Isolation Of Defense Genes Induced By Barley Yellow Dwarf Virus And Aphid And Establishment Of Gene Silencing System In Wheat

Wheat yellow dwarf, one of the most important viral diseases, caused by barley yellow dwarf virus(BYDV). It often causes serious yield loss. BYDV is obligately transmitted by aphids. there is short of germplasm resources that possess BYDV resistance gene in wheat. However, 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 Thinopyrum 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, YW642. 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 is very useful for developing new methods to breeding for disease resistance.Analysis of barley yellow dwarf virus accumulation in wheat is very useful for understanding and studying the mechanism of resistance to BYDV. Semi-quantitative RT-PCR was employed to investigate the different evolution of the content of BYDV-GAV in resistant and susceptible wheat plants. The high resistance to BYDV was confirmed at molecular level in YW642. The results also showed that the defense reaction was actived before 2-3 dpi in resistant wheat.In this thesis the suppression subtraction hybridization(SSH) method was employed to identify BYDV-induced genes from wheat-Thinopyrum intermedium translocation lines YW642 infested with viruliferous aphids. ft was used as the "tester" that the cDNA from seedlings inoculated with viruliferous aphids of BYDV-GAV, while cDNA from untreated leaves was used as the "driver". The subtracted cDNA library was constructed with the PCR-Select Subtraction Kit(CLONTECH). The initial screening of 1534 subtracted clones resulted in 22 candidate clones. After confirmated by semi-quantitative RT-PCR, six clones showed up-regulated expression after inoculation with viruliferous aphids. The six clones also showed the similar expression model after inoculation of nonviruliferous aphids. The above clones were designated as wai(wheat aphid-induced) gene. The results of sequence analysis by BLASTn showed that the six differentially expressed genes shared high sequence homologous with three pieces of sequence of LTR retrotransposons of wheat, PR-4 gene(classified as chitinase), β -1, 3-glucanases and Hordeum vulgare low temperature-responsive RNA-binding protein respectively. The study sets up a fundamention for the elucidation of aphid defense mechanisms and cloning aphid-inducible promoter in wheat.After screening of cDNA-AFLP of differentially expressed gene from four samples, including 2dpi and 3dpi of HW642 inoculated with aviruliferous aphids and viruliferous aphids respectively. One hundred and nine gene fragments were obtained which were induced by BYDV in YW642. In order to getting rid of the genes that were instinctively induced by BYDV under common wheat background. In following cDNA-AFLP analysis, 2dpi and 3dpi samples of susceptible wheat were used as control which were inoculated with viruliferous aphids. we got twelve BYDV-induced fragments that only exist in resistance plants. After confirmed by reverse Northern, two clones were true. One of them was single copy in wheat genome. This one may represent a new gene because there was no matching sequence in GeneBank.Barley stripe mosaic virus (BSMV) that induced successfully PDS gene silencing in barley was used to set up virus-induced gene silencing (VIGS) system in wheat. The results showed that...