Construction And Genetic Transformation Of CrtB+CrtI Tandem Coexpression And Ta?HYD RNA Interference Vectors In Wheat

Carotenoid content in wheat grain is one of the important factors that determine the nutritional quality of wheat.Wheat grain accumulates only a small amount of lutein and zeaxanthin that cannot be converted to vitamin A.At present,although the the outline of carotenoids synthesis has been elucidated,however,we still know little about regulation mechanism of carotenoid metabolism,especially in hexaploid bread wheat.Therefore,identification of carotenoid biosynthetic genes and studying their roles in the synthesis pathway are of great importance to wheat nutrition quality research.In the present study,we first constructed a tandem expression vector containing bacterial phytoene synthase gene(CrtB)and carotene desaturase gene(CrtI)under the control of the wheat endosperm-specific promoter 1Dx5,in order to increase the content of carotenoids in wheat grains and to further study the regulation mechanism of wheat carotene synthesis pathway.Then,the pLRPT-CrtB-2A-CrtI and the TaHYD silencing expression vector pLRPT-TaHYD-RNAi were co-transformed into wheat cultivar Bobwhite via particle bombardment.We obtained 5 CrtB and CrtI co-expressing transgenic lines(OEIB)and 4 TaHYD RNAi transgenic lines(HYDi)through PCR comfirmation,while we did not obtain any transgenic line coexpressing CrtB ?CrtI and TaHYD-RNAi.We also used qPCR to detect the expression of some carotenoid synthetic genes in the seeds of positive transgenic lines.Our results showed that the expression of carotenoid synthetic genes both in OEIB and HYDi transgenic lines were up-regulated,except for TaHYD,suggesting a complicated feedback regulatory mechanism in carotenoid biosynthetic pathway.Finally,we used Western Blotting to detect the expression of CrtB,CrtI and HYD at protein levels in the seeds of transgenic lines.The results showed that the expression of CrtB and CrtI proteins could be detected in transgenic lines OEIB,while the expression of HYD protein in HYDi lines were obviously inhibited.In order to obtain increased carotenoids content,we tried to use the overexpression of upstream gene combined with interfering with the expression of downstream genes to change the metabolic balance of carotenoid synthesis pathway.Although we did not obtaintransgenic lines coexpressing CrtB ?CrtI and TaHYD-RNAi,the study still provides a new idea for improving the content of carotenoids in wheat grain by means of genetic engineering.