菜心BrHsfA3基因克隆及其对高温胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2022-0568

摘要/Abstract

摘要：

为探索菜心对高温胁迫的响应机制，发掘耐热相关基因，本研究采用全基因合成法（PAS）对前期从菜心高温胁迫转录组数据库中筛选到的1个差异表达基因进行克隆，并开展相关生物信息学分析，利用实时荧光定量PCR技术（RT-qPCR）分析该基因在高温胁迫下的表达模式。结果表明，克隆得到开放阅读框（ORF）为906 bp的基因，命名为BrHsfA3。该基因编码301个氨基酸，蛋白相对分子量为34.12 kD，理论等电点为5.00，为无信号肽的亲水性蛋白，存在丝氨酸、苏氨酸和酪氨酸3个磷酸化位点。BrHsfA3蛋白含有1个Hsf保守结构域，具有典型的DNA结合结构域（DBD）、寡聚化结构域（OD）、核定位信号结构域（NLS）和转录激活结构域（AHA），蛋白二级结构以α-螺旋和无规则卷曲为主。进化分析发现，BrHsfA3基因与甘蓝型油菜的亲缘关系最近。RT-qPCR分析结果显示，BrHsfA3在耐热自交系CX1-7叶、花中受高温诱导后表达量大幅上调，且显著高于热敏自交系CX7-3；根中表达量则在热敏自交系CX7-3中较高，在高温诱导后显著上调。本研究结果为进一步探讨BrHsfA3基因在菜心响应高温胁迫应答中的调控作用奠定基础。

关键词: 菜心, BrHsfA3, 高温胁迫, 表达分析

Abstract:

In order to study the response mechanism to heat stress and explore related genes of Chinese flowering cabbage（Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee）, a differentially expressed gene screened from the transcriptome database under heat stress in the early stage was cloned by PCR-based accurate synthesis（PAS）. Then bioinformatics analysis was performed, and the expression profiles of the gene under heat stress were analyzed by RT-qPCR. The results showed that the gene with an ORF of 906 bp was obtained and named as BrHsfA3, encoding 301 amino acid residues, the relative molecular weight was 34.12 kD with a theoretical isoelectric point being 5.00. BrHsfA3 protein was a hydrophilic protein with three phosphorylation sites of serine, threonine and tyrosine. Sequence analysis indicated that BrHsfA3 possessed a Hsf conserved domain, a DNA-binding domain（DBD）, a oligomerization domain（OD）, a nuclear localization signal（NLS）, and an activator domain（AHA）, the secondary structure were mainly alpha helix and random coil. Evolutionary analysis revealed that BrHsfA3 gene had the closest genetic relationship with Brassica napus. The RT-qPCR analysis results showed that the expression of BrHsfA3 was highly induced in the leaves and flowers of heat-resistant self-inbred line CX1-7 by heat stress and significantly higher than in heat-sensitive self-inbred line CX7-3, but the expression in the roots of CX7-3 was high under the same heat treatment, and which was significantly up-regulated after heat stress. These results provide a foundation for the high temperature response mechanism of BrHsfA3 in Chinese flowering cabbage.

Key words: Chinese flowering cabbage, BrHsfA3, heat stress, expression analysis

庞强强, 孙晓东, 周曼, 蔡兴来, 张文, 王亚强. 菜心BrHsfA3基因克隆及其对高温胁迫的响应[J]. 生物技术通报, 2023, 39(2): 107-115.

PANG Qiang-qiang, SUN Xiao-dong, ZHOU Man, CAI Xing-lai, ZHANG Wen, WANG Ya-qiang. Cloning of BrHsfA3 in Chinese Flowering Cabbage and Its Responses to Heat Stress[J]. Biotechnology Bulletin, 2023, 39(2): 107-115.

图/表 11

表1 基因克隆和表达分析引物

Table 1 Primers used for gene cloning and expression analysis

引物名称Primer name	引物序列Primer sequence（5'-3'）	引物用途Primer purpose
BrHsfA3-F1	CGCCAGAATCATACTCCCGC	基因合成 Gene synthesis
BrHsfA3-R1	CCTTAGAGGACAGAAGCATC	基因合成 Gene synthesis
BrHsfA3-F2	GATGTGCTGCAAGGCGATTA	基因合成 Gene synthesis
BrHsfA3-R2	TTATGCTTCCGGCTCGTATG	基因合成 Gene synthesis
BrActin-F	AGCAACTGGGATGACATGGA	RT-qPCR内参基因 RT-qPCR reference genes
BrActin-R	TCACCAGAGTCGAGCACAAT	RT-qPCR内参基因RT-qPCR reference genes
Q-BrHsfA3-F	GTCGACATAACAGACGTGGC	RT-qPCR
Q-BrHsfA3-R	CTGTCTGACGAAGCTGGAGA	RT-qPCR

图1 BrHsfA3的ORF序列及氨基酸序列

Fig. 1 ORF sequences and amino acid sequences of BrHsfA3

图2 BrHsfA3 蛋白疏水性/亲水性预测

Fig. 2 Prediction of the hydrophobic/hydrophilic of BrHs-fA3 protein

图3 BrHsfA3蛋白氨基酸翻译后磷酸化修饰预测

Fig. 3 Prediction of phosphorylation site after amino acid translation of BrHsfA3 protein

图4 BrHsfA3蛋白信号肽预测

Fig. 4 Prediction of the signal peptide prediction of BrHs-fA3 protein

图5 BrHsfA3蛋白的保守结构域及功能结构域

Fig. 5 Conservative domains and functional domain of BrHsfA3 protein

图6 BrHsfA3蛋白二级结构预测横轴表示氨基酸位置；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

Fig. 6 Prediction of BrHsfA3 protein secondary structure Horizontal axis indicates amino acid position. The blue part indicates alpha helix. The purple indicates random coil. The red part indicates extended strand. The green part indicates beta turn

图7 BrHsfA3蛋白三级结构预测

Fig. 7 Prediction of the three-dimensional structure of BrHsfA3 protein

图8 菜心与其他科属植物HsfA3氨基酸序列的分子系统进化树系统树

Fig. 8 Phylogenetic tree of HsfA3 amino acid sequence from flowering Chinese cabbage and other plants

图9 BrHsfA3基因在高温胁迫处理下的FPKM值不同小写字母表示同一类型自交系植株差异显著（P< 0.05），下同

Fig. 9 FPKM value of BrHsfA3 gene under heat stress Different lowercase letters of the same inbred indicate significant difference（P<0.05），the same below

图10 BrHsfA3基因在高温胁迫前后不同组织的相对表达量

Fig. 10 Relative expressions of BrHsfA3 gene in different tissues under heat stress

参考文献 38

[1]	陈汉才, 吴增祥, 林悦欣, 等. 广东菜心、芥蓝研究现状与展望[J]. 广东农业科学, 2021, 48(9):62-71.
	Chen HC, Wu ZX, Lin YX, et al. Research status and prospect of flowering Chinese cabbage and Chinese kale in Guangdong[J]. Guangdong Agric Sci, 2021, 48(9):62-71.
[2]	刘畅. 高温涝渍对菜心农艺性状和生理特性影响的研究[D]. 广州: 广州大学, 2020.
	Liu C. Effect of high temperature and waterlogging on agronomic and physiological traits in flowering Chinese cabbage[D]. Guangzhou: Guangzhou University, 2020.
[3]	陈连珠, 张雪彬, 杨小锋. 根际高温对菜心生长及光合生理的影响[J]. 北方园艺, 2020(14):50-55.
	Chen LZ, Zhang XB, Yang XF. Effects of rhizosphere high temperature on growth and photosynthetic physiology of Chinese flowering cabbages[J]. North Hortic, 2020(14):50-55.
[4]	Yue YZ, Jiang HY, Du JH, et al. Variations in physiological response and expression profiles of proline metabolism-related genes and heat shock transcription factor genes in Petunia subjected to heat stress[J]. Sci Hortic, 2019, 258:108811. doi: 10.1016/j.scienta.2019.108811 URL
[5]	司修洋, 梁文杰, 罗澜, 等. 甜瓜热激转录因子(Hsf)基因家族鉴定及生物信息学分析[J]. 中国蔬菜, 2020(11):23-32.
	Si XY, Liang WJ, Luo L, et al. Identification and bioinformatics analysis of heat shock transcription factor(Hsf)gene family in melon[J]. China Veg, 2020(11):23-32.
[6]	Huang B, Huang ZN, Ma RF, et al. Genome-wide identification and analysis of the heat shock transcription factor family in moso bamboo(Phyllostachys edulis)[J]. Sci Rep, 2021, 11(1):16492. doi: 10.1038/s41598-021-95899-3 pmid: 34389742
[7]	Shen CW, Yuan JP. Genome-wide characterization and expression analysis of the heat shock transcription factor family in pumpkin(Cucurbita moschata)[J]. BMC Plant Biol, 2020, 20:471. doi: 10.1186/s12870-020-02683-y URL
[8]	Li MY, Xie FJ, Li YW, et al. Genome-wide analysis of the heat shock transcription factor gene family in Brassica juncea:structure, evolution, and expression profiles[J]. DNA Cell Biol, 2020, 39(11):1990-2004. doi: 10.1089/dna.2020.5922 URL
[9]	Zhang Q, Geng J, Du YL, et al. Heat shock transcription factor(Hsf)gene family in common bean(Phaseolus vulgaris):genome-wide identification, phylogeny, evolutionary expansion and expression analyses at the sprout stage under abiotic stress[J]. BMC Plant Biol, 2022, 22(1):33. doi: 10.1186/s12870-021-03417-4 pmid: 35031009
[10]	Zhou M, Zheng SG, Liu R, et al. Genome-wide identification, phylogenetic and expression analysis of the heat shock transcription factor family in bread wheat(Triticum aestivum L.)[J]. BMC Genomics, 2019, 20(1):505. doi: 10.1186/s12864-019-5876-x pmid: 31215411
[11]	Wiederrecht G, Seto D, Parker CS. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor[J]. Cell, 1988, 54(6):841-853. doi: 10.1016/s0092-8674(88)91197-x pmid: 3044612
[12]	Scharf KD, Rose S, Zott W, et al. Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF[J]. EMBO J, 1990, 9(13):4495-4501. doi: 10.1002/j.1460-2075.1990.tb07900.x pmid: 2148291
[13]	Sanmiya K, Koja Y, Iguchi A. The cloning of cDNAs for the heat-shock transcription factors HSFA1, HSFA2 and HSFA3 from tobacco[J]. Trop Agric Dev, 2020, 64:34-40.
[14]	Wu Z, Liang JH, Wang CP, et al. Alternative splicing provides a mechanism to regulate LlHSFA3 function in response to heat stress in lily[J]. Plant Physiol, 2019, 181(4):1651-1667. doi: 10.1104/pp.19.00839 URL
[15]	唐锐敏, 贾小云, 朱文娇, 等. 马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析[J]. 作物学报, 2021, 47(4):672-683. doi: 10.3724/SP.J.1006.2021.04114
	Tang RM, Jia XY, Zhu WJ, et al. Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance[J]. Acta Agron Sin, 2021, 47(4):672-683. doi: 10.3724/SP.J.1006.2021.04114 URL
[16]	Larkindale J, Vierling E. Core genome responses involved in acclimation to high temperature[J]. Plant Physiol, 2008, 146(2):748-761. doi: 10.1104/pp.107.112060 pmid: 18055584
[17]	Chen H, Hwang JE, Lim CJ, et al. Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response[J]. Biochem Biophys Res Commun, 2010, 401(2):238-244. doi: 10.1016/j.bbrc.2010.09.038 URL
[18]	Li ZJ, Zhang LL, Wang AX, et al. Ectopic overexpression of SlHsfA3, a heat stress transcription factor from tomato, confers increased thermotolerance and salt hypersensitivity in germination in transgenic Arabidopsis[J]. PLoS One, 2013, 8(1):e54880. doi: 10.1371/journal.pone.0054880 URL
[19]	Zhu MD, Zhang M, Gao DJ, et al. Rice OsHSFA3 gene improves drought tolerance by modulating polyamine biosynthesis depending on abscisic acid and ROS levels[J]. Int J Mol Sci, 2020, 21(5):1857. doi: 10.3390/ijms21051857 URL
[20]	Mittal D, Chakrabarti S, Sarkar A, et al. Heat shock factor gene family in rice:genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses[J]. Plant Physiol Biochem, 2009, 47(9):785-795. doi: 10.1016/j.plaphy.2009.05.003 URL
[21]	吴泽. 百合热激转录因子LlHsfA3调控耐热性和耐盐性的机制解析[D]. 北京: 中国农业大学, 2018.
	Wu Z. Analysis of thermotolerance and salt tolerance mechanism regulated by heat shock transcription factor LlHsfA3 from lily(Lili-um longiflorum)[D]. Beijing: China Agricultural University, 2018.
[22]	Song C, Chung WS, Lim CO. Overexpression of heat shock factor gene HsfA3 increases galactinol levels and oxidative stress tolerance in Arabidopsis[J]. Mol Cells, 2016, 39(6):477-483. doi: 10.14348/molcells.2016.0027 URL
[23]	庞强强, 孙晓东, 周曼, 等. 一种菜心耐热性评价方法:CN113348992A[P]. 2021-09-07.
	Pang QQ, Sun XD, Zhou M, et al. A method for evaluating heat resistance of Chinese flowering cabbage:CN113348992A[P]. 2021-09-07.
[24]	庞强强, 周曼, 孙晓东, 等. 不同菜心品种萌发期和苗期耐热性分析及其鉴定指标筛选[J]. 西北农业学报, 2020, 29(2):295-305.
	Pang QQ, Zhou M, Sun XD, et al. Comprehensive evaluation and indexes screening of heat tolerance at germination and seedling stages in different cultivars of Chinese flowering cabbage[J]. Acta Agric Boreali Occidentalis Sin, 2020, 29(2):295-305.
[25]	庞强强, 周曼, 孙晓东, 等. 菜心耐热性评价及酶促抗氧化系统对高温胁迫的响应[J]. 浙江农业学报, 2020, 32(1):72-79. doi: 10.3969/j.issn.1004-1524.2020.01.09
	Pang QQ, Zhou M, Sun XD, et al. Evaluation of heat tolerance and response of enzymatic antioxidant system to heat stress in Brassica parachinensis L[J]. Acta Agric Zhejiangensis, 2020, 32(1):72-79.
[26]	卢宇鹏. 菜心耐热性评价及耐热基因等位变异分析[D]. 广州: 广州大学, 2020.
	Lu YP. Evaluation of heat tolerance and analysis of allele variation of heat tolerance candidate gene in flowering Chinese cabbage[D]. Guangzhou: Guangzhou University, 2020.
[27]	Rao S, Das JR, Mathur S. Exploring the master regulator heat stress transcription factor HSFA1a-mediated transcriptional cascade of HSFs in the heat stress response of tomato[J]. J Plant Biochem Biotechnol, 2021, 30(4):878-888. doi: 10.1007/s13562-021-00696-8 URL
[28]	Meena S, Samtani H, Khurana P. Elucidating the functional role of heat stress transcription factor A6b(TaHsfA6b)in linking heat stress response and the unfolded protein response in wheat[J]. Plant Mol Biol, 2022, 108(6):621-634. doi: 10.1007/s11103-022-01252-1 URL
[29]	刘栩铭, 李敏, 段琼, 等. 蓖麻RcHSF基因家族鉴定与冷胁迫下的表达模式分析[J]. 华北农学报, 2020, 35(5):62-71. doi: 10.7668/hbnxb.20191312
	Liu XM, Li M, Duan Q, et al. Identification of RcHSF gene family in castor and analysis of expression pattern under cold stress[J]. Acta Agric Boreali Sin, 2020, 35(5):62-71.
[30]	Guo M, Lu JP, Zhai YF, et al. Genome-wide analysis, expression profile of heat shock factor gene family(CaHsfs)and characterisation of CaHsfA2 in pepper(Capsicum annuum L.)[J]. BMC Plant Biol, 2015, 15:151. doi: 10.1186/s12870-015-0512-7 URL
[31]	Rehman A, Atif RM, Azhar MT, et al. Genome wide identification, classification and functional characterization of heat shock transcription factors in cultivated and ancestral cottons(Gossypium spp. )[J]. Int J Biol Macromol, 2021, 182:1507-1527. doi: 10.1016/j.ijbiomac.2021.05.016 pmid: 33965497
[32]	张楠, 王映红, 王志敏, 等. 植物热激转录因子家族的研究进展[J]. 生物工程学报, 2021, 37(4):1155-1167.
	Zhang N, Wang YH, Wang ZM, et al. Heat shock transcription factor family in plants:a review[J]. Chin J Biotechnol, 2021, 37(4):1155-1167.
[33]	焦淑珍, 姚文孔, 张宁波, 等. 园艺植物热激转录因子研究进展[J]. 果树学报, 2020, 37(3):419-430.
	Jiao SZ, Yao WK, Zhang NB, et al. Research progress of heat stress transcription factors(Hsfs)in horticultural plants[J]. J Fruit Sci, 2020, 37(3):419-430.
[34]	Schramm F, Larkindale J, Kiehlmann E, et al. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis[J]. The Plant Jouranl, 2008, 53(2):264-274.
[35]	Yoshida T, Sakuma Y, Todaka D, et al. Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system[J]. Biochem Biophys Res Commun, 2008, 368(3):515-521. doi: 10.1016/j.bbrc.2008.01.134 URL
[36]	孙天晓. 多年生黑麦草热激转录因子HSFAs和HSFCs亚家族基因的功能研究[D]. 武汉: 华中农业大学, 2021.
	Sun TX. Function of heat shock transcription factor HSFAs and HSFCs subfamily genes in perennial ryegrass[D]. Wuhan: Huazhong Agricultural University, 2021.
[37]	Zhu XY, Huang CQ, Zhang L, et al. Systematic analysis of Hsf family genes in the Brassica napus genome reveals novel responses to heat, drought and high CO₂ stresses[J]. Front Plant Sci, 2017, 8:1174. doi: 10.3389/fpls.2017.01174 URL
[38]	庞强强. 茄子HSFs和HSPs基因鉴定及其在高温下的表达模式分析[D]. 广州: 华南农业大学, 2016.
	Pang QQ. Genome wide identification of HSFs and HSPs gene family in eggplant(Solanum melongema L.)and analysis of their expression pattern under high temperature[D]. Guangzhou: South China Agricultural University, 2016.