茶树己糖激酶基因CsHXK2的启动子克隆及表达特性分析

Promoter cloning and expression analysis of the hexokinase gene CsHXK2 in tea plant(Camellia sinensis)

植物己糖激酶是双功能蛋白,具有磷酸化己糖和介导糖信号的关键性作用。前期研究中,我们从茶树中克隆获得4个己糖激酶基因,其中CsHXK2基因编码492个氨基酸残基,与拟南芥AtHXK3、番茄LeHXK4归为Type A类HXKs。利用RT-PCR技术,克隆获得长度为2029 bp的CsHXK2基因启动子。CsHXK2基因可能受到光照、低温、病原菌、糖和多种激素等信号的调控,且可能特异性表达于叶、花、种子、根系、腋芽等组织。CsHXK2蛋白定位于叶绿体内。酵母突变体功能互补试验表明,去除叶绿体转运信号肽的CsHXK2成熟蛋白具有葡萄糖和果糖磷酸化活性。茶树组织特异性表达分析显示,CsHXK2基因在根和茎中表达量最高,而在老叶中表达量最低。CsHXK2基因的表达受低温胁迫而显著下调,经炭疽菌侵染的茶树叶片内CsHXK2基因的表达也受到显著抑制,而外源赤霉素(GA3)处理的茶树叶片内CsHXK2基因表达显著上调。本研究结果表明,CsHXK2基因在茶树的生长发育过程和逆境胁迫响应中发挥重要的调控作用。

Hexokinase is a kind of dual-function protein playing a crucial role in hexose phosphorylation and sugar signaling transduction in plants.In our previous study,four hexokinase genes from tea plant were cloned,and CsHXK2 gene encoded 492 amino acid residues was classified as Type A HXKs together with AtHXK3 from Arabidopsis thaliana and LeHXK4 from tomato.2029 bp promoter of CsHXK2 gene was obtained by RT-PCR.Further sequence analysis showed that CsHXK2 gene may be regulated by light,low temperature,pathogen,sugars and phytohormones,and specifically expressed in leaves,flowers,seeds,roots,axillary buds.CsHXK2 protein was localized in the chloroplast.Functional complementation of the hexokinase-deficient yeast mutant showed that the CsHXK2 mature protein removal of chloroplast transit peptide,had phosphorylation activity of glucose and fructose.Tissue-specific expression analysis found that CsHXK2 had the highest transcriptional levels in roots and stems,whereas the lowest in old leaves.The expression level of CsHXK2 gene was significantly down-regulated in both cold stress treatment and leaves infected treatment by Colletotrichum camelliae.However,the expression level of CsHXK2 was significantly stimulated after exogenous GA3 treatment in tea leaves.In conclusion,the results suggested that CsHXK2 played an important role in the regulation of both development and stress responses in tea plant.