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

毕婉君; 周子维; 武清杨; 郑玉成; 倪子鑫; 柳镇章; 孙云

doi:10.19303/j.issn.1008-0384.2020.04.010

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

doi: 10.19303/j.issn.1008-0384.2020.04.010

福建农林大学园艺学院，福建　福州　350002

基金项目: 国家现代农业（茶叶）产业技术体系建设专项（CARS-19）；福建农林大学科技创新基金项目（cxzx2017178）

详细信息

作者简介:
毕婉君（1997−），女，硕士研究生，研究方向：茶叶加工与品质（E-mail：1013086529@qq.com）

通讯作者:
孙云（1964−），女，博士，教授，研究方向：茶叶加工与品质（E-mail：sunyun1125@126.com）

中图分类号: S 571.1
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- 文章访问数: 1076
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- 被引次数: 0
出版历程
- 收稿日期: 2019-11-20
- 修回日期: 2020-01-24
- 刊出日期: 2020-04-01

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

College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　530002, China

摘要

摘要: 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1 554 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。
- 铁观音 /
- 甘油-3-磷酸酰基转移酶 /
- GPAT基因 /
- 生物信息学分析 /
- 萎凋
Abstract: 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 SWISS-MODEL 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 1 554 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.
- Tieguanyin /
- glycero-3-phosphate acyltransferase /
- GPAT gene /
- bioinformatics analysis /
- withering

HTML全文

图 1 PCR产物凝胶电泳分析

注：M：DL2000 DNA Marker；A：3′-RACE；B：5′-RACE；C：开放阅读框PCR扩增结果。

Figure 1. Gel electrophoresis analysis on PCR products

Note: M: DL2000 DNA marker; A: 3′-RACE; B: 5′-RACE; C: PCR amplification results of open reading frame.

下载: 全尺寸图片幻灯片

图 2 甘油-3-磷酸酰基转移酶蛋白质的信号肽预测结果

Figure 2. Prediction of signal peptide in GPAT protein

下载: 全尺寸图片幻灯片

图 3 甘油-3-磷酸酰基转移酶蛋白质三维结构预测

注：A、B分别表示α-螺旋、β-折叠。

Figure 3. Predicted 3D structure of GPAT protein

Note: A and B represent alpha-helix and beta-folding, respectively.

下载: 全尺寸图片幻灯片

图 4 NJ法构建的GPAT氨基酸序列的系统进化树

Figure 4. Phylogenetic tree of GPAT amino acid sequence constructed by NJ method

下载: 全尺寸图片幻灯片

图 5 甘油-3-磷酸酰基转移酶基因在不同萎凋温度下的相对表达量

注：图中大写字母不同表示差异极显著（P＜0.01），小写字母不同表示差异显著（P＜0.05），XY代表室温处理（24℃）。

Figure 5. Relative expressions of CsGPAT at different withering temperatures

Note: Data with different capital letters mean extremely significant difference（P＜0.01）; and, data with different lowercase letters indicate significant difference（P＜0.05）; XY indicates treatment at room temperature（24℃）.

下载: 全尺寸图片幻灯片

表 1 3′RACE和5′RACE所使用的引物

Table 1. Primers used in 3′ RACE and 5′ RACE

基因　　　　 Gene	引物序列（5′-3′） Primer sequence（5′- 3′）	碱基 Base number	温度 Temperature/℃	GC含量 GC ratio/%
CsGPAT 5′端第一轮引物	AATGTAGTACCGAGGGAAGAAC	22	64	45.5
CsGPAT 5′端第二轮引物	GACAAGCGCTTTTCCCTACTT	21	62	47.6
CsGPAT 3'端第一轮引物	ACAAGAAAGAGAGTTCTTCAAAATGA	26	68	30.8
CsGPAT 3'端第二轮引物	ACAAGAAAGAGAGTTCTTCAAAATGA	26	68	29.6
GeneRacer^TM5′引物	CGACTGGAGCACGAGGACACTGA	23	74	60.9
GeneRacer^TM5′槽式引物	GGACACTGACATGGACTGAAGGAGTA	26	78	50.0
GeneRacer^TM3'引物	GCTGTCAACGATACGCTACGTAACG	25	76	52.0
GeneRacer^TM3'槽式引物	CGCTACGTAACGGCATGACAGTG	23	72	56.5

下载: 导出CSV

表 2 实时荧光定量基因表达分析的引物序列

Table 2. Primer sequence for real-time fluorescence quantitative gene expression analysis

基因 Gene	登录号 Accession number	前端/尾端的引物序列（5′-3′） Front/end primer sequence （5 '- 3′）	扩增长度 Amplification length/bp	退火温度 Annealing temperature/℃
CsEF-1α	KA280301.1	F-TTCCAAGGATGGGCAGAC/R-TGGGACGAAGGGGATTTT	196	59.6
CsGPAT	TEA019813.1	F-TTGGGATTGGGAGACAGAGA/ R-GAAGGTCAAGAGGGCAACAA	177	60.2

下载: 导出CSV

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