Transgenic RNA Interference To Zeaxanthin Epoxidase Gene In Maize

Zeaxanthin, also known as corn flavin (Zeaxanthin,3,3-hydroxy-beta-carotene), which is the most abundant carotenoid in the corn grain, it always coexist with cryptoxanthin, beta-carotene and lutein, becoming a carotenoid mixture. Zeaxanthin is the fat-soluble compound,and its molecule containing40carbons and11conjugated double bonds. Meanwhile zeaxanthin is lutein’s isomer,and lutein is the retina of the eye and the lens indispensable carotenoids. Therefore, they are play a crucial role in maintaining eye health. If old people intake enough carotenoid,they can decrease the risk of age-related macular disease,and both the lutein and zeaxanthin are most important in a variety of carotenoids.Zeaxanthin-rich maize germplasm is expected to breed with the health effects of fresh corn varieties through genetic engineering to create.In corn grain, zeaxanthin synthesized different hydroxylation process such as condensation, dehydrogenation and cyclization, and then experiment oxidation decomposition. Zeaxanthin cyclooxygenase (Zeaxanthin epoxidase, Zep) is a key enzyme in oxidative decomposition of zeaxanthin. In this study, through RNA interference technology to build the Zep gene interference carrier, and use agrobacterium-mediated to transformat maize embryos. In order to foster efficient accumulation of zeaxanthin zeaxanthin-rich genetically modified material.1. Zeaxanthin cyclooxygenase gene RNA interference expression vector was constructed.Maize inbred line "18-599W" mRNA as a template, and through clone the Zep gene547bp fragment. Meanwhile, introduce two restriction endonuclease sites at both ends. Both directions inserted the150bp intron ends that is the RNA interference engineering plasmid pSKintron, then this inverted repeats cloned to pTF101.1M plasmid and constructed monocots RNA interference vector (pTFZepRNAi).2. The transformation of corn callus Through maize inbred line "18-599R" and "18-599W" immature embryos as explants to induced the callus and agrobacterium-mediated to transformed the corn.And it pruduced84regeneration plants after three herbicides gradient screening, and survived79transplants from experiment to field.3. To regeneration plants was detected by PCRThrough design three primers to detected it by PCR, which cover the herbicide screening positive material. Both Fuz/Ruz and ruz/ruz wsa designedthe three primers,which primers Fuz/Ruz is the purpose of cross-promoter fragment-specific amplification and primers Ruz/Ruz is inserted into the vector intron-specific amplification. Using the specific primers, I got nine positive plants from the PCR detection of regenerated plants.The research based on the molecular detection and the phenotype observed, the material of transgenic plants is expected to further nurture enriched the zeaxanthin corn varieties.