Optimization Of Virus Induced Gene Silencing System And Function Analysis Of Wheat(Triticum Aestivum L.) SBEIIb

VIGS technology is a kind of plant defense mechanisms mediated by RNA, which maybe the molecular basis of post-transcriptional gene silencing. When the vectors carrying thegene fragment into the plant, it can induce endogenous gene silencing. VIGS is regarded as apowerful tool for studying gene function. Since Barley stripe mosaic virus has a wide hostrange, it provides an effective tool for studying genes function in wheat. In order to carry outa theoretical basis and technical guidance for gene function study in wheat by VIGS. Weconstructed BSMV vector with PDS gene to explore ways of inoculation, vaccination site,effects of temperature and inoculum culture of BSMV-VIGS system. Eventually weestablished a relatively optimal system. And the system was used to study SBEIIb gene’sfunction. The main results are as follows:1. Wheat PDS gene fragment and SBEIIb fragment was cloned by PCR. We constructedthe VIGS recombinant vectors.2. Spikes were inoculated by injection and rubbing at heading stage. The results showedthat photo bleaching appeared at5-8dpi, distinct at15dpi, and reached its highest intensity at23-26dpi. Only20%plant was infected successfully by injection. But that rate was90%byrubbing. The phenotypic changes in flag leaves whict were inoculated by rubbing were quitesimilar with that in spikes. Photo-bleaching was observed on spikes about20dpi of flag leafbecause of migrate of the gene silencing. Wheat flag leaves were inoculated by rubbing atheading stage and placed under21-24℃and26-29℃. The results showed that around90%of them were infected successly. At higher temperatures, the virus developed too fast whichmade too heavy symptoms to find photobleaching phenotypes. And the plants also got otherserious diseases. That affects the observation and molecular detection of gene silencing.Therefore, the higher temperatures are not conducive to the implementation of the VIGS onwheat. In addition, according to the tolerance range of plants, inoculation times only inducedthe success rate of gene silencing to nearly100%. So the better way is rubbing the spikes at21-24℃3. Determination of pigment content and the qRT-PCR analysis showed that83.6%and91.4%decrease was in Chlorophyll a and carotenoids at20dpi. The content of Chlorophyll bwas less than40%in12dpi-20dpi. Total chlorophyll abundance was less than20%around20dpi.50%decrease in the transcript content of the PDS gene was observed at3dpi ofspikes. The PDS transcript level was always around10%between9dpi and20dpi. PDStranscript level was suppressed until25dpi. So the chlorophyll content decreased caused bythe silencing of PDS gene.4. We infected the wheat with BSMV which carrying SBEⅡb at heading atage, andextracted total RNA of6dpi,9dpi,12dpi,15dpi,20dpi. qRT-PCR results showed thatSBEⅡb gene transcript level was increased at6dpi. Its content was only49%. The ratio wasdropped to43.8%and5%at12dpi and20dpi. The proportion of amylose was increased by10.45%and the proportion of resistant starch was increased by11.45%. So the vectorssilenced the SBEⅡb gene and caused changes in starch. But they showed no significantdifference. In this study, we constructed BSMV-VIGS vectors of PDS and of SBEIIb. We exploredthe VIGS system with the PDS gene as an indicator, such as ways of inoculation, vaccinationsite, the effects of temperature and inoculum culture. Eventually we established a relativelyoptimal system. And the system was used to study SBEIIb gene’s function. We carried out atheoretical basis and technical guidance for gene function study in wheat spikes by VIGS.