Construction Of Exrpession Vectors Of Key Genes In Zeaxanthin Biosynthesis And Apical Transformation Mediated By Agrobacterium Tumefaciens For Zeaxanthin-rich In Maize

Zeaxanthin(β,β-carotene-3,3'-diol) is a kind of fat-soluble compounds, belongs to a class of lutein carotenoids. It is a 40 carbon long molecule, with 11 conjugated double bonds, make zeaxanthin has a distinct color and strong antioxidant activity. Due to its unique chemical structure, zeaxanthin takes a significant effect on preventing blindness and senile cataract caused by macular degeneration of yellow point, inhibiting the occurrence and development of some tumors, as well as reducing the risk of some cardiovascular disease. As one of the Three Main Grain Crops, the corn kernel is rich in content of zeaxanthin. It enjoys great prospects for the development of its health value through breeding zeaxanthin rich fresh corn with Genetic Engineering.The transgenic technology is becoming better and approaching perfection over the past twenty years. Breeding through transgenic technology to get high quality maize enjoys the characteristic of more effective, faster and has stronger pertinences. Currently, callus is the major receptor in large-scale transgenic maize process, however its induction and culture are greatly restricted by corn genotypes and materials draw season, besides, its culture, transformation, screen and regeneration need a long cycle, the transgenic operation scale is relatively small, so a successful transformation is hardly to be ensured. Apical portion as receptor, Agrobacterium-mediated transformation does not depend on callus culture and corn genotypes, has a simple operation and short experimental period, can obtain transgenic plants from the majority of materials. But the positive rate is still need to be improved, and the transformant plants mostly belong to chimaera, the isolation and identification of its later generations is hard. Therefore, this method still needs further research to get improvement.The Biosynthesis of zeaxanthin includes a series of reactions like condensation, dehydrogenation, cyclization, hydroxylation and epoxidation. In this process, (3-Carotene Hydroxylase(BCH) and Zeaxanthin epoxidase(Zep) separately plays a vital role for the synthesis and oxidative decomposition of zeaxanthin. Therefore, this study explores the construction of BCH over expression vector, Zep mRNA antisense expression vector and RNA interference vector, and through the Agrobacterium-mediated apical portion inplanta transformation to regulate the expression of BCH and Zep in corn grain endosperm-up-regulate the expression of BCH and down-regulate the expression of Zep, which finally achieve the purpose of abundant accumulation of Zeaxanthin. Firstly, analyze BCH and Zep gene sequence published in NCBI as well as the Multiple Cloning Site(MCS) in the vector which we used; Then, primers is designed and a appropriate restriction site is introduced at the 5'end using primer5.0, amplifies full-length sequence of BCH mRNA and antisense of Zep mRNA as the target fragment link into the expression vector; Two 547 bp fragments in Zep mRNA conserved region are amplified as RNAi target sequence using two pairs of specific primers with the same combining site and different restriction sites, legates the two amplified fragments into pSKintron MCS in forward and reverse direction respectively, obtains the inverted repeat structure whith 150 bp maize intron which can formed hairpin structure after transcription; At last, legates BCH mRNA full length sequence, Zep mRNA antisense and the inverted repeat structures into the expression vector pTF101.1M, which has Ubiquitin as a promoter, T-NOS as a terminator, Bar gene as selection marker, Constructed the three vectors in this study:BCH over-expression vector(BCHOE), Zep mRNA antisense and RNA interference expression vector (ZepAE and ZepRNAi).Transform the three expression vectors plasmid into the Agrobacterium strain EHA105 one by one through Freeze-thaw method, and use inplanta transform the apical portion of "18 bai", three vector BCHOE,ZepAE and ZepRNAi obtain transformed plants 717,809 and 750 respectively through the Agrobacterium-mediated transform, after phosphinothricin 0.2 g/L screening, there are 32,21 and 37 resistant plants with the resistance rates are 3.94%,4.04% and 5.25%.The above results indicate that compared to the transformation depends on callus as receptors, Agrobacterium-mediated apical portion inplanta transform method operation simple, significantly reduced experimental period, is feasible, the transgenic plants after the further identification can be used for zeaxanthin-rich genetically modified varieties' cultivation.