length sequence of the promoter and construct the promoter - GUS chimeric gene expression vector. We will design a transient expression test in the plant tissue.Then single-cotyledon efficient expression vector from the promoter and disease-resistant genes will be transformed into a susceptible wheat variety to obtain a new resistant wheat variety.

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CLC: > Agricultural Sciences > Crop > Cereal crops > Wheat > Wheat
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