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

Abstract

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

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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Table 4

Genes and functional annotations significantly correlated with StHSP37.2, StHSP37.0, and StHSP22.6"

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

Table 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

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

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