Maize Transformation With Key Enzyme Genes Of β-carotene Biosynthetic Pathway

P-carotene (C40H56) is one of the carotenoids, which is an orange fat-soluble compounds. It is a stable natural pigment in the nature. Many natural foods contain β-carotene, such as:green vegetables, sweet potatoes, carrots, spinach. β-carotene plays an important role in physiological functions:it is the precursor of vitamin A, has the role of the immune and antioxidant, can prevent cancer and delay the development of cancer and so on.But the human body can not synthesize carotenoids, mainly dependent on diet. The content of carotenoid is relatively low in many plants, especially the β-carotene. Corn as the largest food in the world and China, corn endosperm has relatively low levels of carotenoids. Improve the amount of β-carotene can significantly improve the nutritional value of corn.In higher plants, β-carotene is synthesized by the isoprenoid pathway in plastids. The biosynthesis process includes condensation, dehydrogenation, cyclization, hydroxy, and epoxy, etc. At present, the key enzymes’genes of the catalytic reactions have been cloned and identified successively; it opened the way to product P-carotene by recombinant DNA technology and genetic engineering. This study cloned the key enzymes’ genes of P-carotene biosynthetic pathway:the gene of phytoene synthase (PSY) and phytoene dehydrogenase (CrtI), the two genes was connected fusion gene by the overlapping PCR method, the fusion gene was inserted into the monocot plant expression vector, the plant expression vector was shifted to the maize of "18-599" by Agrobacterium-mediated transformation, in order to get the plants with massive accumulation of P-carotene.1. The plant expression vector was constructedAccording to the PST and CrtI gene sequences which published on NCBI, the primers were designed. The PSY gene was cloned from the corn cDNA, the CrtI gene was cloned from the Erwinia uredovora DNA, the pea ribulose small subunit transit peptide gene sequences were syntheticed.We connected the three genes into a fusion gene by the overlapping PCR, at the same time introducted the restriction sites, Finally the fusion gene was inserted into the monocot plant expression vector pTF101.1.2. Cultivation and transformation of maize embryonic calli The explants is the immature embryo of "18-599", then was induced embryogenic callus,the callus was transformed by Agrobacterium-mediated.Finally got150To generation regenerated plants after herbicide resistance medium gradient screening.3. Molecular detection of regenerated plantsDesign specific PCR primers to amplify the target gene1315bp fragment,The To generation150regenerated plants were detected; there were10plants which were certified to be positive by PCR detection of target genes.Based on the results of PCR, the positive plants were identified for further; we expect to provide material for the new breed which is rich of P-carotene.