Cloning Of CSAG Gene And Analysis Of AG Gene System In Tea Plant

Tea plant(Camllia sinensis) is a hermaphrodite 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 important woody crops. Growth of tea from the seeds of the flower bud differentiation began to form about a year and a half to go through the time, This resulted with in a competitive relationship, on the one hand is the tea flower development and last year fertilized seeds develop into fruits, secondly, fresh tea leaf development also need to have enough nutrients, If nutritional deficiencies will be greatly reduced, the quality of the finished product the economic benefits of tea will be impacted. Pass long time, the main method of practice to improve the yield of fresh tea leaves picked tea buds is a method to control the reproductive growth of tea. So, how much more effective inhibition of the growth of tea to promote reproductive vegetative growth of tea production of tea has become a raise fundamental core issue. AGAMOUS gene is one of the major regulatory genes of plant hair organ development, if they can get the detailed structure and function of the regulatory mechanism, by means of molecular biology and effective control of tea from AGAMOUS gene transcription regulation of flower development that suppress reproductive growth of tea can be fundamentally resolved.In this study, the research material was local tea plant germplasm (C. sinensis cv ZiYangzhong) in shaanxi, First, total RNA was extracted to the tea plant through improved SDS acid phenol law system; Homologous amplified by RT-PCR and combined RACE method to obtain full length cDNA of CsAG gene in tea plant; By semi-quantitative RT-PCR method to study the gene expression of CsAG various tissues in tea plant, Finally, bioinformatic analysis on CsAG gene structure and function in tea plant and AG gene system evolution.Experimental results were reduced as follow:1. By modified SDS acid phenol to extracted total RNA from different organizations in tea plant, Finally the CDS of AGAMOUS homologous genes was cloned from tea plant by conjunction with RT-PCR homologous amplification, RACE fragment and subcloned verification method, Full-length sequence of1263bp,5’-UTR length of208bp,3’-UTR of315bp, encoding reading frame sequence length of738bp, the start codon was ATG, the termination codon was TAA. Homology comparison showed that sequence homology of up to90%with camellia japonica AGAMOUS, named CsAG in tea plant(GenBank Aaccession Number, KJ630566).2. Semi-quantitative RT-PCR results showed that:CsAG genes are expressed in the differentiation mid-buds and flowering sepals, petals, stamens, pistils and young fruits and shoots of tea plant, But different expression levels of various tissues.Expressed strongest in the stamen, pistil, followed by flower buds, sepals and fruit, expressed weak in petals, CsAG gene expressed almost undetectable in shoots, indicating that CsAG was a functional gene involved in tea flower development.3. CsAG structure and function prediction result display:CsAG encoding a254amino acid sequence, comprising60amino acids and100amino acid region of MADS and K-box region, be classified typical MADS-box family gene. Predicted protein formula is C1216H1972N370O383S11, relative molecular weight was28255.9isoelectric point PI was9.38, unstable protein parameters was56.76, be classified a hydrophilic and unstable protein, Phosphorylation sites forecasts show that the protein has multiple phosphorylation sites, includ tryptophan (Ser) of13, tyrosine (Tyr) locus of3. Protein structure prediction results show CsAG similar protein with other plants such as tobacco, grapes, etc. Camellia tertiary structure, the a-helix (100aa,40.82%), extended chain (23aa,9.03%) and random coil (122aa,49.80%) constituted.4. CsAG gene phylogenetic analysis showed that:CsAG and other plants AG homologous genes have motif I and motif II this two co-existence conserved regions, which was unique Characteristics of AG family, further proved CsAG was MADS-box family members and belonged to AG gene family; build CsAG phylogenetic tree was found, CsAG are accurately categorized into Class C gene family, which have highest similarity with AG1gene in camellia japonica, indicated that CsAG gene have recently affinity with camellia japonica, followed by Vicia sepium and Hydrangea macrophylla.