玉米大斑病菌小分子热激蛋白基因克隆与表达分析

doi:10.3864/j.issn.0578-1752.2024.17.006

中国农业科学 ›› 2024, Vol. 57 ›› Issue (17): 3384-3397.doi: 10.3864/j.issn.0578-1752.2024.17.006

玉米大斑病菌小分子热激蛋白基因克隆与表达分析

张淑红¹(), 张运峰¹, 高凤菊¹, 武秋颖¹, 许可¹, 李亚子¹, 李艳梅¹, 谷守芹², 范永山¹(), 巩校东²()

¹ 唐山师范学院生命科学系/河北省植物生物技术研究与应用重点实验室/唐山市农业病原真菌与毒素重点实验室，河北唐山 063000
² 河北农业大学生命科学学院/河北省农业微生物生物信息利用技术创新中心，河北保定 071000

收稿日期:2024-04-26 接受日期:2024-06-16 出版日期:2024-09-01 发布日期:2024-09-04
通信作者:
范永山，E-mail：fanyongshan@126.com
巩校东，E-mail：gxdjy@126.com
联系方式: 张淑红，E-mail：zsh3535@163.com。
基金资助:
国家自然科学基金(22078171); 中央引导地方科技发展资金(236Z6508G); 河北省省级科技计划(21374301D); 唐山师范学院重点培育项目(ZDPY06); 唐山师范学院重点培育项目(ZDPY08)

Cloning and Expression Analysis of Genes of Small Heat Shock Protein in Setosphaeria turcica

ZHANG ShuHong¹(), ZHANG YunFeng¹, GAO FengJu¹, WU QiuYing¹, XU Ke¹, LI YaZi¹, LI YanMei¹, GU ShouQin², FAN YongShan¹(), GONG XiaoDong²()

¹ Department of Life Sciences, Tangshan Normal University/Hebei Key Laboratory of Plant Biotechnology Research and Application/ Tangshan Key Laboratory of Agricultural Pathogenic Fungi and Toxins, Tangshan 063000, Hebei
² College of Life Sciences/Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, Hebei Agricultural University, Baoding 071000, Hebei

Received:2024-04-26 Accepted:2024-06-16 Published:2024-09-01 Online:2024-09-04

摘要/Abstract

摘要：

【目的】克隆玉米大斑病菌（Setosphaeria turcica）小分子热激蛋白（small heat shock protein，sHSP）基因，分析其结构及在病菌发育和HT-毒素诱导过程中的表达模式。【方法】利用隐马尔可夫模型（hidden Markov model，HMM）筛选玉米大斑病菌全基因组范围内的sHSP家族成员，采用PCR技术克隆玉米大斑病菌01-23菌株的sHSP，通过生物信息学方法进行sHSP的理化性质分析、亚细胞定位、结构预测和系统发育分析，RNA-Seq和RT-qPCR分析sHSP在玉米大斑病菌不同发育阶段和HT-毒素诱导过程中的表达情况。【结果】从玉米大斑病菌基因组筛选到3个sHSP家族成员（StHSP37.2、StHSP37.0和StHSP22.6），克隆了01-23菌株中3个sHSP的DNA序列，编码的sHSP蛋白均属于弱酸、亲水蛋白，无跨膜结构域和信号肽；二级结构中无规则卷曲占58.97%—60.35%，而β-转角仅为2.69%—7.83%；亚细胞定位预测StHSP37.2和StHSP37.0位于细胞核，而StHSP22.6位于细胞核和细胞质；均含有近C端的ACD_sHSP-like结构域，StHSP37.2、StHSP37.0和StHSP22.6分别有2、3和5个保守基序；利用SWISS-Model和AlphaFill构建了sHSP单体的三级结构模型；系统发育分析结果表明，StHSP22.6与链格孢（Alternaria alternata）、StHSP37.2和StHSP37.0与玉米小斑病菌（Bipolaris maydis）的sHSP亲缘关系较近；玉米大斑病菌sHSP在菌丝中表达量最高，其次为芽管、附着胞和侵入钉，在分生孢子中表达量最低；StHSP22.6和StHSP37.2与玉米大斑病菌HT-毒素诱导显著负相关，在14 d时相对基因表达量分别上调6.45和18.12倍，21、28 d时StHSP37.2表达量仅上调2.56、1.78倍，而StHSP22.6表达量与WT无显著差异；StHSP37.0与HT-毒素诱导显著正相关，14、21和28 d时相对基因表达量分别下调59.23%、86.30%和88.11%；通过与玉米大斑病菌sHSP显著相关的表达基因挖掘，推测StHSP37.2和StHSP22.6主要与HSP90、HSP104、分解代谢及线粒体Mg²⁺转运有关，而StHSP37.0主要与液泡碱性氨基酸转运、有机合成和分泌有关。【结论】玉米大斑病菌sHSP家族成员既具有高度保守性，又与其他sHSP有结构和系统发育的差异性；不仅与玉米大斑病菌菌丝、芽管、附着胞和侵入钉发育相关，在HT-毒素诱导过程中也发挥重要调控作用。

关键词: 玉米大斑病菌, 小分子热激蛋白, 基因克隆, 结构分析, HT-毒素诱导

Abstract:

【Objective】The objective of this study is to clone the small heat shock protein (sHSP) genes in Setosphaeria turcica, elucidate their structural characteristics, and explore their expression profiles during pathogen development and in response to HT-toxin induction.【Method】The hidden Markov model (HMM) was used to identify sHSP family members across the entire S. turcica genome. PCR technology was used to clone sHSP genes from S. turcica strain 01-23. Bioinformatics methods were then applied for the analysis of physicochemical properties, subcellular localization, structural prediction, and phylogenetic analysis of the sHSP genes obtained. Additionally, RNA-Seq and RT-qPCR were performed to determine the expression of sHSP genes across different developmental stages and during HT-toxin induction in S. turcica.【Result】Three sHSP family members (StHSP37.2, StHSP37.0 and StHSP22.6) were identified from the genome of S. turcica. The corresponding DNA sequences were successfully cloned from strain 01-23. The encoded sHSP proteins were weakly acidic and hydrophilic proteins, without transmembrane domain or signal peptide. Random coil in the secondary structure accounted for 58.97% to 60.35%, and β-turn ranged from 2.69% to 7.83% only. Subcellular localization prediction indicated that StHSP37.2 and StHSP37.0 were located in the nucleus, while StHSP22.6 was located in both nucleus and cytoplasm. Conserved ACD_sHSP-like domains were identified near C-terminus, with 2, 3, and 5 conserved motifs in StHSP37.2, StHSP37.0, and StHSP22.6, respectively. The monomer tertiary structure models of sHSP were constructed using SWISS-Model and AlphaFill. Phylogenetic analysis indicated close relationships between StHSP22.6 and sHSP in Alternaria alternata, and between StHSP37.2/StHSP37.0 and sHSP in Bipolaris maydis. The sHSP genes of S. turcica had the highest expression levels in hyphae, followed by germ tubes, appressoria, and penetration pegs, with the lowest expression levels in conidia. StHSP22.6 and StHSP37.2 showed significant negative correlations with HT-toxin induction, and the relative gene expression was upregulated by 6.45 and 18.12 folds on day 14, respectively. On day 21 and 28, StHSP37.2 showed modest upregulations of 2.56 and 1.78 folds, respectively, while StHSP22.6 did not differ from the wild-type (WT). StHSP37.0 exhibited significant positive correlations with HT-toxin induction, with a significant downregulation by 59.23%, 86.30%, and 88.11% on day 14, 21, and 28, respectively. Exploration of expressed genes significantly associated with sHSP of S. turcica suggested that StHSP37.2 and StHSP22.6 were mainly related to HSP90, HSP104, catabolism, and mitochondrial Mg²⁺ transport, while StHSP37.0 appeared to be associated with vacuolar alkaline amino acid transport, organic synthesis, and substance secretion.【Conclusion】The sHSP family members in S. turcica demonstrate a high degree of conservation yet exhibit structural and phylogenetic differences from other sHSPs. They are integral to the development of hyphae, germ tubes, appressoria, and penetration pegs, and also exert significant regulatory functions during HT-toxin induction.

Key words: Setosphaeria turcica, small heat shock protein (sHSP), gene cloning, structure analysis, HT-toxin induction

张淑红, 张运峰, 高凤菊, 武秋颖, 许可, 李亚子, 李艳梅, 谷守芹, 范永山, 巩校东. 玉米大斑病菌小分子热激蛋白基因克隆与表达分析[J]. 中国农业科学, 2024, 57(17): 3384-3397.

ZHANG ShuHong, ZHANG YunFeng, GAO FengJu, WU QiuYing, XU Ke, LI YaZi, LI YanMei, GU ShouQin, FAN YongShan, GONG XiaoDong. Cloning and Expression Analysis of Genes of Small Heat Shock Protein in Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2024, 57(17): 3384-3397.

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表4

与StHSP37.2、StHSP37.0和StHSP22.6显著相关的基因及功能注释"

靶基因 Target gene	正相关基因及功能注释 Positive correlation gene and functional annotation		负相关基因及功能注释 Negative correlation gene and functional annotation
StHSP37.2	117863*	2.A.1.44.1 L-氨基酸转运蛋白-3 The L-amino acid transporter-3, LAT3; r=0.98, P=4.0e-16	25166	1.A.35.5.1线粒体内膜Mg²⁺通道蛋白Mitochondrial inner membrane Mg²⁺ channel protein, Mrs2; r=-0.88, P=1.0e-08
	150187	KOG0546 HSP90共伴侣HSP90 co-chaperone CPR7/ cyclophilin; r=0.98, P=3.1e-17	89201	PF09349OHCU脱羧酶Decarboxylase; r=-0.88, P=1.3e-08
	162245	KOG2825亚砷酸盐转运ATP酶 Putative arsenite- translocating ATPase; r=0.98, P=7.2e-16	157713	PF08241甲基转移酶Methyltransferase; r=-0.90, P=1.6e-09
	165581	KOG1667 Zn²⁺结合蛋白Zn²⁺-binding protein Melusin/ RAR1; r=0.98, P=1.6e-16	165190	KOG2393转录起始因子IIF Transcription initiation factor IIF, large subunit (RAP74); r=-0.88, P=1.8e-08
	165808	KOG0156细胞色素P450 CYP2亚家族 Cytochrome P450 CYP2 subfamily; r=0.98, P=1.1e-16	166884	KOG0234果糖-6-磷酸2-激酶Fructose-6-phosphate 2-kinase/fructose-2,6-biphosphatase; r=-0.87, P=3.0e-08
	166021	KOG0019分子伴侣（HSP90家族）Molecular chaperone (HSP90 family); r=0.98, P=3.0e-18	1337606	PF08427 Armadillo类螺旋结构域蛋白3 Armadillo-like helical domain-containing protein 3, C-terminal; r=-0.91, P=1.2e-09
StHSP37.0	31815	PF00545核糖核酸酶Ribonuclease; r=0.96, P=3.1e-14	28099	KOG1969 DNA复制检查点蛋白DNA replication checkpoint protein CHL12/CTF18; r=-0.83, P=5.0e-07
	148030	PF10503酯酶PHB解聚酶Esterase PHB depolymerase; r=0.93, P=7.5e-11	43017	PF11754 Velvet因子Velvet factor; r=-0.83, P=6.9e-07
	164754	PF06108未知功能蛋白Protein of unknown function (DUF952); r=0.94, P=2.0e-11	43464	KOG1974 DNA拓扑异构酶I互作蛋白DNA topoisomerase I-interacting protein; r=-0.84, P=2.6e-07
	164808	KOG1614外泌体3′-5′核糖核酸酶复合物Exosomal 3′-5′ exoribonuclease complex, subunit Rrp45; r=0.93, P=6.0e-11	145658	未知蛋白Unknown protein; r=-0.85, P=1.2e-07
	166696	PF13419卤酸脱卤酶样水解酶Haloacid dehalogenase-like hydrolase; r=0.97, P=6.9e-15	183743	KOG2483上游转录因子Upstream transcription factor 2/L-myc-2 protein; r=-0.89, P=7.8e-09
	1467493	2.A.1.3.27液泡碱性氨基酸转运蛋白The vacuolar basic amino acid (Arg, Lys, His) transporter, Vba3; r=0.93, P= 5.4e-11	1116535	未知蛋白Unknown protein; r=-0.84, P=3.4e-07
StHSP22.6	91583	KOG1051分子伴侣HSP104及相关ATP-依赖Clp蛋白酶Chaperone HSP104 and related ATP-dependent Clp proteases; r=0.93, P=4.1e-11	28352	KOG2645核苷酸焦磷酸酶Nucleotide pyrophosphatase/Type I phosphodiesterase; r=-0.88, P=2.1e-08
	163286	KOG0730 AAA+型ATP酶AAA+-type ATPase; r=0.95, P=2.3e-12	46176	IPR029069 HotDog域超家族HotDog domain superfamily; r= -0.85, P=1.1e-07
	165491	KOG1051分子伴侣HSP104及相关ATP-依赖Clp蛋白酶Chaperone HSP104 and related ATP-dependent Clp proteases; r=0.95, P=1.5e-12	86594	KOG0960线粒体加工肽酶Mitochondrial processing peptidase, beta subunit, and related enzymes; r=-0.85, P=1.9e-07
	174460	PF11951真菌特异性转录因子Fungal specific transcription factor domain; r=1.00, P=1.4e-28	99139	KOG0465线粒体延伸因子Mitochondrial elongation factor; r=-0.85, P=1.9e-07
	1367638	KOG1192 UDP-葡糖醛酸基和葡糖基转移酶UDP-glucuronosyl and UDP-glucosyl transferase; r=0.94, P=4.5e-12	117439	GO:0000981 II型RNA聚合酶RNA polymerase II-specific; r=-0.86, P=6.3e-08
	1465755	KOG0548分子伴侣Molecular co-chaperone STI1; r=0.93, P=4.4e-11	165190	KOG2393 转录起始因子Transcription initiation factor IIF, large subunit (RAP74); r=-0.84, P=2.1e-07

表4

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引物名称 Primer name	克隆用引物序列 Sequence for gene cloning (5′-3′)	引物名称 Primer name	表达分析用引物序列 Sequence for gene expression analysis (5′-3′)
StHSP37.2 F	ACACCAGCAACTCCACAGAT	StHSP37.2 F’	TATCGCGTTTCACGACCTCA
StHSP37.2 R	CATTTACTACTCATAATCAACCATCA	StHSP37.2 R’	GCTTTCTTTGCAGCATCCTTT
StHSP37.0 F	GTAGGCAGTAGGATTCCGC	StHSP37.0 F’	CCATTGTCGTTTGCGTCATTC
StHSP37.0 R	CTCAACACACCATCCTCCAA	StHSP37.0 R’	CCAAGATCCCTTTGCTCCAT
StHSP22.6 F	ATCACTGCGCTCAACAATCTC	StHSP22.6 F’	TCGCTAAGCAGTCGCAAGAG
StHSP22.6 R	ACCACGATGCTGAGGATGC	StHSP22.6 R’	GAAGGCAAAGGAGCGGTGG
		β-tubulin F	CAACGAAGCCTCCAACAACA
		β-tubulin R	CTCGGTGTAGTGACCCTTTGC

项目Item	StH<BOLD>SP</BOLD>22.6	StH<BOLD>SP</BOLD>37.2	StH<BOLD>SP</BOLD>37.0
分子式Formula	C₁₀₀₀H₁₅₄₈N₂₉₀O₃₀₈S₂	C₁₆₁₉H₂₅₂₂N₄₇₄O₅₃₂S₄	C₁₅₉₅H₂₃₈₄N₅₁₀O₅₀₉S₇
氨基酸数目Number of amino acids	196	334	342
等电点pI	6.19	5.04	5.74
疏水性系数Grand average of hydropathicity	-0.997	-0.912	-1.030
不稳定系数Instability index	52.62	63.02	39.50
亚细胞定位 Subcellular localization	细胞核Nucleus 细胞质Cytoplasm	细胞核Nucleus	细胞核Nucleus

家族成员 Family member	α-螺旋α-helix		β-折叠β-sheet		β-转角β-turn		无规则卷曲Random coil
家族成员 Family member	数目 Number	比例 Ratio (%)	数目 Number	比例 Ratio (%)	数目 Number	比例 Ratio (%)	数目 Number	比例 Ratio (%)
StHSP37.2	91	27.25	37	11.08	9	2.69	197	58.98
StHSP37.0	67	16.92	59	14.90	31	7.83	239	60.35
StHSP22.6	59	18.91	54	17.31	15	4.81	184	58.97

玉米大斑病菌小分子热激蛋白基因克隆与表达分析

Cloning and Expression Analysis of Genes of Small Heat Shock Protein in Setosphaeria turcica

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