Gene Cloning Of Castanea Mollissima’s Two Flower Bud Differentiation Genes And Construction Of Its RNAi Expression Vector

Chinese chestnut(Castanea mollissima. BL., Castanea., Fagaceae) is one of importanthorticultural plant, which is widely distributed all over the world, and it has highlyeconomic cultivation value. As deciduous trees, Chestnut is one of the ancient trees ofChinese origin, which is also one of very important woody grain and economic forest treespecies in China, and in the high-value export. It is a monoecism and diclinous plant withunisexual flowers. The order of the development of the staminate flowers are catkin andthe female flowers spica, Flowering(Staminate flowers and female flowers) of chestnut isdifferent in space and time, and the quantity of female flowers is much lower than thestaminate ones, that the male one consume a large amount of nutrients, which directlyaffect the production of chestnut. Study on the characteristics and floral of the chestnut, wehad mastered differentiation and development period of female and male flower aboutchestnut through morphology, but floral genes regulation of chestnut should be for furtherresearch from molecular biology. Vernalization-related gene FLC might play role in theplants flowering transition and regulation inhibit flower development by inhibitiondownstrram target gene. This experiment choose about luotian chestnut, whose cultivationvariety rose as the research material, through reverse transcription and RACE technique,FLC and MADS of Chinese chestnut bud differentiation related gene were separated bycDNA cloning. For two genes’ cDNA sequence, and the sequences of bioinformaticsanalysis, provide the basis for later research of the gene expression analysis. At the sametime to build the Chinese chestnut FLC plant expression vector of RNA interference, themain research contents and results are as follows:(1)Molecular cloning and Bioinformatics analysis of the cDNA sequence of CmFLC.In this study, we cloned and characterized a Flowering Locus C gene from Castaneamollissims. The full-length cDNA sequence of CmFLC was968bp containing an openreading frame(ORF) of681bp, which encoded a227amino acids with a predictedmolecular mass of25.62kDa and the theoretical isoelectric point(PI) of5.80. CmFLC wasan intro-free gene, and its deduced polypeptide contained a M box of61amino acids in theN terminal. The secondary structure of CmFLC was mainly composed of alpha helix and random coil. Comparative analysis showed that CmFLC had a high similarity to other plantFLC proteins, and contained all the M box and K box. The homology based structuralmodeling showed that CmFLC has the typical structure of Populus tomentosa FLC.Phylogenetic tree revealed that CmFLC and CcFLC were assigned to the same clade.(2)Molecular cloning and Bioinformatics analysis of the cDNA sequence of CmMADSand Plant Expression Vector of RNA interference pcl301-ubi-CMFLC-dsRNAi. In thisstudy, we cloned and characterized a MADS gene from Castanea mollissims. Thefull-length cDNA sequence of CmMADS was922bp containing an open readingframe(ORF) of681bp, which encoded a227amino acids with a predicted molecular massof25.87kDa and the theoretical isoelectric point(PI) of6.27. CmMADS was an intro-freegene, and its deduced polypeptide contained a M box of58amino acids in the N terminal.The secondary structure of CmMADS was mainly composed of alpha helix and randomcoil. Comparative analysis showed that CmMADS had a high similarity to other plantMADS proteins, and contained all the M box and K box. The homology based structuralmodeling showed that CmMADS has the typical structure of Malus domestica MADS.Phylogenetic tree revealed that CmMADS and TrMADS3were assigned to the same clade.Use BanH Ⅰ and Kpn Ⅰ and enzyme intermediate carrier pBluescript SK plus-FR andplant expression vector pcl301-ubi, recycling pBluescript SK plus-FR enzyme cut intosmall pieces, connected pcl301-ubi in larger pieces, a plant expression vector pcl301-ubi-CmFLC-dsRNAi.Next next build good carrier of RNA interference and conversion ofagrobacterium mediated by its putting the recombinant plasmid into tobacco, the functionof the carrier for further study of the interference and CmFLC gene function.