盐肤木中UDP-葡萄糖合成关键UGPase基因的克隆与功能预测OA

In order to investigatethe key UGPase genes responsible for UDP-glucose biosynthesis,uridine diphosphate glucose pyrophosphorylase(UGPase)family genes in Rhus chinensis were cloned relied on third-generation transcriptome data,and protein sequence,prokaryotic protein expression and in vitro enzyme catalytic activity,gene expression pattern,promoter cloning and sequence,etc.were analyzed.The results were as follows:(1)Four RcUGPase family genes were identified,their encoded proteins were subjected to bioinformatics analysis,and their protein sequences were further compared with the homology genes in other species by constructing a phylogenetic tree,which suggested that RcUGPase1 and RcUGPase3 belonged to UGPase-A class and UGPase-B class,respectively.(2)RcUGPase1,RcUGPase2,and RcUGPase4 were successfully cloned and recombined onto pET28a vector,and recombinant proteins were obtained via the prokaryotic expression vector.In vitro enzyme activity analysis revealed that the RcUGPase1 exhibited UGPase enzymatic activity.(3)The in vivo UGPase activity was significantly increased during the early developmental stages of Chinese gallnut formation(21 d and 47 d),which was consistent with the gradually rising gallotannin content reported in previous studies.(4)During the early developmental stages of Chinese gallnut formation(21 d and 47 d),the expression of RcUGPase1 was dramatically up-regulated,the expression of RcUGPase2 was down-regulated,and no significant changes were observed in the expression of RcUGPase3 and RcUGPase4.(5)A2334bp upstream promoter fragment of RcUGPase1 gene was cloned,this fragment was predicted to contain multiple environmental and hormone response elements that may involved in the upstream regulatory network.In summary,based on in vitro enzyme activity analysis and the correlation between gene expression with in vivo RcUGPase enzyme activity and gallotannins contents in Chinese gallnut,RcUGPase1 may be the key enzyme gene catalyzing the synthesis of UDP-glucose in R.chinensis,and the elevated UGPase enzyme activity during the early developmental stages of Chinese gallnut might play a key role in the accumulation of gallotannins in Chinese gallnut.