Cloning And Functional Characterization Of A Putative Galur Gene In Kiwifruit

L-ascorbic acid (AsA) which is generally called Vitamin C by people plays essential roles in maintaining human health and improving plant biochemistry reaction, especially when plants are in adversity stress. Therefore, it is significant to research AsA. GalUR gene is a key enzyme in conversion pathway of uronic acids which is a possible pathway about AsA biosynthesis. We hope we can provide theoretic reference for the pathway through our study.In this study, we cloned GalUR gene from Actinidia deliciosa cv.'Qinmei', and constructed three express vectors of the gene, which lay the foundation for lucubrating the function of GalUR gene. The main results in the study were as follows:1.Obtaining cDNA overall length of GalUR gene from kiwi fruit. The cDNA sequence length is 1028 bp , having an whole open reading frame of 990 bp, encoding 329 amino acid. It has a homology of the nucleotide sequences of 100% with Actinidia deliciosa (FG475088.1) through BLAST in Genebank, while the homology of amino acid sequences is above 70% with other plants.2.Constructing plant excessive express vector pWR/GalUR. Then we transformed it into EHA105 (Agrobacterium tumefacien) competent cells successfully.3.Constructing plant RNAi express vector pHGRV/GalUR and transforming it into EHA105 competent cells.4.We tested several indicators relating to AsA content and observed the changes of GalUR gene expression level with quantitative real-time RT-PCR in terms of different growth phases of kiwi fruits and different tissues of kiwi. Firstly, 30d after anthesis, both AsA content and the gene expression level in kiwi fruit were the highest, and then they maintained the certain levels. Secondly, in mature leaf, AsA content and the gene expression level were both higher than other tissues. The results indicated the expression of GalUR gene has close relation with AsA biosynthesis.5.Under the basis of restriction enzyme digestion, we built prokaryotic expression vector pET/GalUR and transformed into E.coli BL21 competent cells. After being induced by IPTG, an approximately 57kD specific protein band was obtained. According to the optimal expression conditions, polyclonal antibody was prepared.6.The total proteins of different tissues in kiwi extracted were used to be western blot. In the process, polyclonal antibody prepared was primary one. The protein expression level in mature leaf was the highest, which corresponded with the result of quantitative real-time RT-PCR. The polyclonal antibody could be distinguished specifically by antihelion in kiwi.