玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析

doi:10.3864/j.issn.0578-1752.2022.16.006

中国农业科学 ›› 2022, Vol. 55 ›› Issue (16): 3134-3143.doi: 10.3864/j.issn.0578-1752.2022.16.006

玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析

郝玉彬^1,²(),李海笑^1,²,张赛¹,刘宁^1,²,刘英姿³,曹志艳^1,²(),董金皋^1,²()

¹河北农业大学植物保护学院,河北保定 071001
²华北作物改良与调控国家重点实验室/河北省植物生理与分子病理学重点实验室,河北保定 071001
³河北省承德市园林管理中心,河北承德 067000

收稿日期:2022-03-11 接受日期:2022-04-02 出版日期:2022-08-16 发布日期:2022-08-11
通讯作者: 曹志艳,董金皋
作者简介:郝玉彬,E-mail： 839970427@qq.com。
基金资助:
国家玉米产业技术体系(CARS-02);国家自然科学基金(32072370);国家自然科学基金(31901827);河北省自然科学基金(C2020204183)

Identification and Functional Analysis of StSCD Family in Setosphaeria turcica

HAO YuBin^1,²(),LI HaiXiao^1,²,ZHANG Sai¹,LIU Ning^1,²,LIU YingZi³,CAO ZhiYan^1,²(),DONG JinGao^1,²()

¹College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
²State Key Laboratory of North China Crop Improvement and Regulation/Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei
³Garden Management Centre of Chengde City, Hebei Province, Chengde 067000, Hebei

Received:2022-03-11 Accepted:2022-04-02 Online:2022-08-16 Published:2022-08-11
Contact: ZhiYan CAO,JinGao DONG

摘要/Abstract

摘要：

【目的】小柱孢酮脱水酶（scytalone dehydratase,SCD）是黑色素合成过程中的关键酶。本文旨在鉴定玉米大斑病菌（Setosphaeria turcica）小柱孢酮脱水酶基因（StSCD）家族,并分析玉米大斑病菌附着胞发育过程中StSCD基因家族表达量差异及SCD抑制剂对黑色素合成的影响,为进一步研究StSCD基因家族在黑色素合成和附着胞发育中的作用打下基础。【方法】利用玉米大斑病菌野生型菌株01-23的全基因组数据,获得StSCD基因家族的全序列,并与玉米小斑病菌（Cochlibolus heterostrophus）、稻瘟病菌（Pyricularia oryzae）、瓜类炭疽病菌（Colletotrichum lagenaria）等真菌的SCD进行序列比对;收集玉米大斑病菌附着胞不同发育时期的材料进行实时荧光定量PCR（qRT-PCR）分析,获得不同时期、不同StSCD的表达量,从而确定与病菌侵染和附着胞黑色素化密切相关的脱水酶基因;使用SCD抑制剂环丙酰菌胺处理玉米大斑病菌,测定菌落生长速度、黑色素合成量、附着胞膨压等,确定StSCD在附着胞发育过程中的作用。【结果】玉米大斑病菌全基因组中共鉴定到4个StSCD,其编码蛋白具有SCD保守结构域及保守的催化及底物结合氨基酸残基。StSCD3与灰葡萄孢（Botrytis cinerea）中功能冗余的SCD2蛋白具有较高同源性,StSCD4与多主棒孢（Corynespora cassiicola）中参与黑色素合成的SCD蛋白具有较高同源性。通过分析玉米大斑病菌生长发育过程中不同时期StSCD的表达量发现,在附着胞时期4个StSCD表达量均上调,其中StSCD3、StSCD4表达量上调尤为显著,附着胞再生菌丝时期StSCD3、StSCD4的表达量均显著下降,并且在附着胞诱导整个时期StSCD4的表达量较高。环丙酰菌胺处理玉米大斑病菌后,黑色素合成受阻,附着胞膨压显著降低。【结论】玉米大斑病菌含有4个StSCD,推测StSCD4参与DHN（1,8-间苯二酚）黑色素的合成,并进而影响附着胞膨压积累。

关键词: 玉米大斑病菌, StSCD家族, 黑色素, 环丙酰菌胺

Abstract:

【Objective】Scytalone dehydratase (SCD) is a key enzyme in melanin synthesis. The objective of this study is to identify the StSCD gene family of Setosphaeria turcica, and to analyze the differences in the expression of StSCD gene family during the appressorium development of S. turcica and the effect of SCD inhibitors on melanin synthesis, which will lay a foundation for further study on the important role of StSCD gene family in melanin synthesis and appressorium development. 【Method】Using the whole genome data of the wild-type strain 01-23 of S. turcica, the complete sequence of the StSCD gene family was obtained, and the protein sequences were compared with the SCD of Cochlibolus heterostrophus, Pyricularia oryzae, Colletotrichum lagenaria and other fungi. Materials from different appressorium developmental stages were collected for qRT-PCR analysis to obtain the expression levels of different StSCD genes at different stages, so as to determine the important dehydratase genes closely related to pathogen infection and appressorium melanization. Carpropamid, an inhibitor of SCD, was used to treat S. turcica, and the colony growth rate, melanin production and appressorium turgor pressure were measured, so as to determine the important function of SCD in appressorium development. 【Result】Four StSCD genes were found by searching the whole genome of S. turcica, the encoded protein of them has SCD conserved domain and conserved catalytic and substrate binding amino acid residues. StSCD3 protein has high homology with the functionally redundant SCD2 protein of Botrytis cinerea, and StSCD4 has high homology with SCD protein of Corynespora cassiicola. By analyzing the expression levels of StSCD genes at different stages during the growth and development of the pathogen, it was found that the expression levels of four StSCD genes were all up-regulated at the appressorium stage, and the expression levels of StSCD3 and StSCD4 were particularly up-regulated. The expression levels of StSCD3 and StSCD4 in the regenerated hyphae of the appressorium were significantly decreased, and the expression levels of StSCD4 were higher during the whole period of appressorium induction. The melanin synthesis was blocked and the appressorium turgor pressure was significantly reduced after carpropamid treatment. 【Conclusion】There are four StSCD genes in S. turcica. It is speculated that StSCD4 is involved in the synthesis of DHN melanin, and then affects the accumulation of appressorium turgor pressure.

Key words: Setosphaeria turcica, StSCD family, melanin, carpropamid

郝玉彬,李海笑,张赛,刘宁,刘英姿,曹志艳,董金皋. 玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析[J]. 中国农业科学, 2022, 55(16): 3134-3143.

HAO YuBin,LI HaiXiao,ZHANG Sai,LIU Ning,LIU YingZi,CAO ZhiYan,DONG JinGao. Identification and Functional Analysis of StSCD Family in Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2022, 55(16): 3134-3143.

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引物名称 Primer name	序列 Sequence (5′-3′)
StSCD1DL-F	GCTCTCAAAGACGCTTGCAC
StSCD1DL-R	TACCAAGCCATGACCGTGTC
StSCD2DL-F	CATCCAGCCCCAAGTTCCTT
StSCD2DL-R	CCAGCAAATTTCCACTCGCC
StSCD3DL-F	GGTGTTCAGTCTTTCACGCG
StSCD3DL-R	TCCATCGCTCTCCTCTCCAA
StSCD4DL-F	AGCTTCAACACGCACTGGTA
StSCD4DL-R	CTAGGCCTTTTCCGGGATCC
Tubulin-F	GGGAACTCCTCACGGATGTTG
Tubulin-R	TAACAACTGGGCAAAGGGTCA

基因名称 Gene name	基因编号 Gene number	基因长度 Gene length (bp)	ORF长度 ORF length (bp)	氨基酸数量 Number of amino acid (aa)	基因组位置 Genomic location
StSCD1	gene_12136	688	525	175	scaffold_29:99576-100263 (-)
StSCD2	gene_9587	639	486	162	scaffold_14:439746-440384 (-)
StSCD3	gene_4774	590	531	177	scaffold_6:14708-15297 (-)
StSCD4	gene_41	865	540	180	scaffold_1:164410-165274 (+)

	环丙酰菌胺浓度 <BOLD>C</BOLD>arpropamid concentration (μg·mL^-1)
	0	5.0	10.0
膨压 Turgor pressure (MPa)	5.60±0a	5.32±0.16b	5.06±0.16c

玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析

Identification and Functional Analysis of StSCD Family in Setosphaeria turcica

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