The Effect Of OsCCD1 On Carotenoids Accumulation In Rice Seeds

Cultivation of the "Golden Rice" with high carotenoid content is one of the most important ways to combat vitamin A deficiency.The phytoene synthase from maize(ZmPSY)and the Erwinia uredovora carotene desaturase(CRTI)are two key enzymes in carotenoid biosynthesis pathway,Which have been successfully applied to develop "Golden Rice".Recent years studies found that carotenoid cleavage dioxygenases(CCDs)plays an important role in degradation of carotenoids in seeds.There are six members of the CCD family in rice,of which only OsCCD1 is highly expressed in rice endosperm and OsCCD1 gene is supposed to play an important role in regulating the accumulation of carotenoid in rice seeds.Previously,to improve the content of carotenoid in rice seeds,two vectors named pGYGC(referred to as GC,two genes of ZmPSY and CRTI drived by GluC and Gt1 promoter respectively were inserted into it)and pGTPCC(referred to as PCC,the "GluC-OsCCDl RNAi" segment inserted into the GC vector),have been constructed by the project coorperation unit of the Chinese university of Hong Kong professor Baocai Tan research team.On this basis,two vectors were transformed into rice in this project to analyze the effect on the accumulation of carotenoids through knockdown the expression of OsCCD1 in rice seeds,and lay the foundation for the development of the next generation of super golden rice.The main results are summarized as follows:1.Genetic transformation of rice.After PCC and GC cassettes were transferred into calli of rice,the resistant calli generated by the two vectors showed orange and bright yellow respectively,while as the non-transformed calli showed yellowish.Speculating that the content of carotenoids were significantly improved in the resistant calli generated by PCC and GC cassettes,and the content of carotenoid in the resistant callus of PCC is higher than the that of GC.After differentiation of the resistant callus,38 and 47 transgenic lines were obtained by PCC and GC respectively.2.Expressional analysis of OsCCD1.The qRT-PCR analysis showed that the expression of OsCCD1 in transgenic rice leave tissue of PCC was apparently higher than in transgenic rice of GC,and the expression of OsCCD1 have differences among individuals from both transgenic plants.Further speculated that knockdown expression of OsCCD1 enhanced the accumulation of carotenoid content in the resistant callus and transgenic seeds of PCC,and OsCCD1 gene expression differences lead to differences in carotenoid accumulation.3.Phenotypic characteristics of transgenic rice.No obvious phenotype difference showed between the transgenic plants of PCC and GC and the non-transgenic rice,such as rice growth,development and seed morphology.However,just as the similar colors of calli,the endosperm color in seed showed obvious difference among them.Among them,the endosperm in non-transgenic rice is white,cassette tansgenic rice of GC is golden,and tansgenic rice of PCC cassette is dark golden.It is suggested that almost no carotenoids accumulation in the endosperm of non-transgenic rice,and the carotenoid accumulation in transgenic rice endosperm of PCC cassette is significantly higher than that of GC.4.Determination the content of carotenoids in rice endosperm.Using the optimized HPLC analysis method detects the content of carotenoid.The composition of carotenoids detected including lutein,zeaxanthin,beta-cryptoxanthin,alpha-carotene and beta-carotene which is the main composition of carotenoid in transgenic rice seeds.The results showed that,in T1 and T2 generation of transgenic rice,the carotenoid content of seeds in the transformation PCC cassette was significantly higher than that of transformation GC cassette.These results indicated that enhanced caroteniods biosynthesis in conjunction with knockdown the expression of OsCCD1 can significantly elevated the accumulation of carotenoids in rice endosperm.The result also confirmed that OsCCD1 is an important gene in the carotenoid catabolism of rice seeds.