小麦热激转录因子TaHsfA1亚家族基因的生物学特性及耐热性分析

doi:10.3969/j.issn.1674-7968.2022.01.001

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摘要植物热激转录因子(heat shock transcription factors, Hsf)存在于大多数植物中,在热胁迫和多种其他逆境胁迫响应过程中发挥重要作用。本研究从冬小麦(Triticum aestivum) '沧6005'中同源克隆获得3个同源基因TaHsfA1-1 (GenBank No. MW756130)、TaHsfA1-2 (GenBank No. MW756131)和TaHsfA1-3 (GenBank No. MW756132),cDNA长度分别是1 590、1 566和1 569 bp。3个蛋白质序列都含有典型的DNA结合结构域(DNA binding domain, DBD)、相同的核定位信号序列(nuclear localization signal, NLS)RRKP/KKRR和转录激活结构域序列(aromaromatic, large hydrophobic and acidic amino residues, AHA) DSFWEQFLCA。TaHsfA1-1、TaHsfA1-2和TaHsfA1-3与节节麦(Aegilops tauschii) AetHsfA1的氨基酸序列相似性均高达96%。通过染色体定位分析确定TaHsfA1-1、TaHsfA1-2和TaHsfA1-3分别定位于染色体4A、5B和5D上。通过瞬时转化烟草(Nicotiana tabacum)表皮细胞并观察发现,正常条件下TaHsfA1蛋白质都定位于细胞核。qPCR分析显示,3个基因的表达均被37 ℃热胁迫(heat stress, HS)和水杨酸(salicylic acid, SA)上调,而脱落酸(abscisic acid, ABA)处理明显下调其表达,TaHsfA1-3同时被H₂O₂上调。通过在酿酒酵母(Saccharomyces cerevisiae) AH109中鉴定发现TaHsfA1-1、TaHsfA1-2和TaHsfA1-3均具有转录激活活性。依据3个基因对热胁迫响应程度,选取TaHsfA1-1遗传转化拟南芥(Arabidopsis thaliana)并观察表型发现,TaHsfA1-1能够提高热胁迫下拟南芥幼苗的基础耐热性和获得耐热性,不同转基因株系的叶绿素含量与其表型及存活率相一致,正常及热激后均引起相关热激蛋白基因的表达。该研究表明小麦HsfA1亚族基因具有耐热性调控功能,为进一步探究小麦Hsf家族基因特性及耐热性功能提供了理论基础。

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关键词 ：小麦, 热激转录因子A1 (TaHsfA1), 生物学特性, 亚细胞定位, 耐热性功能

Abstract：Heat shock transcription factor (Hsf) exists in most plants and plays an important role in the response to heat tolerance and other stresses. Family genes showed diverse characteristics and functions. In this study, 3 homoeologous genes TaHsfA1-1 (GenBank No. MW756130), TaHsfA1-2 (GenBank No. MW756131) and TaHsfA1-3 (GenBank No. MW756132) were obtained by homologous cloning technology from wheat (Triticum aestivum), their cDNA lengths were 1 590, 1 566 and 1 569 bp, respectively. Three protein sequences all contained the typical DNA binding domain (DBD), the same nuclear localization signal (NLS) sequence RRKP/KKRR and transcription activation domain sequence (aromaromatic, large hydrophobic and acidic amino acid residues, AHA) DSFWEQFLCA. The similarity of TaHsfA1-1, TaHsfA1-2 and TaHsfA1-3 to Aegilops tauschii AetHsfA1 was as high as 96%. Chromosome location analysis confirmed that TaHsfA1-1, TaHsfA1-2 and TaHsfA1-3 were located on chromosomes 4A, 5B and 5D, respectively. Through transient reporter assay with tobacco (Nicotiana tabacum) epidermal cells, it was found that the proteins was localized in the nucleus under the normal conditions. The qPCR analysis showed that the expression levels of the 3 genes were up-regulated by heat stress (HS) and salicylic acid (SA) treatment, respectively and down-regulated by abscisic acid (ABA) treatment, and TaHsfA1-3 expression was up-regulated by H₂O₂. Through assay in Saccharomyces cerevisiae AH109, TaHsfA1-1, TaHsfA1-2 and TaHsfA1-3 all had transcriptional activation activity. According to the expression level responsive to heat stress, TaHsfA1-1 was selected and transformed into Arabidopsis thaliana. Through observation the phenotype, it was found that TaHsfA1-1 could improve the basic thermotolerance and acquired thermotolerance of Arabidopsis thaliana seedlings and the chlorophyll contents of different transgenic lines under high temperature stress. The chlorophyll contents were consistent with the phenotype and survival rate. Hsp genes were induced to expression under the normal conditions and after heat stress in TaHsfA1-1 transgenic A. thaliana plants. The results indicated that TaHsfA1 could regulate thermotolerance, providing a theoretical basis and technical support for further exploring the characteristics and function of heat tolerance of wheat Hsf family.

Key words： Wheat Heat shock transcription factor A1 (TaHsfA1) Biological characteristics Subcellular location Thermotolerance function

收稿日期: 2021-04-01

ZTFLH:

S512.1

基金资助:河北省自然科学基金(C2019301133); 河北省现代农业科技创新工程项目(494-0402-YBN-S2XB; 494-0402-YBN-C7GQ); 河北省农林科学院基本业务费项目(2018110102)

通讯作者: **myhf2002@163.com;zhn.8888-@163.com

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引用本文:

刘然, 孟祥照, 苑赛男, 李国良, 杨阳, 段硕楠, 张华宁, 郭秀林. 小麦热激转录因子TaHsfA1亚家族基因的生物学特性及耐热性分析[J]. 农业生物技术学报, 2022, 30(1): 1-14.
LIU Ran, MENG Xiang-Zhao, YUAN Sai-Nan, LI Guo-Liang, YANG Yang, DUAN Shuo-Nan, ZHANG Hua-Ning, GUO Xiu-Lin. Biological Characteristics and Thermotolerance Analysis of Heat Shock Transcription Factor TaHsfA1 Subfamily Genes in Wheat (Triticum aestivum). 农业生物技术学报, 2022, 30(1): 1-14.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.01.001 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I1/1

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