Real-Time Pcr Detection Of Wheat Yellow Mosaic Virus And Transgenic Wheat Mediated By Rna-Intcrference

Wheat yellow mosaic disease is an important disease in different regions of China. The virus,Wheat yellow mosaic virus is classified in the genus Bymovirus, family Potyviridae. Filamentousviruses transmitted by the plasmodiophoraceous fungus P. graminis cause typical symptoms includingstreaking, mosaic, dwarfing and reduced heading in wheat. The genome of WYMV is comprised of two(+) single-stranded RNAs, RNA1encodes for coat protein (CP) and six others: P3,7K, nuclearinclusion protein a (NIa), nuclear inclusion protein b (NIb), cytoplasmic inclusion protein (CI),14K;RNA2encodes for a polyprotein that contains P1and P2.Symptoms of WYMV infection are similar to those caused by WSSMV and other biotic andabiotic agents. For this reason and the fact that there are several virus species existed together in thefield, detection and diagnosis of WYMV is an important area of study. Here, we report for the first timea sensitive and reliable real-time PCR procedure for WYMV detection in infected wheat tissues. Theaccuracy of normalized data is highly dependent on the internal control genes β-actin. Primers andprobe for specific detection of WYMV were designed within the conserved region of coat protein gene.All TaqMan probes were labeled with FAM at the5’ end and TAMRA at the3’ end.The curve slope andamplification efficiency which established of the standard sample conform to the quantitativerequirements. Comparing the results, real time RT-PCR was close104-fold more sensitive than indirectELISA.RNAi which introduced by double-stranded RNA (dsRNA), leads to gene silencing phenomenon.It is a defense mechanism, to adjust the expression of genes. In this study, we designed WYMV CPmediated dsRNA precursor accordance with the mechanism of RNAi, inserted into the pWMB006plasmid, transformed into callus of wheat embryo and then eliminated the co-transformation markergenes. We want to obtain transgenic wheat lines with resistance to WYMV.The interference vector pWMB006DsCP and three control vectors were constructed, and thentransformed into callus of wheat embryo. By PCR analysis in T0generation,15,8,3and5transgenicplants were obtained from pWMB006DsCP, pWMB006sCP, pWMB006asCP and pWMB006plasmids,respectively. The high transgenic rate of the pWMB006DsCP was0.73%, the other vector transgenicrate were0.34%,0.25%and0.15%, respectively.The separation of T1generation do not follow theMendel’s law. The infected plants show obvious yellowing, dwarfing and stem lodging phenomenon,while disease-resistant varieties growth vigorous. The integration of the exogenous gene causes thechanges of plants phenotype.