玉米大斑病菌Septin基因家族的鉴定与表达模式分析

doi:10.3864/j.issn.0578-1752.2020.24.005

Abstract

Abstract:

【Background】Septin, which is widely found in all eukaryotes except plants, is a highly conserved GTP binding protein family and is considered to be the fourth cytoskeletal protein after microtubules, microfilaments and intermediate fibers. Septin proteins of pathogenic fungi are involved in cell polarity determination, morphological shaping and morphological transformation associated with pathogenicity.【Objective】The objective of this study is to identify Septin gene family of Setosphaeria turcica, analyze its expression pattern at different developmental stages, and to lay a foundation for clarifying the relationship between the membrane protein Septin and fungal infection structure development.【Method】The amino acid sequences of six Septin proteins in Bipolaris maydis were used as probe sequences, online Blastp alignment and keyword search were carried out in the S. turcica database to obtain S. turcica candidate Septins. Then bioinformatics analyses including gene structure, physical and chemical properties and transmembrane region structure of the Septins were conducted. The materials of S. turcica that were induced by artificial hydrophobic media at different stages of infective structures development and infected host leaves at different times were collected, and RT-qPCR (real-time fluorescence quantitative PCR) technology was used to analyze the transcription levels of Septins at different stages of infective structures development.【Result】Six candidate Septins were obtained, and among them, four typical Septins contained G1, G3, and G4 motifs, were named by StSep1, StSep2, StSep3 and StSep4, respectively. The expression level of Septins increased slowly under the induction on artificial hydrophobic media. StSep1 was active in the late stage of germ tube formation, and its expression level was 25.69 times of that in the conidia period (P<0.01). The expression level of StSep4 reached a peak in appressorium anaphase, and then the expression level was down-regulated. The change trend of Septin expression in the process of pathogen infection was basically consistent with that under the induction of artificial hydrophobic material. StSep1 expression was significantly up-regulated at 6 h after inoculation (P<0.01). As time went on, StSep4 actively expressed during the period of appressorial formation, and the expression reached a peak at 18 h after inoculation. After that, the expression was down-regulated, but it was still higher than that in the early stage of germination. StSep2 and StSep3 were active at 18 h and 24 h after inoculation, exceeding the initial stage of germination.【Conclusion】There are 4 core Septins in the genome of S. turcica. StSep1 and StSep4 are actively expressed during the formation of germ tube and appressoria, respectively. The results will provide a theoretical basis for further clarifying the function of Septin and the infection regulation mechanism of S. turcica.

Key words: Setosphaeria turcica, Septin gene family, RT-qRCR, gene expression

LONG Feng,WANG Qing,ZHU Hang,WANG JianXia,SHEN Shen,LIU Ning,HAO ZhiMin,DONG JinGao. Identification and Expression Pattern Analysis of Septin Gene Family of Setosphaeria turcica[J].Scientia Agricultura Sinica, 2020, 53(24): 5017-5026.

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References 34

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名称Primer	序列Sequence (5′ to 3′)
β-Tubulin-F	GGGAACTCCTCACGGATGTTG
β-Tubulin-R	TAACAAVTGGGCAAAGGGTCA
StSep1F	ACTGCTGCCTGTTCTTCA
StSep1R	TGGAACTCCTCCTTTATCC
StSep2F	GATTCGGAGACCAGATTGA
StSep2R	AGTAGGCGTGATGAAGTAGAG
StSep3F	TGGCTCGGAGAAGGATGT
StSep3R	GGGTTCGGATGAGGATGG
StSep4F	GCTCATCCGCACCCACATG
StSep4R	CATCTTCTGGAGCTTGGC

反应组分 Reaction component	反应体系 Reaction system	终浓度 Final concentration
2×Fast Super EvaGreen Master Mix	10 μL	1×
F, R primers	1 μL	0.1-0.5 μmol·L^-1 each
cDNA (500 ng·μL^-1) template	1 μL
H₂O	补足至20 μL Up to 20 μL

基因 Gene	基因登录号 Database accession number	基因长度 Gene length (bp)	蛋白质ID Protein ID	大小 Size (aa)	基因组定位 Genomic mapping
StSep1	XP_008021140.1	1588	162710	341	scaffold_1:3755824-3757411 (+)
StSep2	XP_008027140.1	1883	163440	375	scaffold_3:81842-83724 (+)
StSep3	XP_008028687.1	1406	93639	381	scaffold_4:2483454-2484859 (+)
StSep4	XP_008026163.1	1424	110191	432	scaffold_21:190124-191547 (+)
StSep5	XP_008030793.1	1977	165795	475	scaffold_8:185341-187317 (-)
StSep6	XP_008030826.1	2554	99177	793	scaffold_8:379528-382081 (-)

特征蛋白 Characteristic protein	StSep1	StSep2	StSep3	StSep4
相对分子质量 Relative molecular mass (kD)	39.1454	42.6070	44.0992	49.7026
PI	7.20	5.07	8.54	6.56
(Asp+Glu)	44	72	60	68
(Arg+Lys)	44	53	63	66
分子式Molecular formula	C₁₇₃₃H₂₇₃₅N₄₉₅O₅₂₀S₁₀	C₁₈₅₁H₂₉₈₅N₅₃₅O₅₉₅S₁₁	C₁₉₃₈H₃₁₀₇N₅₇₃O₅₈₀S₁₂	C₂₁₇₉H₃₅₁₄N₆₂₄O₆₇₁S₁₆
不稳定性系数 Coefficient of instability	49.91	47.37	48.84	42.73
脂肪族氨基酸指数 Aliphatic amino acid index	84.31	86.53	78.87	81.62
总平均亲水性 Total average hydrophilicity	-0.498	-0.608	-0.175	-0.681

Identification and Expression Pattern Analysis of Septin Gene Family of Setosphaeria turcica

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