The Expression Of Rice Serrate Protein In The Infecting Model Of Rice Ragged Stunt Virus

Rice ragged stunt disease is spread by Nilaparvata Lugens, and its pathogen isRRSV (Rice ragged stunt virus).Its typical symptoms mainly showed dwarf,rolledleaf,leaf margin serrate.Some studies have found that the formation of dwarf, rolledleaf and leaf margin serrate have most important relationship with serrated proteingene(SE). In our study, Real-time PCR technology was used to detect the expressionlevel of SE gene in rice plants infecting RRSV, and compared with healthy rice,wefound that SE-1gene expression quantity is obviously up-regulated when riceinfecting RRSV, while SE-2and SE-3gene expression quantity showed no obviousdifference when rice infecting RRSV.To study the fuction of SE promoter in rice,In this experiment, We constructedthe transformation vector Pro-SE-1, Pro-SE-2and Pro-SE-3, which were transferredsuccessfully into Oryza sativum cv Nipponbare and Arabidopsis via Agrobacteriumtumefaciens-mediated transformation and Floral dipping method.We obtained75positive T0transgenic rice in all Plants, of these,27positive of Pro-SE-1,43positiveof Pro-SE-2,5positive of Pro-SE-3and obtained T1resistant Arabidopsis plant wereselected by PCR detection and resistance screening.GUS histochemical staining method were used for analysis localization and tissueexpression in transgenic rice, and the result showed that GUS gene can expressed inthe transgenic rice leaves compared with wild type plants.GUS gene expressedweakly in Pro-SE-1transgenic rice,and less in root and stem.Howere,GUS geneexpression under the Control of Pro-SE-2and Pro-SE-3analysis indicated that GUSwas detected in roots and leaves,and the activity of GUS was more prominent in theroot hair and leaf margin, and it also demonstrated that the target gene wassuccessfully transformed into rice plant.The analysis of expression of GUS gene intransgenic rice by qPCR indicated that GUS gene were expressed in a different degreecompared with wild type rice.The expression of GUS gene by driving Pro-SE-1werepositively correlated with the expression of SE-1in Pro-SE-2promoter transgenic rice.In Arabidopsis thaliana, the result revealed the expression of SE promoter genein different tissues and organs is specific compared with wild plant,and its juvenileleaves were expressed clearly. GUS gene expression under the Control of these SEpromoter analysis indicated that GUS activity was detected in roots, leaves,petal,silique,but no found GUS gene expression in stem,GUS activity of Pro-SE-2andPro-SE-3were more strongly in the anthers,stigma and juvenile leaf.We found that the phenotype of rice plant with Pro-SE-2gene showed leafrolling, leaves serrating, and the leaves of rice that was transformed into Pro-SE-3genes showed shrivel, and the phenotype is similar to that of symptoms of riceinfecting RRSV.No specific phenotype was found in rice plants which was transferedinto Pro-SE-1.The results of expression levels analysis of SE-1,SE-2,SE-3gene inPro-SE-1,Pro-SE-2, Pro-SE-3transgenic rice separately showed that the expressionlevels of SE-1,SE-2and SE-3gene were down-regulated compared with Control intransgenic rice.But the expression levels of SE-1was up-regulated and had significantdifferences,the expression quantity of SE-2,SE-3have no obvious difference inPro-SE-2transgenic rice,while the expression quantity of SE-1was down-regulated inPro-SE-3transgenic rice. The expression quantity of SE-2and SE-3would riseslightly, but it has not significant difference in Pro-SE-3transgenic rice.To further study the relationship between SE promoter and SE gene in transgenicrice, we obtained the antiserum of serrated protein, and the results showed that theserum polyclonal antibody titer of three serrated protein their excellent by use ofELISA and Western blot.So the polyclonal antibody can be used to detect theexpression quantity of serrate protein in rice Plant.From the above,our result showed that Pro-SE-2promoter could enhance overexpression of SE1endogenous gene and Pro-SE-3promoter could enhance overexpression which endogenous gene in transgenic rice.The analysis of GUS stainingshowed that GUS expression of these three promoter were more strongly in thejuvenile leaf and reproductive organs of mature period, and it indicated that theexpression of promoter have temporal-spatial and tissue specificity.