Development Of New Wheat Germplasm With Resistance To BYDV And WDV Mediated By RNA Interference

Wheat is one of the most important food crop in the world and plays a vital role in food securityand national economy. Wheat yellow dwarf disease caused by Barley yellow dwarf virus (BYDVs) isone of the most destructive viral disease of wheat crop not only in China but also in the world. Theepidemics of BYDVs generally leads to a yield loss around20%to30%, even up to50%or completeproduction failure compared to the normal production level.In recent year, wheat dwarf disease caused by Wheat dwarf virus (WDV) has been an increasingdestructive viral disease in Europe, North Africa, Asia and Oceania. Especially in Europe, the yield losscaused by WDV has been over40%to80%. Wheat dwarf disease disease was firstly detected andreported by our research group in2007in China. Subsequently, it was found in12provinces includingShaanxi, Gansu, Hebei and Yunnan. This disease, which caused serious yield loss in Hancheng city,north of Shaanxi province, has been a potential threat to the wheat in northwestern of China.The most efficient and economic method to control viral disease is the application of resistantvarities. However, it is very hard to obtain a new resistant wheat varity with high yield and goodquantity through traditional breeding. The recently discovered RNA interference (RNAi) is an importantmechanism of host against pathogens by specific degradation of the mRNA mediated bydouble-stranded RNA. The transgenic plant could show the resistance to the viral disease when the virusgenes or their homologous fragment were degraded by RNA interference. It gives us a opportunity thatwheat lines with resistance to BYDVs and WDV could be obtained by transgenic breeding based on theprinciple of RNAi.The purpose of this research is to obtain the transgenic wheat lines with high resistance toBYDV-GAV and WDV. Firstly, we developed a series of RNAi vectors containing conserved coatprotein gene sequences of BYDV-GAV and WDV, which are suitable for wheat transformation. Theseplasmid constructs were transformed into callus of wheat immature embryo by particle bombardment.The genetically modified regeneration were obtained after the tissue culture. The seedlings were testedand selected by PCR, Southen blotting, Q-RT-PCR and bioassay. In addition, the linear minimumexpression box for particle bombardment transformation was designed for biosafety of transgenic wheat.The expression box was transformed into callus of wheat immature embryo via particle bombardmentand the positive seedlings were obtained by PCR. Among of them, the resistance plants without othervector sequence and antibiotic gene could be screened for the next application. The main results arelisted as follows:1. Construction of RNAi expression vector: We have developed RNAi expression vectors for wheattransformation including pMCG161+/-BW, pMCG161+BW, pMCG161-BW, pMCG161+/-W,pMCG161+W, pMCG161+/-W, pMCG161-W, pWMB006+/-HBW, pWMB006+HBW,pWMB006-HBW and gene expression casseett+/-HBW.2. Biolistic transformation and molecular detection of foreign genes: Those vectors had beentransfered into immature wheat embryos. Then transgenic tissues were selected by herbicide resistance and detected by molecular methods. PCR results showed36plants positive for pMCG161+/-BW,5plants positive for pMCG161+BW,5plants positive for pMCG161-BW,20plants positive forpWMB006+/-HBW,5plants positive for pWMB006+HBW,5plants positive for pWMB006-HBW,10plants positive for pMCG161+/-W,4plants positive for pMCG161+W,4plants positive forpMCG161-W,11plants positive for gene expression casseett+/-HBW. respectively. The averagetransformation rate and the highest transformation rate were1.2%and2.0%respectively. Southenblotting analysis showed that the foreign sequence had been integrated into the transgenic wheatgenome with1-3copies. Real time quantitative PCR analysis revealed that the expression levels of thetarget genes in transgenic plants was relatively lower than them in wild type. The gene expression ofpositive plants which transformed with pMCG161+/-BW1is0.3of the controled ones, withpMCG161+/-BW2is0.24and with pMCG161+/-HBW is0.15. It indicated that the gene silencingeffect played a role in transgenic plants.3. Bioassay of transgenic plants: By observation of their agronomic characters of T1plant, they werethe same as controls. The evaluation of the lines T2and T3resistant to BYDVs by artificial inoculationshowed that disease resistant GM wheats had been obtained.