Cloning,Vector Construction And Transgenic Plant Development Of Glycosyltransferase Genes Of Arabidopsis Thaliana

Glycosyltransferases are widely present in plants, catalyzing the formation of the glycoside bond. They can transfer activated sugars from nucleotide sugar donors to a wide range of molecular acceptors including hormones, secondary metabolites and abiotic chemicals and toxins from the environment. The transfer of a sugar onto an acceptor usually changes acceptor in its bioactivity, stability, transport properties, subcellular localization and its interaction with the receptor. It also can reduce or remove the toxicity of endogenous and exogenous substances. Glycosylation of small molecule acceptors is a very common physiological phenomenon in plant, which is thought to be one of the main mechanisms to maintain metabolic balance. Glycosylation of plant molecules is involved in balancing hormones, defense response, detoxification, plant secondary metabolism and signal transduction, which is of great significance in maintaining normal growth and development in plants.Arabidopsis contains a large number of glycosyltransferases, most of their functions are not clear. In order to study the function of these genes and to develop their breeding value, cloning and genetic transformations of three Arabidopsis glycosytransferases were done in this study by cell engineering and genetic engineering technology. The transgenic Arabidopsis and transgenic tobacco of those genes were obtained, thus laying a foundation for the further study of the functions and practical application of those genes. The main contents and results of this research were summarized as follows.1. Three glycosyltransferase genes of Arabidopsis were cloned and their plant expression vectors were constructed.The full-length cDNA of UGT71B2, UGT75C1, and UGT78D1were cloned from Arabidopsis thaliana by RT-PCR. These three genes were cloned into pBluescript II SK (+) respectively, and sequencing results showed the accuracy of cloned sequences. Plant expression vectors of three glycosyltransferase genes were constructed by replacing the GUS gene of vectoe pBI121respectively. These plant expression vectors can be used for plant genetic transformation.2. The transgenic Arabidopsis and transgenic tobacco of three genes were obtained.Through the Agrobacterium-mediated genetic transformation, transgenic research was conducted on Abrabidopsis and tobacco. The over-expression transgenic Arabdopsis lines of these three genes were obtained by kanamycin resistance screening, PCR and RT-PCR detection. Also the over-expression transgenic tobacco lines of UGT71B2were obtained.3. The function of glycosyltransferase gene UGT78D1was preliminarily analyzed.Using three-prime PCR method, the T-DNA insertion mutation homozygote of UGT78D1was identified. It was found that the primary root of the ugt78d1mutant is obviously shorter compared with wide-type Arabdopsis, presumably due to the changed dynamic balance of endogenous hormones in plants as a consequence of the gene mutation. The analysis of auxin level and distribution through DR5::GUS transgenics and the functional complementation experiments are in progress.4. Through this study, the basic theory and technical methods of cell engineering and genetic engineering were mastered, such as gene cloning, vector construction, tissue culture technique, plant transgenic technique, gene expression analysis technology and so on, which laid a good foundation for engaging in crop molecular design or drug development through gene engineering in the future.