Cloning Of CsDEF Gene And Analysis Of AP3 / DEF Gene System In Tea Plant

Tea plant(Gamllia sinensis) is a monoecious and cross pollinated plants belongs to theaceae camellia tea plants, The case for many years and has a warm and humid growing fear of cold often characterized by shrub or small tree with green leaves, which is one of the Chinese important economic trees. Tea is widely preferred beverage, is an important commodity in the international trade, the economic value is mainly reflected in the leaf. Tea fruit mature period of about a year and a half, the reproductive growth and vegetative growth compete for nutrients, thereby affecting the quality of tea. Farmers after spraying hormone, pruning flower and other ways to reduce the reproductive growth, In this way not only of the high cost and the tea will be affected the food security. Use molecular genetics can provide the basic theory for the safety of solve this problem. In this study, the results of previous studies on tea plant flower development, the class B gene CsDEF cloning and functional analysis of flower development, the molecular mechanism of tea plant, and further improve the yield and quality of tea to provide the basis.In this study, the research material was local tea plant germplasm (Camllia sinensis cv. Ziyangzhong) in shaanxi.The extraction of tea flower organs in different tissues of RNA using improved SDS-acid phenol method;Combined with RT-PCR and RACE to obtain full length cDNA of CsDEF gene in tea plant; By semi-quantitative RT-PCR method to study the gene expression of CsDEF various tissues in tea plant; Analyzed by bioinformatics the function of CsDEF gene. The following results were obtained:(1) The full length sequence 912bp of CsDEF gene was cloned from tea plant, 5’-UTR length of 34bp length,3’-UTR of 197bp length, there was 30bp Poly (A) tail. Encoding open reading frame sequence length of 681 bp, the start codon was ATG, the termination codon was TAG GenBank login No. KP772271.(2) The result of semi quantitative RT-PCR showed that CsDEF gene is expressed in all the tissues or organs of tea plant., but different expression levels of various tissues. The strongest expression in differentiation of flower bud, the petals and stamens; followed by pistils and fruit; the lowest in the leaf and sepal. Suggest that CsDEF gene expression is the typical feature of B gene.(3) CsDEF gene sequence analysis showed that it encodes a protein of 226 amino acids, is a MIKC type gene. MADS region of 58 amino acids, K-box 98 amino acids. The molecular formula of protein was C1131H1836N338O350S10, the relative molecular weight of 26089.6 PI, isoelectric point was 9.03, protein stability coefficient is 43.20, the fat soluble index of 84.51 and the average hydrophilicity of -0.781. The CsDEF protein contains multiple phosphorylation sites, including tryptophan (Ser) in 15 sites, followed by threonine (Thr) 9. The protein had no transmembrane structure, and it mainly distributed in the nucleus and the cytoplasm. Analysis second-level structure showed that the α-helix 58% and no random coil 31% constituted. second-level structure analysis results are consistent with the three level.(4) CsDEF gene amino acid sequence multiple alignment results showed that:the CsDEF gene and DEF gene of other plants are specific to AP3/DEF gene family of PI-derived motif and euAP3-modif region in the C end, indicated that CsDEF is a member of the AP3/DEF family MADS-box family.(5) To construct CsDEF gene phylogenetic tree, CsDEF gene of tea plant was classified into the B family of genes, including CsDEF gene and Camellia japonica CjDEF similarity was highest, that of CsDEF gene and phylogenetic tree of Camellia oleifera and recently, followed by the Stewartia pseudocamellia.