Gene Cloning And Enzyme Activity Identification Of Glucosyltransferases Of Auxins And Phenylpropanoids

Glycosylation is a wide-spread physiological phenomenon, and is thought to be one of the most important mechanisms in maintaining plant cell homeostasis. Glycosyltransferases (GTs) are the enzymes responsible for glycosylation. They can typically transfer single or multiple activated sugars from nucleotide sugar donors, especially uridine5’-diphosopho glucose (UDP-glucose), to a wide range of small molecular acceptors, thus change their bioactivity, solubility, stability, subcellular localization and binding property to the acceptors. This study is focused on the gene cloning, prokaryotic expression and enzyme activity identification of glucosyltransferase genes of auxins and phenylpropanoids from rice and poplar. The main contents and results of this study are listed as follows.1. The cloning and identification of auxin glucosyltransferase from riceAuxin is one of the most important phytohormones in plant, which controls the development of roots, apical sominance, gravitroism, phototropism, leaf morphology. Auxin combines with other small molecules, such as glucose and methyl. The glycosylation of auxin is an mportant form of bound auxin. Previous sdudies have revealed that the overexpression of auxin glycosyltransferase genes in plants exhibits phenomenon of weak gravitropism, influencing plant branches,leaves morphogenesis. The auxin glucosytransferase has been sdudied in the model plant Arabidopsis, but the gene of auxin glucosyltransferase in Oryza sativa has not yet been isolated now. In this study, an auxin glycosyltransferase of rice was cloned and characterized for the first time.(1) Eleven candidate genes of auxin glycosyltransferases were predicted, cloned, and expressed using prokaryotic system and their recombinant proteins were purified. A phylogenetic tree was constructed using eleven putative auxin glycosyltransferases of rice and all published Arabidopsis and Zea may family1GTs. It was found that rice llgenes were located on a unique branch with Arabidopsis and zea may genes which glycosylate auxins to their glucosides in Arabidopsis and Zea may, suggesting that the rice11genes might be involved in the glycosylation modification of auxin. These eleven genes were constructed to pGEX prokaryotic expression vectors, expressed in E.coli XL1-Blue, and then recombinant proteins were purified.(2) Glycosyltransferase OsIAGT1was found to have high enzyme activity responsible for the glycosylation of auxins. The enzyme activities of eleven recombinant proteins were analyzed by incubating them separately with six auxin. Following HPLC and LC-MS analyses for the reaction products, it was found that OsIAGT1glycosylated6sbustance to produce their corresponding glucose ester. These6substrates include IAA, IBA, ICA, IP A, NAA,2,4-D.(3) The enzymatic properties of glycosyltranferase OsIAGT1were studied. Relative conversion rates of these6substrates were calculated under the same condition, and it was found that OsIAGTl had a higher enzymatic activity toward IAA, IBA and IP A than toward ICA, NAA, and2,4-D. Taking IAA as an example, the influence factors of enzymatic activity including temperature, pH were tested. The results showed that30degrees centigrade was the best temperature for enzyme activity. The pH optimum was8.0. Finally, the kinetic parameters of this enzyme catalyzing IAA, IBA were also calculated. Results showed that the Km values of these two auxins were almost identical, indicating that they may competitively bind glycosyltransferase OsIAGTl in planta.In summary, this study identified, for the first time, a rice glycosyltransferase OsIAGT1responsible for the glycosylation of auxins. This discovery lays a foundation for the further study on the function of OsIAGTl in vivo and its role in regulating auxin metabolism.2. The cloning and identification of glucosyltransferase genes of phenylpropanoids in Oryza sativa and Poplus tometentosa.Phenylpropanoids can be converted into a broad spectrum of O-ester conjugates. The abundant sinapate esters in Brassica napus and Arabidopsis thaliana reflect a well-known metabolic network, including UDPglucose:sinapate glucosyltransferase (SGT), sinapoylglucose:choline sinapoyltransferase (SCT), sinapoylglucose:L-malate sinapoyltransferase (SMT) and sinapoylcholine (sinapine) esterase (SCE).1-O-Sinapoylglucose, produced by SGT during seed development, is converted to sinapine by SCT and hydrolyzed by SCE in germinating seeds. The released sinapate feeds via sinapoylglucose into the biosynthesis of sinapoylmalate in the seedlings catalyzed by SMT. Sinapoylmalate is involved in protecting the leaves against the deleterious enflects of UV-B radiation. Sinapine might function as storage vehicle for ready supply of choline for phosphatidylcholine biosynthesis in young seedlings. And the sinapate has same pathway with the synthsis of lignins.The A. thaliana brtl (bright trichome1) mutant impaired in UGT84A2enhances resistance of Soybean Rust. In this study, the glucosyltransferase genes of phenylpropanoids in Oryza sativa and Poplus tometentosa were cloned and identified for the first time.(1) Three rice candidate genes of phenylpropanoid glycosyltransferases were cloned and expressed using prokaryotic system and recombinant proteins were purified. A phylogenetic tree was constructed using three putative rice glycosyltransferases of phenylpropanoids, and all published Arabidopsis family1GTs about phenylpropanoids.It was found that rice three rice genes were located on a unique branch with Arabidopsis genes which glycosylate phenylpropanoids to their glucose ester suggesting that the three genes might be involved in the glycosylation modification of phenylpropanoids. These three genes were constructed to pGEX prokaryotic expression vectors, expressed in E.coli XL1-Blue, and then recombinant proteins were purified.(2) Glycosyltransferase OsSGT was found to have high enzyme activity responsible for the glycosylation of phenylpropanoids. The enzyme activities of three recombinant proteins were analyzed by incubating them separately with sinapate. Following HPLC and LC-MS analyses for the reaction products, it was found that OsSGT glycosylated sinapate acid to produce its corresponding glucose ester.(3) The cloning and identification glucosyltransferase genes of phenylpropanoids in Populus tometentosa. The candidate genes of phenylpropanoid glycosyltransferases were cloned from poplar and expressed using prokaryotic system and recombinant proteins were purified. Glycosyltransferase PtSGT1and PtSGT3were found to have high enzyme activity responsible for the glycosylation of phenylpropanoids.3. In addition, the relationship of Arabidopsis UGT76C2glycosyltransferase activity and the single nucleotide substitution was studied in this thesis. The effects of mutated gene on the enzyme activity and in vivo physiological role were also investigated.