小麦热激蛋白基因TaHSP90-1的克隆与表达分析

doi:10.13304/j.nykjdb.2022.0393

摘要/Abstract

摘要：

热激蛋白（heat shock protein，HSPs）是一类广泛存在于植物体内的应激蛋白，在植物响应逆境过程中发挥重要作用。为进一步了解小麦HSP90-1基因并探究其相关生物学功能，搜索小麦基因组序列数据库，获得了小麦A、B、D 3个基因组上的同源序列，根据其染色体位置分别命名为TaHSP90-1-A、TaHSP90-1-B和TaHSP90-1-D，通过同源克隆从12个小麦品种中得到TaHSP90-1 DNA序列。序列分析表明，TaHSP90-1-A、TaHSP90-1-B和TaHSP90-1-D分别含有2 121、2 136和2 130 bp的完整开放阅读框，分别编码707、712和710个氨基酸；其蛋白序列C端均含有1个HSP90结构域，为HSP家族HSP90亚家族成员。TaHSP90-1在不同小麦品种中存在SNP位点，分别位于TaHSP90-1-B第2个内含子区域和TaHSP90-1-D启动子区域。系统进化分析表明，TaHSP90-1-A与二粒小麦处于同一分支；TaHSP90-1-B和TaHSP90-1-D处于同一分支。顺式作用元件分析发现，TaHSP90-1-A、TaHSP90-1-B和TaHSP90-1-D启动子区域均含有干旱响应元件（MBS element）和热响应元件（HSE element）。RT-PCR结果显示，TaHSP90-1-A、TaHSP90-1-B和TaHSP90-1-D在苗期和成熟期的叶片中表达量较高。在干旱胁迫条件下，TaHSP90-1表达量在耐旱小麦品种中较高，为旱敏感小麦品种表达量的150倍；在热胁迫条件下，TaHSP90-1在热敏感与耐热小麦品种中均上调表达，其中，TaHSP90-1-A和TaHSP90-1-D在耐热小麦品种中上调表达倍数更高。

关键词: 小麦, HSP90, 干旱胁迫, 热胁迫, SNP

Abstract:

Heat shock proteins （HSPs） are a kind of stress proteins widely exist in plants and play important role in the response of plants to stress. In order to further understand the wheat HSP90-1 gene and explore its related biological functions，this paper searched the wheat genome sequence database， and obtained the homologous gene sequences on wheat A， B and D subgenomes， which were named as TaHSP90-1-A， TaHSP90-1-B and TaHSP90-1-D， respectively. TaHSP90-1 DNA sequences were obtained from 12 wheat varieties through homologous cloning and sequence analysis showed that the complete open reading frames of TaHSP90-1-A， TaHSP90-1-B and TaHSP90-1-D were 2 121， 2 136 and 2 130 bp， encoding 707， 712 and 710 amino acids， respectively. The C-terminal sequence of TaHSP90-1-A， TaHSP90-1-B and TaHSP90-1-D contained an HSP90 domain， which were members of HSP90 subfamily in HSP family. SNP loci were found among different wheat varieties， which were located in the second intron region of TaHSP90-1-B and the promoter region of TaHSP90-1-D. Phylogenetic analysis showed that TaHSP90-1-A and two grain wheat were in the same branch， and TaHSP90-1-B and TaHSP90-1-D were in the same branch. Cis acting element analysis showed that the promoter regions of TaHSP90-1-A， TaHSP90-1-B and TaHSP90-1-D contained drought response element （MBS element） and thermal response element （HSE element）. RT-PCR showed that TaHSP90-1-A， TaHSP90-1-B and TaHSP90-1-D had high expression in leaves at seedling and mature stages of wheat. Under drought stress， the expression level of TaHSP90-1 was higher in drought tolerant wheat varieties， which was 150 times compared with varieties of drought sensitive. Under heat stress， TaHSP90-1 was up-regulated in both heat sensitive and heat resistant varieties， while TaHSP90-1-A and TaHSP90-1-D were significantly up-regulated in heat-resistant varieties of wheat.

Key words: wheat, HSP90, drought stress, heat stress, SNP

中图分类号:

S512.1

陆青, 梁婷, 王伟伟, 汪德州, 吴娴, 王小燕, 唐益苗. 小麦热激蛋白基因TaHSP90-1的克隆与表达分析[J]. 中国农业科技导报, 2022, 24(8): 44-54.

Qing LU, Ting LIANG, Weiwei WANG, Dezhou WANG, Xian WU, Xiaoyan WANG, Yimiao TANG. Cloning and Expression Analysis of Wheat Heat Shock Protein Gene TaHSP90-1[J]. Journal of Agricultural Science and Technology, 2022, 24(8): 44-54.

图/表 8

表1 引物名称及序列

Table 1 Primer name and sequence

引物名称 Primer name	引物序列 Primer sequence （5’-3’）	用途 Purpose	扩增产物长度PCR product length/bp	退火温度 Temperature/℃
TaHSP90-1-A-F	TCGAGAAGTGGCAGCGGAGACGGCA	克隆基因Gene cloning	2 318	62
TaHSP90-1-A-R	AGGAAATGAGACTCTTCTTCAATCT
TaHSP90-1-B-F	CCAATCTTCCGGCGAAAGAGAGGCC		2 236	60
TaHSP90-1-B-R	TGAAATGAGATTCTTCAAGAGAT
TaHSP90-1-D-F	TGACAGCCATGGCGGACGTGCAG		2 230	60
TaHSP90-1-D-R	GAGGTCGACTGAAGAATTTCAGT
TaHSP90-1-A-qPCR-F	GGGGGGAGAGACCACAAG	荧光定量PCR Real-time PCR	117	60
TaHSP90-1-A-qPCR-R	CGGAGATGGGATCGCTAGG
TaHSP90-1-B-qPCR-F	GCAGTATGTGTGGGAGTCGC		194	60
TaHSP90-1-B-qPCR-R	TCCAGAGGTAGATGGGGTAG
TaHSP90-1-D-qPCR-F	AGCAGTACGTGTGGGAGTCGC		199	60
TaHSP90-1-D-qPCR-R	TCGGTCCAGAGGTAGATGGGG
TaHSP90-1-A-promoter-F	CAAGTACATGGACCATCTAGCG	克隆启动子Cloning promoter	2 115	64
TaHSP90-1-A-promoter-R	GACCTTAGCAGAAATGGGGAGGC
TaHSP90-1-B-promoter-F	TAACATTGTGAAACTGATTATTT		2 234	60
TaHSP90-1-B-promoter-R	AATGGACCTCAGAAGAAACGGG
TaHSP90-1-D-promoter-F	GAAAAAGATGTTGAATTTATTGT		2 178	60
TaHSP90-1-D-promoter-R	AGGGAAGCGGTTCGCCTTGGCCT
Actin-F	TACTCCCTCACAACAACCG	内参 Quantitative reference	317	60
Actin-R	AGAACCTCCACTGAGAACAA	内参 Quantitative reference

图1 TaHSP90-1同源蛋白多序列比对注：深蓝色背景的氨基酸序列表示序列完全匹配；浅蓝色背景的氨基酸序列表示氨基酸序列匹配有差异；蓝色和红色下划线表示HATPase_C结构域；黑色下划线表示HSP90结构域。

Fig. 1 Multi sequence alignment of TaHSP90-1 homologous proteinNote：The amino acid sequence on the dark blue background indicates that the sequence is completely matched； the amino acid sequence on the light blue background indicates that the amino acid sequence matching is different； the blue and red underline indicates HATPase_C domain； the black underline indicates HSP90 domain.

图2 小麦TaHSP90-1基因全长及启动子序列扩增结果与分析A：小麦TaHSP90-1基因全长序列扩增结果；B：小麦TaHSP90-1启动子扩增结果；C：小白麦、皖麦33、矮抗58 TaHSP90-1基因结构和序列变异；D：TaHSP90-1基因启动子的结构和序列变异。M—DL 2 000 分子量标记；UTR—非翻译区；CDS—编码序列

Fig. 2 Amplification results and analysis of the full-length sequence and promoter of wheat TaHSP90-1 geneA：Full length amplification of wheat TaHSP90-1 gene； B：Amplification results of wheat TaHSP90-1 promoter； C：Sketch map of structure and sequence variation of gene TaHSP90-1 in Xiaobaimai，Wanmai 33 and Aikang 58； D：Sketch map of structure and sequence variation of gene TaHSP90-1 promoter in Xiaobaimai，Wanmai 33 and Aikang 58. M—DL 2 000 marker； UTR—Untranslated region； CDS—Coding sequence

图3 小麦TaHSP90-1蛋白与其他植物的同源蛋白序列的系统进化分析注：At—拟南芥；Os—水稻； Tu—乌拉尔图小麦；Hv—大麦；Td—二粒小麦；Sl—番茄；St—马铃薯；Ta—小麦。

Fig.3 Phylogenetic analysis of wheat TaHSP90-1 protein and other plant homologous protein sequencesNote：At—Arabidopsis thaliana； Os—Oryza sativa； Tu—Triticum urartu； Hv—Hordeum vulgare； Td—Triticum dicoccoide； Sl—Solanum lycopersicum； St—Solanumtuberosum； Ta—Triticum aestivum.

表2 TaHSP90-1顺式作用元件统计

Table 2 Cis-acting element statistics of TaHSP90-1

基因 Gene	顺式作用元件序列 Cis-acting element sequence （5’-3’）	顺式作用元件 Cis-acting element	长度 Length/bp
TaHSP90-1-A	CAACTG	MBS	6
TaHSP90-1-D	CAACTG	MBS	6
TaHSP90-1-D	CAACTG	MBS	6
TaHSP90-1-A	ACATCCTCCG	HSE	10
TaHSP90-1-A	AGATCCTCTAG	HSE	11
TaHSP90-1-A	GAATCCTTGTG	HSE	11
TaHSP90-1-A	TTTTTCCTTT	HSE	10
TaHSP90-1-A	GGCATCCTGGG	HSE	11
TaHSP90-1-B	AGATTGGAACTCCC	HSE	14
TaHSP90-1-B	CTCACGAATCGCA	HSE	13
TaHSP90-1-B	CTCACGAATCGCA	HSE	13
TaHSP90-1-D	ACGAGAAAATGCA	HSE	13
TaHSP90-1-D	ACAACTCCCCAGT	HSE	13
TaHSP90-1-D	ACAAATCCATGAAC	HSE	14
TaHSP90-1-D	TTTTGAACCGGT	HSE	12
TaHSP90-1-D	ATGGTCCGTATGT	HSE	13
TaHSP90-1-D	CAAGTAGAAGCTTCG	HSE	15

图4 小麦TaHSP90-1在不同组织中的相对表达量A：苗期；B：成熟期

Fig. 4 Relative expression of TaHSP90-1 in different tissues of wheatA： Seedling stage； B： Mature stage

图5 干旱胁迫下小麦TaHSP90-1在不同品种中的表达分析A：旱胁迫12 h生长情况；B：旱胁迫后TaHSP90-1基因表达

Fig. 5 Expression analysis of wheat TaHSP90-1 in different varieties under drought stressA： 12 h growth of Xiaobaimai and Wanmai 33 under drought stress； B： Expression of TaHSP90-1 gene in Xiaobaimai and Wanmai 33 after drought stress

图6 热胁迫下小麦TaHSP90-1在不同品种中的表达分析A：小白麦和矮抗58热胁迫12 h后的表型；B：小白麦和矮抗58热胁迫后TaHSP90-1基因表达

Fig. 6 Expression analysis of wheat TaHSP90-1 in different varieties under heat stressA： Phenotype of Xiaobaimai and Aikang 58 after heat stress 12 h； B： Expression of TaHSP90-1 gene in Xiaobaimai and Aikang 58 after heat stress

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