盐肤木中UDP-葡萄糖合成关键UGPase酶基因的克隆与功能预测

Cloning and functional prediction of the key UGPase genes responsible for UDP-glucose synthesis in Rhus chinensis

关键词:  盐肤木，五倍子，UGPase，UDP-葡萄糖，没食子单宁合成

Key words:  Rhus chinensis, Chinese gallnut, UGPase, UDP-glucose, biosynthesis of gallotannins

摘要:

克隆盐肤木（Rhus chinensis）尿苷二磷酸葡萄糖焦磷酸化酶（uridine diphosphate glucose pyrophosphorylase, UGPase）家族基因，探究参与催化UDP-葡萄糖生物合成的关键基因，为进一步解析五倍子中没食子单宁的高含量积累机制奠定基础。该研究依托三代转录组数据，开展基因同源克隆、蛋白序列分析、原核表达及体外酶活分析、基因表达模式分析、启动子克隆及序列分析等。结果表明：（1）鉴定到4个RcUGPase家族基因，并对其编码蛋白进行生物信息学分析，与其他物种的同源基因构建系统进化树，发现RcUGPase1与RcUGPase3分属于UGPase-A与UGPase-B类型。（2）克隆到RcUGPase1、RcUGPase2和RcUGPase4三个基因，利用pET28a载体构建了其重组原核表达载体，并成功诱导纯化重组蛋白，体外酶活分析发现RcUGPase1具有UGPase酶催化活性。（3）五倍子发育早期（21 d和47 d），体内RcUGPase酶活性显著上升，与前人报道的单宁含量的逐渐升高是一致的。（4）转录组数据显示在五倍子发育早期（21 d和47 d），RcUGPase1的表达显著上调，RcUGPase2的表达下降，RcUGPase3和RcUGPase4变化不显著。（5）克隆RcUGPase1基因上游2 334 bp的启动子片段，预测出其上有多种环境和激素响应元件可能参与上游的调控网络。综上认为，结合体外酶活、基因表达与五倍子中RcUGPase酶活性和单宁含量的相关性等结果，推测RcUGPase1可能是五倍子中催化UDP-葡萄糖合成的关键酶基因，五倍子发育初期RcUGPase酶活性的上调可能对五倍子中单宁的积累起到重要作用。

Abstract:

The uridine diphosphate glucose pyrophosphorylase (UGPase) family genes in Rhus chinensis were cloned, and the key UGPase genes responsible for UDP-glucose biosynthesis were investigated. The study will lay the foundation for further analysis of the mechanisms of gallotannins over-accumulation in Chinese gallnut. This study relied on third-generation transcriptome data to conduct gene homologous cloning, protein sequence analysis, prokaryotic protein expression and in vitro enzyme catalytic activity analysis, gene expression pattern analysis, promoter cloning and sequence analysis, etc.. The results were as follows: (1) Four RcUGPase family genes were identified, 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 reconstituted onto pET28a vector, and recombinant proteins were obtained by the prokaryotic expression system. In vitro enzyme activity analysis revealed that the RcUGPase1 had a UGPase enzymatic activity. (3) The in vivo UGPase enzyme activity was significantly increased during the early developmental stages of Chinese gallnut formation (21 d and 47 d), when the endogenous contents of gallotannins were gradually gained. (4) During this process, the expression of RcUGPase1 was dramatically up-regulated, the expression of RcUGPase2 was down-regulated, and no significant changes were observed on the expression of RcUGPase3 and RcUGPase4. (5) A 2 334 bp promoter sequence upstream of RcUGPase1 gene was cloned and multiple cis-acting elements in response to environments and hormone signaling were predicted. In summary, based on in vitro enzyme activity analysis and the correlation between gene expression with in vivo UGPase enzyme activity and gallotannins contents, RcUGPase1 may be the key enzyme gene catalyzing the synthesis of UDP-glucose in Rhus chinensis, and the elevated UGPase enzyme activity during the early developmental stages of Chinese gallnut might play a key role in over-accumulation of gallotannins in Chinese gallnut.