铁观音茶树甘油-3-磷酸酰基转移酶(CsGPAT)基因克隆及萎凋过程中的表达分析

Cloning and Expression of Glycerol-3-phosphate Acyltransferase Gene in Tieguanyin Tea Leaves during Withering

【目的】以铁观音茶树鲜叶为试验材料,对参与甘油磷脂代谢途径的茶树甘油-3-磷酸酰基转移酶(Glycerol-3-phosphate acyltransferase,CsGPAT)进行克隆、生物信息学分析以及不同萎凋温度的表达量分析,了解GPAT基因在茶叶萎凋过程中的重要意义,以期为茶叶萎凋过程中温度调控提供理论基础。【方法】基于乌龙茶加工(萎凋)转录组数据,筛选获得茶树GPAT同源序列。利用ExPASy Protparam、SMART、SignalP 4.1Server、PSORT、prediction protein等在线软件进行生物信息学分析,利用SWISS-MODEL在线工具编辑GPAT蛋白质三维结构;在NCBI Blastp进行氨基酸序列同源比对。提取铁观音叶片RNA进行qRT-PCR,检测实时表达情况,克隆茶树GPAT基因全长。【结果】克隆得出CsGPAT(IDcsa:CSA000941.1/IDcss:TEA019813.1)基因序列全长1554 bp,编码497个氨基酸;GPAT蛋白属于稳定疏水性蛋白,不含信号肽,具有磷脂生物合成功能的PlsC域;进化树分析表明CsGPAT基因与油茶的亲缘关系最近。qRT-PCR分析结果显示CsGPAT基因在20℃温度萎凋时表达量最高。【结论】CsGPAT基因在茶叶萎凋受到相对低温胁迫时,表达量上调,表明CsGPAT表达与茶叶萎凋过程中的温度调控密切相关。

【Objective】Glycerol-3-phosphate acyltransferase(CsGPAT)involving the glycerolipid metabolic pathway was cloned from fresh leaves of Tieguanyin tea(Camellia sinensis)plants for bioinformatics analysis and expression analysis of different withering temperature.To understand the significance of GPAT gene in the process of tea withering,in order to provide a theoretical basis for temperature regulation in the process of tea withering.【Method】By screening the transcriptome data on withering oolong tea,the homologous GPAT sequence was obtained.Software including ExPASy Protparam,SMART,SignalP 4.1 Server,PSORT,prediction protein,etc.were used to obtain its bioinformatics;the SWISSMODEL online tool applied to arrive at its 3D protein structure;and,NCBI Blastp employed to analyze its homologous alignment on the amino acid sequence.RNA from the tea leaves was extracted for qRT-PCR to determine the real-time expression as well as for cloning the full length of GPAT.【Result】The cloned CsGPAT(IDcsa:CSA000941.1/IDcss:TEA019813.1)had a full length of 1554 bp encoding a predicted protein of 497 amino acids.The bioinformatics showed CsGPAT not a signal peptide but a stable hydrophobic protein with a PlsC domain of phospholipid biosynthetic function that closely related to Camellia oleifera.Its expression peaked at the withering temperature of 20℃.【Conclusion】The expression of CsGPAT in the tea leaves was up-regulated when the withering temperature was relatively low.It suggested a crucial role the gene played in oolong tea processing.