火龙果<em>HpTCP9</em>基因克隆及其在干旱胁迫下的表达分析

doi:10.3969/j.issn.1674-7968.2024.07.006

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摘要 TCP (teosinte branched1/cycloidea/proliferating cell factor)是植物特有的转录因子,广泛参与植物的生长发育和应答环境胁迫过程。TCP9是TCP基因家族Ⅰ类成员,在植物应答干旱非生物胁迫中发挥重要作用。火龙果(Hylocereus)具有很强的抗旱性,是研究植物抗旱的理想材料。为探究TCP9基因在火龙果抗旱性中的生物学功能,本研究以红肉火龙果(Hylocereus polyrhizus)品种'大红'为供试材料,利用RT-PCR技术对红肉火龙果HpTCP9基因进行克隆。克隆出的HpTCP9 (GenBank No. OR145855.1)基因ORF长729 bp,编码242个氨基酸,分子量为24.86 kD,等电点为5.04,亲水指数为1.021。结构域分析表明,HpTCP9蛋白含有1个典型的TCP保守结构域。蛋白结构预测结果表明,HpTCP9蛋白的氨基酸中α螺旋占比12.40%,无规则卷曲占比66.12%,延伸链占比12.81%,β转角占比8.68%。构建携带GFP标签的pCAMBIA2300-HpTCP9-GFP过表达载体,通过拟南芥(Arabidopsis thaliana)原生质体转化法检测出HpTCP9为核定位蛋白。实时荧光定量PCR分析HpTCP9在干旱处理下的表达模式,结果表明,HpTCP9基因在20% PEG6000模拟干旱处理诱导下显著上调表达,表达量与处理时长呈正相关。综上,HpTCP9属于TCP家族Ⅰ类亚家族成员,具有TCP保守结构域,是定位于细胞核的疏水蛋白,且在火龙果的干旱耐受性方面发挥功能。本研究为深入探究火龙果HpTCP9基因功能提供理论依据。

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	倪芮
	罗悠悠
	黄渺
	蔡骏一
	何奕含
	刘文霖
	姚依妮
	肇瑾

关键词 ：红肉火龙果, HpTCP9, 基因克隆, 干旱胁迫

Abstract：TCP (teosinte branched1/cycloidea/proliferating cell factor) is a plant specific transcription factor that is widely involved in plant growth and development and response to environmental stress. TCP9 is a class Ⅰ member of TCP gene family and plays an important role in respose to plant drought stress. Pitaya (Hylocereus sp.) is an ideal material for drought resistance research because of the strong drought resistance. Hylocereus polyrhizus variety 'Da Hong' were used to isolated the HpTCP9 gene by RT-PCR technology in order to investigate the biological function of the TCP9 gene in the drought resistance of pitaya. Bioinformatics analysis showed that the ORF length of the HpTCP9 gene was 729 bp, encoding 242 amino acids, with a molecular weight of 24.86 kD, an isoelectric point of 5.04, and a hydrophilicity index of 1.021. Domain analysis showed that the HpTCP9 protein sequence had a typical TCP conserved domain. The protein structure prediction results indicated that α spiral was 12.40%, irregular curls were 66.12%, and extended chains were 12.81%, The β-turn was 8.68%. To detect the subcellular localization of HpTCP9, a pCAMBIA2300-HpTCP9-GFP expression vector carrying a GFP tag was constructed. HpTCP9 was detected as a nuclear localization protein through Arabidopsis protoplast transformation. Real time fluorescence quantitative PCR analysis showed that the HpTCP9 gene was significantly upregulated by 20% PEG6000 simulated drought treatment, and the expression level was positively correlated with the treatment duration. The above results indicated that the HpTCP9 belonged to the ClassⅠsubfamily of the TCP family, had a TCP conserved domain, was a hydrophobic protein located in the nucleus, and played a role in the drought stress defense mechanism of H. polyrhizus. This study provides a theoretical basis for further exploring the function of the HpTCP9 gene in H. polyrhizus.

Key words： Hylocereus polyrhizus HpTCP9 Gene clone Drought stress

收稿日期: 2023-08-02

中图分类号： S667.9

基金资助:贵州省高等学校科技拔尖人才项目(黔教合KY字KY[2021]028); 国家自然科学基金(32260799); 贵州大学重点培育项目([2019]42号)

通讯作者: * zhaojin7094@163.com

引用本文:

倪芮, 罗悠悠, 黄渺, 蔡骏一, 何奕含, 刘文霖, 姚依妮, 肇瑾. 火龙果HpTCP9基因克隆及其在干旱胁迫下的表达分析[J]. 农业生物技术学报, 2024, 32(7): 1533-1542.
NI Rui, LUO You-You, HUANG Miao, CAI Jun-Yi, HE Yi-Han, LIU Wen-Lin, YAO Yi-Ni, ZHAO Jin. Cloning and Expression Analysis of HpTCP9 Gene in Pitaya (Hylocereus polyrhizus) Under Drought Stress. 农业生物技术学报, 2024, 32(7): 1533-1542.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.07.006 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I7/1533

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