辣椒海藻糖-6-磷酸合酶基因<i>CaTPS9</i>的克隆及表达分析

黄立娟; 魏敏; 苟秉调; 段盼盼; 郭娜纳; 魏兵强

doi:10.19303/j.issn.1008-0384.2023.07.005

辣椒海藻糖-6-磷酸合酶基因CaTPS9的克隆及表达分析

doi: 10.19303/j.issn.1008-0384.2023.07.005

甘肃农业大学园艺学院，甘肃　兰州　730070

基金项目: 国家自然科学基金项目（31760572）；甘肃省重点研发计划项目（21YF5NA091）；兰州市人才创新创业项目（2021-RC-65）；甘肃农业大学青年导师扶持基金项目（GAU-QDFC-2020-07）

详细信息

作者简介:
黄立娟（2000 —），女，硕士研究生，主要从事蔬菜遗传育种与分子生物学研究，E-mail：hk1234561018@163.com

通讯作者:
魏兵强（1980 —），男，博士，教授，主要从事蔬菜遗传与分子育种研究，E-mail：bqwei@gsau.edu.cn

中图分类号: S641.3
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出版历程
- 收稿日期: 2022-12-20
- 修回日期: 2023-04-10
- 网络出版日期: 2023-08-16
- 刊出日期: 2023-07-28

Bioinformatics and Expression of CaTPS9 in Chili Peppers

College of Horticulture, Gansu Agricultural University, Lanzhou, Gansu　730070, China

摘要

摘要: 目的明确辣椒中海藻糖-6-磷酸合酶（TPS）基因CaTPS9的表达特性和生物学功能，进一步了解TPS对辣椒生长中调控非生物胁迫的作用。方法以辣椒品种强丰101为试验材料，克隆CaTPS9基因，并对辣椒CaTPS9的理化性质、蛋白结构、顺式作用元件、系统进化树等进行分析；通过qRT-PCR分析CaTPS9基因在不同组织（商品果果肉、幼果果肉、成熟果果肉、商品果胎座、幼果胎座、成熟果胎座、叶、根、茎、花）和胁迫处理（低温和植物生长调节剂处理）中的表达模式。结果 CaTPS9基因CDS序列全长2604 bp，编码867个氨基酸。CaTPS9蛋白包含Glyco_transf_20和Trehalose_PPase两个保守结构域，分子质量为97.60 kDa，不稳定指数为44.27，理论等电点为5.63，亚细胞定位预测CaTPS9蛋白位于细胞质中。生物信息学分析表明，CaTPS9蛋白属于亲水性蛋白，且不存在跨膜结构和信号肽序列，蛋白结构主要由α-螺旋和无规则卷曲组成。系统进化关系分析表明，CaTPS9与烟草（Nicotiana tabacum L.）、番茄（Solanum lycopersicum L.）和马铃薯（Solanum tuberosum L.）中的同源基因亲缘关系较近。启动子顺式作用元件分析表明，CaTPS9启动子区域含有与激素、胁迫及植物生长发育相关的顺式作用元件。此外，CaTPS9在叶片中表达量最高，在商品果胎座中表达量最低。在水杨酸（Salicylic acid，SA）处理12 h后CaTPS9基因的表达量被显著提升，而低温、吲哚乙酸（3-indoleacetic acid，IAA）、脱落酸（Abscisic acid，ABA）、赤霉素（Gibberellin acid，GA₃）和茉莉酸甲酯（Methyl jasmonate，MeJA）处理能够显著抑制CaTPS9基因表达量。结论 CaTPS9基因可能通过海藻糖生物合成途径响应逆境胁迫。
- 辣椒 /
- 海藻糖-6-磷酸合酶 /
- 生物信息学 /
- 非生物胁迫 /
- 表达分析
Abstract: Objective Bioinformatics and expression of trehalose-6-phosphate synthase (TPS) gene in chili peppers,CaTPS9, were studied to understand its role in response to abiotic stress during the plant growth. Method CaTPS9 was cloned from Qiangfeng 101 to analyze the physicochemical properties, protein structure, cis-regulatory element, and phylogenetic tree. Expressions of the gene in the pericarp and placenta of a commercial pepper as well as the young pericarp, ripened pericarp, young placenta, ripened placenta, leaves, roots, stems, and flowers of chili pepper plants under varied stresses including low temperature, 3-indoleacetic acid(IAA), abscisic acid(ABA), salicylic acid(SA), gibberellin A₃(GA₃), and methyl jasmonate(MeJA) were determined using qRT PCR. Result The full-length cDNA sequence of CaTPS9 was 2604 bp encoded 867 amino acids. Located in the cytoplasm, the protein contained two conserved domains, Glyco_transf_20 and Trehalose_PPase, with a molecular weight of 97.60 kDa, an instability index of 44.27, and a theoretical isoelectric point of 5.63. It was a hydrophilic protein free of transmembrane structure and signal peptide sequence with a structure consisting mainly of alpha helixes and random coils. Phylogenetically, CaTPS9 was close to the homologous genes in Nicotiana tabacum, Solanum lycopersicum and Solanum tuberosum. It had hormones response, stress response, and plant growth and development cis−elements and was expressed most highly in the leaves, lowly in the placentas of a commercially available pepper, and significantly by 12 h SA induction. Stress, such as low temperature, IAA, ABA, GA₃, and MeJA, significantly inhibited the expression. Conclusion It appeared that CaTPS9 might regulate the response of chili pepper plants to various abiotic stresses through the trehalose biosynthetic pathway.
- Chili pepper /
- TPS /
- bioinformatics /
- abiotic stress /
- expression analysis

HTML全文

图 1 CaTPS9基因扩增产物

M：GL DNA Marker 5000，1～2：PCR产物。

Figure 1. Amplification products of CaTPS9

M: DNA marker 5000; 1-2: amplified cDNA product of CaTPS9.

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图 2 CaTPS9蛋白疏水性和亲水性预测

Figure 2. Predicted hydrophobicity and hydrophilicity of CaTPS9 protein

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图 3 CaTPS9蛋白磷酸化位点预测

Figure 3. Predicted phosphorylation sites on CaTPS9 protein

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图 4 CaTPS9蛋白的二级结构预测

蓝色区域表示α-螺旋；紫色区域表示无规则卷曲；红色区域表示延伸链；绿色区域表示β-转角。

Figure 4. Predicted secondary structure of CaTPS9 protein

Blue region: alpha helix; purple color: random coil; red shade: extended strand; green area:beta turn.

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图 5 CaTPS9蛋白的三级结构预测

Figure 5. Predicted tertiary structure of CaTPS9 protein

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图 6 CaTPS9系统进化关系分析

Figure 6. Phylogenetic relationship of CaTPS9

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图 7 CaTPS9基因在辣椒不同组织的表达模式

PECP：商品果果肉；YPE：幼果果肉；RPE：成熟果果肉；PLCP：商品果胎座；YPL：幼果胎座；RPL：成熟果胎座；L：叶；R：根；S：茎；F：花；不同小写字母表示CaTPS9基因表达量在辣椒不同组织间差异显著（P＜0.05）。图8同。

Figure 7. Expressions of CaTPS9 in different tissues of pepper plant

PECP: pericarp of commerical pepper; YPE: young pericarp; RPE: ripened pericarp; PLCP: placenta of commercial pepper; YPL: young placenta; RPL: ripened placenta ; L: leaf; R: root; S: stem; F: flower; those with different lowercase letters significantly different between different tissues of pepper at P＜0.05. Same for Fig. 8.

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图 8 辣椒CaTPS9基因在不同胁迫条件下的表达模式

Figure 8. Expressions of CaTPS9 under abiotic stresses

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表 1 CaTPS9基因引物信息及功能

Table 1. Information and function of CaTPS9 primer

引物名称 Primer name	引物序列 Primer sequence（5′-3′）	用途 Purpose
CaTPS9-F	ATGGCATCAAGATCTAGTGCA	基因扩增 Gene amplification
CaTPS9-R	TTACCCACTCAAATTAACAGATGAG	基因扩增 Gene amplification
qCaTPS9-F	GCATTGGAGATGACAGGTCGGATG	荧光定量PCR qRT-PCR
qCaTPS9-R	ACTTGGCTTTGCTTGGCTTTTGC	荧光定量PCR qRT-PCR
qActin-F	AGAGATTCCGTTGCCCAGAGGTC	内参基因 Reference gene
qActin-R	AGCCACCACTGAGCACAATGTTAC	内参基因 Reference gene

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表 2 CaTPS9启动子顺式作用元件预测分析

Table 2. Putative cis−element analysis on promoter regions of CaTPS9

分类 Classification	元件名称 Element name	序列 Sequence	功能预测 Function prediction
激素响应元件 Hormone response element	脱落酸响应元件 ABRE	ACGTG	参与响应脱落酸的顺式作用元件 cis-acting element involved in abscisic acid responsiveness
	生长素响应元件 AuxRR-core	GGTCCAT	参与响应生长素的顺式作用元件 cis-acting regulatory element involved in auxin responsiveness
	赤霉素响应元件 TATC-box	TATCCCA	参与响应赤霉素的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
	水杨酸响应元件 TCA-element	CCATCTTTTT	参与响应水杨酸的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
胁迫响应元件 Stress response element	干旱诱导元件 MBS	CAACTG	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
胁迫响应元件 Stress response element	厌氧诱导元件 ARE	AAACCA	参与厌氧诱导的顺式作用元件 cis-acting regulatory element essential for the anaerobic induction
植物生长发育元件 Plant growth and development elements	玉米蛋白代谢调节元件 O2-site	GATGA（C/T）（A/G）TG（A/G）	参与玉米蛋白代谢调节的顺式作用元件 cis-acting regulatory element involved in zein metabolism regulation
	种子特异调控元件 RY-element	CATGCATG	参与种子特异调控的顺式作用元件 cis-acting regulatory element involved in seed-specific regulation
	分生组织表达相关元件 CAT-box	GCCACT	与分生组织表达相关的顺式作用元件 cis-acting regulatory element related to meristem expression

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