Cloning And Sequence Analysis Of CsDAM2 Gene And Its Promoter In Huangjin Tea (Camellia Sinensis)

Tea plant (Camellia sinensis) is a kind of seasonal perennial economic crops, which bud dormancy would directly impact on the economic benefits of tea production due to the starting and stopping point of dormant period. Two influencing factors of tea bud dormancy are low temperature and short day sunshine, either of which would activate its dormancy when below the critical value, and it is the result that gene expression regulation changed in vivo. Promoter, the key to gene transcription and protein expression, has a directly influence on the growth and development of tea plant as well as the yield and quality of tea leaves. Therefore, it has a scientific importance on bud dormancy regulation, yield and quality enhancement, new early-budding varieties cultivation through mechanism analysis of dormancy and germination in gene transcription and protein expression level. Huangjin tea, native to Baojing County, west of Hunan Province, has a trait of budding earlier 10-15 days than other tea varieties, however, the early germinantion mechanism of Huangjin tea remains unclear. Previous experiments have separated candidate gene:Dormancy Associated MADS-box (CsDAM2), a gene from Huangjin tea might be associated with early germination, when compared with the local tea variety Xiangfeicui. In this study, the cDNA gene and its promoter were cloned and analyzed by modern molecular biology technology, in order to provide scientific basis for functional analysis and transgenic plants establishment of CsDAM2 gene. The main results were listed as follows:(1) CsDAM2 gene full-length cDNA sequence were successfully being cloned by using Huangjin tea extracted RNA through modified Tri-Reagent method. Sequence analysis showed that the promoter sequence of CsDAM2 gene with 1386 bp. It contained an open reading frame (ORF) of 657 bp coding region which encoding a polypeptide of 218 amino acid residues with a predicable molecular mass of 24.88 KDa. The molecular formula of CsDAM2 protein is C1075H1779N323O341S6 while the theoretical isoelectric point PI value is 8.96. The deduced amino acid sequence showed 71%,68%,61%,57%,49% homology with CsDAM2 from Populus tomentosa, Theobroma cacao, Coffea arabica, Dioscorea esculenta (Lour.) Burkill, Gossypiumspp, and the similarity of MADS-box protein in tea plant was 35%. Online prediction software indicated that CsDAM2 protein were hydrophilic and non secretory, and its protein peptide contains 1 phosphorylation sites and 53% helix,39% random coil,8% fold structures.(2) Obtained a promoter sequence of 478 bp long of Huangjin tea I young leaves by using enhanced CTAB method via specific primers design of CsDAM2 gene cDNA sequence, genomic gene clone and amplification by chromosome walking technology, eukaryotic expression vector construction (pMDTM19-T Vector) and to golden tea, a young leaves as raw material, total DNA was extracted by improved CTAB method, according to CsDAM2 gene cDNA sequence specific primers, using chromosome walking technology of golden tea genomic gene were cloned and amplified, and E.coli DH5a transformation. Bioinformatics analysis showed the A/T content of CsDAM2 promoter sequence was 71.13%, which was consistent with the characteristics of the high A/T content of plant promoter. Furthermore, CsDAM2 gene promoter contains lots of cis-acting elements, including light response associated elements of G-box, I-box, GAG-motif and I-box; abscisic acid responsive element of ABRE, gibberellin responsive element of P-box and other hormone stress related components; heat stress related element of HSE; defense and stress response element TC-rich repeats as well as some unnamed, specific function or unknown cis-acting elements etc..