Full-length CDNA Cloning Of UBC Gene From Camellia Oleifera And Construction Of Its RNAi Vector

Camellia oleifera is a perennial woody oil plants,and is a principal species of woody oil plants in the South of China. Camellia oil contains oleic acid and linoleic acid UFA mostly, and the average content of UFA is above 90%. So it is one which of the most popular high nutritional value edible oil in the current. Since the development process of Camellia oleifera seed is in the environment of high temptory, dry spell and soil depletion, the process of the lipids biosynthesis is refer to varieties of abiotic stress,including drought,heavy metals, nutritional deficiencies, high salt, pests and diseases, high or low temperature. The Ubiquitin/26S proteasome pathway mediate the pross of specific degradation of protein,and it Significantly affect plant's growth and development. As a key enzyme in the pathway, ubiquitin-conjugating enzyme produce a significant impact on plant cell cycle regulation, senescence, cell differentiation, hormone signaling response, photomorphogenesis and abiotic stress response.In this research, the author cloned full-length cDNA and made bioinformatic analysis of E2 gene on the base of the cDNA library and the EST library of Camellia oleifera. And then constructed RNAi vector. The main results are as follows:1. Molecular clone and bioinformatics analysis of E2 Gene from C. oleifera. In the constructed cDNA library of Camellia oleifera by our laboratory,we identifyed the length cDNA of E2 gene, which has 804bp. After we compared with the nucnic acid database in other species,we believed that we have get the full length of E2 encoding 152 amino acids. Using a series of bioinformatics analysis,such as isoelectric point, molecular weight, membrane structure, hydrophobicity, functional secondary structure prediction analysis. We found that the hydrophobic index of E2 was from-3.633 to 2.122, and in the protein of E2, the whole polypeptide chain showed hydrophilic except the 37,102 and 105 amino acids showed hydrophobic.E2 protein has two obsolete transmembrane domain. Looking at the secondary structure of E2 protein, we found that a helix was the main structural elements of E2 from Camellia oleifera, at the same time,random coil were scattered throughout the protein.The active site of Camellia oleifera ubiquitin-conjugating enzyme is from 77 to 92 amino acids,and the cysteine site is on the 88 amino acids.2.The constract of RNAi vector of E2 Gene from C. oleifera.Constracted the intermediate vector pMD19-Dimer using fragment replacement method.Cloned the fragment of RNAi vector using the primer designed in accordance with the information of E2 gene.Insert the interference segment into the intermediate vector pMD19-Dimer ordinally.Then digested plant vector pBI 121 and vetor pMD19-Dimer-E2-RNAi by Xba I and Sac I separatelly, and purificated, connected, at last constructed the vector pBI-E2-RNAi...