玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析

doi:10.3864/j.issn.0578-1752.2024.05.006

中国农业科学 ›› 2024, Vol. 57 ›› Issue (5): 900-908.doi: 10.3864/j.issn.0578-1752.2024.05.006

玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析

张博文¹^,⁴(), 赵丽雯², 徐璐²^,⁴, 李盼¹^,⁴, 曾凡力²^,³^,⁴, 孟亚南²^,³^,⁴(), 董金皋¹^,³^,⁴()

¹ 河北农业大学植物保护学院，河北保定 071001
² 河北农业大学生命科学学院，河北保定 071001
³ 华北作物改良与调控国家重点实验室，河北保定 071001
⁴ 河北省植物生理与分子病理学重点实验室，河北保定 071001

收稿日期:2023-10-12 接受日期:2023-11-05 出版日期:2024-03-01 发布日期:2024-03-06
通信作者:
董金皋，E-mail：shmdjg@hebau.edu.cn。
孟亚南，E-mail：mengyananMN@163.com。
联系方式: 张博文，E-mail：2693312895@qq.com。
基金资助:
河北省自然科学基金(C2022204138); 河北省教育厅青年拔尖人才计划(BJ2020003); 华北作物改良与调控国家重点实验室自主课题(NCCIR2021ZZ-5)

Identification and Gene Function Analysis of StCks1, a Cyclin- Dependent Kinase Subunit of Setosphaeria turcica

ZHANG BoWen¹^,⁴(), ZHAO LiWen², XU Lu²^,⁴, LI Pan¹^,⁴, ZENG FanLi²^,³^,⁴, MENG YaNan²^,³^,⁴(), DONG JinGao¹^,³^,⁴()

¹ College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
² College of Life Science, Hebei Agricultural University, Baoding 071001, Hebei
³ State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei
⁴ Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Baoding 071001, Hebei

Received:2023-10-12 Accepted:2023-11-05 Published:2024-03-01 Online:2024-03-06

摘要/Abstract

摘要：

【目的】Cks1（cyclin-dependent kinase subunit 1）是细胞周期蛋白依赖性激酶复合体CDK（cyclin-dependent kinase）的结构亚基，是细胞周期调控过程中的关键基因。论文旨在鉴定玉米大斑病菌（Setosphaeria turcica）StCks1，分析玉米大斑病菌附着胞发育过程中StCks1表达量差异及其互作蛋白，为进一步研究StCks1在附着胞发育中的作用打下基础。【方法】通过对玉米大斑病菌野生型菌株01-23全基因组数据分析，获取StCks1蛋白序列；利用软件MEGA 5.0和在线数据库Pfam、SMART对酿酒酵母、裂殖酵母和拟南芥等物种Cks1蛋白进行系统发育树构建和保守结构域分析；收集玉米大斑病菌附着胞发育不同时期样品进行转录组测序以及表达量分析。利用原核诱导表达系统，构建原核表达载体pGEX-6p-3-StCks1，并转化大肠杆菌表达菌株BL21，对重组蛋白GST-StCks1诱导表达纯化；通过GST pull-down、Western blot技术和酵母双杂交试验，鉴定StCks1的互作蛋白。【结果】玉米大斑病菌全基因组中鉴定到唯一StCks1蛋白编码基因，该蛋白由130个氨基酸组成；其三级结构主要由4股β折叠和3个α螺旋构成，含有HxPEPH（His-any- Pro-Glu-Pro-His）保守位点和Cks保守结构域；通过转录组测序和表达量分析发现，StCks1在附着胞发育不同时期表达量显著上调；重组蛋白GST-StCks1在1 mmol·L^-1 IPTG，30 ℃诱导2 h可获得大量可溶性蛋白；通过GST pull-down技术获取大量互作蛋白并进行质谱鉴定，通过Western blot和酵母双杂交试验，验证StCks1与细胞周期蛋白依赖性激酶CDK1存在互作。【结论】玉米大斑病菌中含有唯一的StCks1，通过与细胞周期蛋白依赖性激酶CDK1互作调控附着胞的发育。

关键词: 玉米大斑病菌, 细胞周期调控, StCks1, 原核诱导表达纯化, GST pull-down

Abstract:

【Objective】Cks1 is a structural subunit of the cyclin-dependent kinase complex CDK, and is a key gene in the process of cell cycle regulation. The aim of this article is to identify the StCks1 of Setosphaeria turcica, analyze the differences in StCks1 expression and its interacting proteins during the development of appressorium of S. turcica, and to lay the foundation for further research on the role of StCks1 in the development of appressorium.【Method】By analyzing the entire genome data of the wild-type strain 01-23 of S. turcica, the StCks1 protein sequence was obtained. Using software MEGA 5.0 and online databases Pfam and SMART, phylogenetic tree construction and conserved domain analysis of Cks1 proteins from different species such as Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, and Arabidopsis thaliana were conducted. Samples were collected from different stages of appressorium of S. turcica for transcriptome sequencing and expression analysis. Using a prokaryotic induction expression system, a prokaryotic expression vector pGEX-6p-3-StCks1 was constructed, and transformed the E. coli expression strain BL21 to induce expression and purification of the recombinant protein GST-StCks1. The interaction protein of StCks1 was identified through GST pull-down, Western blot, and yeast two-hybrid assay.【Result】The only StCks1 protein coding gene was identified in the entire genome of S. turcica, which is composed of 130 amino acids. Its tertiary structure mainly consists of four strands of β-fold and three α helices, containing HxPEPH (His-any-Pro-Glu-Pro-His) conserved sites and Cks conserved domains. Through transcriptome sequencing and expression analysis, it was found that the expression level of StCks1 was significantly up-regulated at different stages of appressorium development. A large amount of soluble protein could be obtained by inducing recombinant protein GST-StCks1 at 1 mmol·L^-1 IPTG and 30 ℃ for 2 h. A large number of interacting proteins were obtained using GST pull-down technology and identified by mass spectrometry. Western blot and yeast two-hybrid experiments were conducted to verify the interaction between StCks1 and the cyclin-dependent kinase CDK1.【Conclusion】There is a unique StCks1 in S. turcica, which regulates the development of appressorium through interaction with the cyclin-dependent kinase CDK1.

Key words: Setosphaeria turcica, cell cycle regulation, StCks1, prokaryotic induced expression and purification, GST pull-down

张博文, 赵丽雯, 徐璐, 李盼, 曾凡力, 孟亚南, 董金皋. 玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析[J]. 中国农业科学, 2024, 57(5): 900-908.

ZHANG BoWen, ZHAO LiWen, XU Lu, LI Pan, ZENG FanLi, MENG YaNan, DONG JinGao. Identification and Gene Function Analysis of StCks1, a Cyclin- Dependent Kinase Subunit of Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2024, 57(5): 900-908.

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引物<BOLD>P</BOLD>rimer	序列Sequence (5′-3′)	酶切位点Restriction site
oMYN1: GST-StCks1-F	GCGGATCCATGTTTCCACTGCATCTTTT	BamH I
oMYN2: GST-StCks1-R	CACTCGAGTCAGTGCATAGGAGGCTGAT	Xho I

玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析

Identification and Gene Function Analysis of StCks1, a Cyclin- Dependent Kinase Subunit of Setosphaeria turcica

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