玉米大斑病菌SC35同源基因表达规律与互作分析

doi:10.3864/j.issn.0578-1752.2021.04.006

中国农业科学 ›› 2021, Vol. 54 ›› Issue (4): 733-743.doi: 10.3864/j.issn.0578-1752.2021.04.006

玉米大斑病菌SC35同源基因表达规律与互作分析

李天聪¹(),朱行¹(),魏宁¹,龙凤¹,武建颖¹,张燕¹,董金皋^2,³,申珅¹(),郝志敏^1,²()

¹河北农业大学生命科学学院/河北省植物生理与分子病理学重点实验室,河北保定 071001
²“华北作物改良与调控”国家重点实验室,河北保定 071001
³河北农业大学植物保护学院,河北保定 071001

收稿日期:2020-05-12 接受日期:2020-05-28 出版日期:2021-02-16 发布日期:2021-02-16
通讯作者: 申珅,郝志敏
作者简介:李天聪,E-mail： 905351819@qq.com。|朱行,E-mail： 1175593328@qq.com。
基金资助:
国家现代农业产业技术体系(CARS-02-25);河北省自然科学基金(C2018204120);河北省高等学校青年拔尖人才科学技术研究项目(BJ2014349Y)

The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica

LI TianCong¹(),ZHU Hang¹(),WEI Ning¹,LONG Feng¹,WU JianYing¹,ZHANG Yan¹,DONG JinGao^2,³,SHEN Shen¹(),HAO ZhiMin^1,²()

¹College of Life Sciences/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Hebei Agricultural University, Baoding 071001, Hebei
²State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei
³College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei

Received:2020-05-12 Accepted:2020-05-28 Online:2021-02-16 Published:2021-02-16
Contact: Shen SHEN,ZhiMin HAO

摘要/Abstract

摘要：

【目的】获得玉米大斑病菌（Setosphaeria turcica）SC35类剪接因子的基因,并分析该基因家族之间的相互作用及在病菌不同生长发育时期与侵染过程中的表达规律,为明确剪接因子SC35家族与真菌生长发育的关系打下基础。【方法】以拟南芥（Arabidopsis thaliana）SC35编码的氨基酸序列为探针序列,在玉米大斑病菌全基因组数据库进行同源比对,获得玉米大斑病菌中潜在的SC35蛋白;利用生物信息学方法对其保守结构域、系统进化关系进行分析;分别收集接种在感病玉米叶片表面不同时间及玉米大斑病菌菌丝、分生孢子、芽管、附着胞及侵入丝等不同发育时期的材料,利用实时荧光定量PCR（qRT-PCR）技术分析在对玉米叶片侵染不同时间及不同发育时期可变剪接因子的转录水平;利用酵母双杂交技术明确玉米大斑病菌可变剪接因子之间的相互作用。【结果】在玉米大斑病菌中获得了8个潜在的SC35类蛋白基因,其编码的氨基酸具有典型的SR蛋白（Ser-Arg rich protein）结构域,而StSC1 C端具有2个RRM基序。8个基因位于染色体组的不同物理位置,不具有连锁关系。系统进化分析表明8个剪接因子分布于不同进化支,同源性较低。在病菌侵染寄主叶片过程中,StSC1、StSC2、StSC3、StSC4、StSC5、StSC6、StSC8在侵染18 h时表现为表达上调趋势,StSC7呈下调趋势,StSC4在6—18 h表现出较高表达活性;不同生长时期中,StSC1在病菌附着胞及侵染丝形成时期表达水平极显著上调（P＜0.001）,是分生孢子时期的24.44、8.25倍,其余7个可变剪接因子在病菌的生长过程中表达水平均呈下调趋势。酵母双杂交结果表明StSC4与StSC6、StSC3与StSC8、StSC3与StSC4、StSC8与StSC4有体外互作关系。【结论】在玉米大斑病菌侵染过程中及不同发育时期可变剪接因子的表达模式不同,StSC4在病菌整个侵染过程均活跃表达;StSC1、StSC4和StSC6对病菌附着胞和侵入丝的形成发挥比较重要的调控作用;StSC4与StSC6、StSC3与StSC8、StSC3与StSC4、StSC8与StSC4通过互作,调控剪接复合体的形成。

关键词: 玉米大斑病菌, 可变剪接因子, 实时荧光定量PCR, 酵母双杂交

Abstract:

【Objective】The objective of this study is to obtain the homologous genes of the splicing factor SC35 in Setosphaeria turcica, and to analyze the interaction among them and the expression profiles during the different growth and development stages and infection process of the pathogen. It would lay the foundation for illustrating the relationship between the SC35 family members and fungal pathogenicity.【Method】Based on amino acid sequences of SC35 protein in Arabidopsis thaliana as probe sequences, online Blastp alignment was carried out in the S. turcica genome database to obtain candidate SC35 homologues. Then they were analyzed for conserved domain and phylogenetic relationship through bioinformatics procedures. The materials of S. turcica were collected at different infection stages on maize leaves and multiple developmental stages, such as hyphae, conidia, germ tubes, appressorium and penetration hyphae, to analyze the transcription levels of SC35 homologues through real-time quantitative PCR (qRT-PCR). And their interactions were verified in vitro by the yeast two-hybrid test.【Result】Eight SC35 genes of S. turcica were obtained, named as StSC1, StSC2, StSC3, StSC4, StSC5, StSC6, StSC7, and StSC8, respectively. All of them owned typical SR protein domains, besides StSC1 showed two RRM domains. They located at different physical locations of the genome and had no linkage. Phylogenetic analysis showed that the eight alternative splicing factors were distributed in different clades, with low homology. In the process of infection on maize leaves, gene StSC1, StSC2, StSC3, StSC4, StSC5, StSC6 and StSC8 were up-regulated, while StSC7 was down-regulated at 18 h after inoculation. StSC4 showed high expression activity at 6-18 h. In different development periods, the expression levels of StSC1 were extremely significant up-regulated (P＜0.001) in the appressorium and the formation of penetration hyphae, which were 24.44 and 8.25 times higher than those in the conidial period, but the others were down-regulated during the development. The yeast two-hybrid proved that StSC4 interacted with StSC6, StSC3 with StSC8, StSC3 with StSC4, and StSC8 with StSC4. 【Conclusion】The expression patterns of SC35 are different in the infection process and multiple developmental stages of S. turcica. StSC4 is actively expressed throughout the infection process. StSC1, StSC4 and StSC6 play important regulatory roles during the formation of appressorium and penetration hyphae. StSC4 and StSC6, StSC3 and StSC8, StSC3 and StSC4, StSC8 and StSC4 interact to regulate the formation of splicing complexes.

Key words: Setosphaeria turcica, alternative splicing factor, qRT-RCR, yeast two-hybrid

李天聪,朱行,魏宁,龙凤,武建颖,张燕,董金皋,申珅,郝志敏. 玉米大斑病菌SC35同源基因表达规律与互作分析[J]. 中国农业科学, 2021, 54(4): 733-743.

LI TianCong,ZHU Hang,WEI Ning,LONG Feng,WU JianYing,ZHANG Yan,DONG JinGao,SHEN Shen,HAO ZhiMin. The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2021, 54(4): 733-743.

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引物Primer	序列Sequence (5′-3′)
Tubulin-F/R	GGGAACTCCTCACGGATGTTG/TAACAACTGGGCAAAGGGTCA
StSC1-F/R	AGAGCCAGAAACACTGAGCC/AAGCGAGAAAAGACACGCCT
StSC2-F/R	GAAAATCAAGACGACCCGCC/CTTTGCCCATTGCTTTCGCT
StSC3-F/R	AGCGCAAGGAGAAAGAGAGG/CCGTCTTCCTTGATACGCCT
StSC4-F/R	CACTGGAGATGGTTGGCGAT/TTGCACACAAACAGCACGTT
StSC5-F/R	GAACCTCTTCGTCACAGGCA/GCATGATGTTGCACTGCTCC
StSC6-F/R	GCTTTGGCTTTGTCCGCTAC/ACGCGGATTCTACGTCCATC
StSC7-F/R	GCGACTTTGGCACCGTTAAG/TCGAGCGTGGCATACTCAAT
StSC8-F/R	GGCCTTGACCAAGAACGTCA/GCGGACATCCTTGATGACAC

引物Primer	序列Sequence (5′-3′)
StSC4-F/R	GGCCATGGAGGCCAGTGAATTCATGTCTGTGCTTCTAGAAACATCAC/TTCATCTGCAGCTCGAGCTCGATGGATCCTCATCGTCGACGGTCCCGGT
StSC6-F/R	CATATGGCCATGGAGGCCAGTGAATTCATGTCGAAACTCTTCATTG/CATCTGCAGCTCGAGCTCGATGGATCCCTACCATTGCTGCTG
StSC8-F/R	TATGGCCATGGAGGCCGAATTCATGCCGTCCCGTTCGC/CTAGTTATGCGGCCGCTGCAGGTCGACGGATCCTCATCTGCGCTCCCTACTTG
StSC3-F/R	ATATGGCCATGGAGGCCGAATTCATGAACTCGATCCGCGCCATCCAGGAG/GTTATGCGGCCGCTGCAGGTCGACGGATCCTTACACGCGAGTGTCTCTCT
StSC7-F/R	CATATGGCCATGGAGGCCGAATTCATGGAGGACGACGCCAAGATG/TTATGCGGCCGCTGCAGGTCGACGGATCCCTACTCGTCGTCCATATCGTC

名称 Name	JGI标号 ID in JGI	NCBI登录号 Accession number in NCBI	大小 Size (aa)	基因长度 Gene length (bp)	基因定位 Gene mapping
StSC1	46444	XP_008023357.1	391	1505	scaffold_14:633653-635474 (+)
StSC2	21139	XP_008027986.1	282	1031	scaffold_4:463120-464150 (-)
StSC3	171745	XP_008027251.1	304	915	scaffold_3:679211-680616 (-)
StSC4	113046	XP_008020610.1	477	1555	scaffold_1:618088-619811 (+)
StSC5	99605	XP_008030866.1	111	336	scaffold_8:693429-693764 (+)
StSC6	168126	XP_008023544.1	161	835	scaffold_14:724868-726377 (-)
StSC7	88128	XP_008025196.1	159	537	scaffold_2:109463-109999 (-)
StSC8	156630	XP_008029952.1	343	1157	scaffold_6:1923760-1924916 (+)

玉米大斑病菌SC35同源基因表达规律与互作分析

The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica

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