Analysis Of Related Genes Of Carotenoids Synthesis Of Erwinia Uredovora In Transgenic Rice

Rice(Oryza sativa L) is one of the most important crops in the world,high and stable grain yield of rice is very valuable for our national food security.However,rice often suffers from a variety of stressful conditions during its growth and development.The abiotic stresses which increase the reactive oxygen species in cells and make oxidative damage to cells,eventually result in the decreased grain yield and depresses quality of rice.Increasing rice yield and improving rice quality are the most important aspects in rice breeding.The exotic antioxidant genes are introduced into the rice genome through plant genetic engineering to produce new super rice with high yield and strong stress tolerance.Carotenoids are components of photosynthetic membranes in plants and microbe, which can protect the photosynthetic system from oxidative damage.Many kinds of carotenoids are important precursor of vitamin A,which are strong antioxidants.Erwinia uredovora,as a kind of microorganism,has a much more simple and energy-saving carotenoid synthesis pathway than plants.And crtB,crtI,crtY are key enzymes ofβ-carotene synthesis process in Erwinia uredovora.In our experiments,the key genes crtB,crtI,crtY were cloned through PCR from the plasmid pACCAR16â–³crtX,a plasmid containing all the genes involved in theβ-carotene synthesis process in Erwinia uredovora.The CBN(Cab-crtB-NOS),CIN(Cab-crtI-NOS) and CYN(Cab-crtY-NOS) expression cassettes were constructed throughout ligating the three key genes crtB,crtI,crtY under the control of Cab prometer and then the expression cassettes were constructed into pC1301,a binary vector for plant transformation.The recombinant vectors were introduced respectively into rice varieties Zhonghua11(Japonica) and 9311(Indica) via Agrobacterium-mediated genetic transformation with the generation of 40 transgenic rice plants.Additionally,the three recombinant vectors were introduced together into rice variety Zhonghua11(Japonica) through gene gun-mediated genetic transformation with the generation of 20 transgenic rice plants.GUS activities were detected in transgenic callus,and the roots and seeds of T₀ generatic plants obtained for 80%positive rate.And PCR analyses were performed using the DNA extracted from leaves of the transgenic rice plants as template to amplify the hygromycin,crtB/I/Y and gus gene to further test whether the foreign fragments were successfully introduced into rice genome.The results showed that the foreign genes transformations were successful and the transgenic rice plants had been obtained.Besides,prokaryotic expression was also performed by constructing the key genes into pet32a,induced by IPTG.The proteins were expressed in inclusion bodies.Then antibodies of crtB,crtI,crtY were prepared through imunizing rabits with the purified antigen seperated by SDS-PAGE.And the antiserums were detected by Western bolt with strong specificity and high titer of 1:10000000,which were very valuable for testing the expression proteins in transgenic rice plants.Therefore,a system for stable expression of key enzymes ofβ-carotene synthesis process from Erwinia uredovora in vivo has been established by genetic engineering in rice(Oryza sativa L),and the role of increased carotenoid contents in the transgenic rice plants for protecting photosynthesis from oxidative stress needs to be investigated further to elucidate the molecule mechanism of carotenoids in antioxidation and to create new rice germplasm resources with high tolerance to oxidative stress.