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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.05.006

https://www.chinaagrisci.com/CN/Y2024/V57/I5/900

图/表 10

表1

图1

图2

图3

图4

图5

图6

图7

图8

图9

参考文献 24

[1]	VAN INGHELANDT D, MELCHINGER A E, MARTINANT J P, STICH B. Genome-wide association mapping of flowering time and northern corn leaf blight (Setosphaeria turcica) resistance in a vast commercial maize germplasm set. BMC Plant Biology, 2012, 12: 56. doi: 10.1186/1471-2229-12-56 pmid: 22545925
[2]	HUMAN M P, BERGER D K, CRAMPTON B G. Time-course RNAseq reveals Exserohilum turcicum effectors and pathogenicity determinants. Frontiers in Microbiology, 2020, 11: 360. doi: 10.3389/fmicb.2020.00360
[3]	MENG Y N, ZENG F L, HU J J, LI P, XIAO S L, ZHOU L H, GONG J G, LIU Y W, HAO Z M, CAO Z Y, DONG J G. Novel factors contributing to fungal pathogenicity at early stages of Setosphaeria turcica infection. Molecular Plant Pathology, 2022, 23(1): 32-44. doi: 10.1111/mpp.13140
[4]	GONG X D, FENG S Z, ZHAO J, TANG C, TIAN L, FAN Y S, CAO Z Y, HAO Z M, JIA H, ZANG J P, ZHANG Y F, HAN J M, GU S Q, DONG J G. StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in northern corn leaf blight pathogen Setosphaeria turcica. Microbiological Research, 2017, 201: 30-38. doi: 10.1016/j.micres.2017.04.009
[5]	EBBOLE D J. Magnaporthe as a model for understanding host-pathogen interactions. Annual Review of Phytopathology, 2007, 45: 437-456. doi: 10.1146/phyto.2007.45.issue-1
[6]	DOHLMAN H G. G proteins and pheromone signaling. Annual Review of Physiology, 2002, 64: 129-152. pmid: 11826266
[7]	ZHANG S R, HAO Z M, WANG L H, SHEN S, CAO Z Y, XIN Y Y, HOU M L, GU S Q, HAN J M, DONG J G. StRas2 regulates morphogenesis, conidiation and appressorium development in Setosphaeria turcica. Microbiological Research, 2012, 167(8): 478-486. doi: 10.1016/j.micres.2012.02.009
[8]	SHEN S, HAO Z M, GU S Q, WANG J J, CAO Z Y, LI Z Y, WANG Q, LI P, HAO J, DONG J G. The catalytic subunit of cAMP-dependent protein kinase A StPKA-c contributes to conidiation and early invasion in the phytopathogenic fungus Setosphaeria turcica. FEMS Microbiology Letters, 2013, 343: 135-144. doi: 10.1111/femsle.2013.343.issue-2
[9]	MA S X, CAO K K, LIU N, MENG C, CAO Z Y, DAI D Q, JIA H, ZANG J P, LI Z Y, HAO Z M, GU S Q, DONG J G. The StLAC 2 gene is required for cell wall integrity, DHN-melanin synthesis and the pathogenicity of Setosphaeria turcica. Fungal Biology, 2017, 121(6/7): 589-601. doi: 10.1016/j.funbio.2017.04.003
[10]	ZHANG M M, ZHANG S Q. Mitogen-activated protein kinase cascades in plant signaling. Journal of Integrative Plant Biology, 2022, 64(2): 301-341. doi: 10.1111/jipb.13215
[11]	ZENG F L, MENG Y N, HAO Z M, LI P, ZHAI W B, SHEN S, CAO Z Y, DONG J G. Setosphaeria turcica ATR turns off appressorium- mediated maize infection and triggers melanin-involved self- protection in response to genotoxic stress. Molecular Plant Pathology, 2020, 21(3): 401-414. doi: 10.1111/mpp.v21.3
[12]	MENG Y N, ZHANG X J, ZENG F L, ZHAI W B, LI P, HU J J, XIAO S L, HAO Z M, CAO Z Y, CHEN C, DONG J G. Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica. Journal of Integrative Agriculture, 2023, 22(4): 1068-1081. doi: 10.1016/j.jia.2022.07.002
[13]	OSÉS-RUIZ M, SAKULKOO W, LITTLEJOHN G R, MARTIN- URDIROZ M, TALBOT N J. Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(2): E237-E244.
[14]	SAUNDERS D G, AVES S J, TALBOT N J. Cell cycle-mediated regulation of plant infection by the rice blast fungus. The Plant Cell, 2010, 22(2): 497-507. doi: 10.1105/tpc.109.072447 pmid: 20190078
[15]	KCIUK M, GIELECIŃSKA A, MUJWAR S, MOJZYCH M, KONTEK R. Cyclin-dependent kinase synthetic lethality partners in DNA damage response. International Journal of Molecular Sciences, 2022, 23(7): 3555. doi: 10.3390/ijms23073555
[16]	KAISER P, MONCOLLIN V, CLARKE D J, WATSON M H, BERTOLAET B L, REED S I, BAILLY E. Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets. Genes and Development, 1999, 13(9): 1190-1202. doi: 10.1101/gad.13.9.1190
[17]	刘雪琴, 任凭, 张择, 曾凡力. 酿酒酵母Cdk1抑制蛋白的研究进展. 生物化学与生物物理进展, 2018, 45(5): 512-518.
	LIU X Q, REN P, ZHANG Z, ZENG F L. The progress of cell cycle Cdk1 inhibitors in budding yeast. Progress in Biochemistry and Biophysics, 2018, 45(5): 512-518. (in Chinese)
[18]	COOLS T, DE VEYLDER L. DNA stress checkpoint control and plant development. Current Opinion in Plant Biology, 2009, 12(1): 23-28. doi: 10.1016/j.pbi.2008.09.012 pmid: 19010080
[19]	CHEN N, GONG J, CHEN X, XU M, HUANG Y, WANG L, GENG N, ZHOU Q. Cytokeratin expression in malignant melanoma: Potential application of in situ hybridization analysis of mRNA. Melanoma Research, 2009, 19(2): 87-93. doi: 10.1097/CMR.0b013e3283252feb
[20]	INUI N, KITAGAWA K, MIWA S, HATTORI T, CHIDA K, NAKAMURA H, KITAGAWA M. High expression of Cks1 in human non-small cell lung carcinomas. Biochemical and Biophysical Research Communications, 2003, 303(3): 978-984. pmid: 12670508
[21]	SHAPIRA M, BEN-IZHAK O, LINN S, FUTERMAN B, MINKOV I, HERSHKO D D. The prognostic impact of the ubiquitin ligase subunits Skp2 and Cks1 in colorectal carcinoma. Cancer, 2005, 103(7): 1336-1346. pmid: 15717322
[22]	HINDLEY J, PHEAR G, STEIN M, BEACH D. Suc1⁺ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe. Molecular and Cellular Biology, 1987, 7(1): 504-511.
[23]	YUE X, QUE Y, DENG S, XU L, OSES-RUIZ M, TALBOT N J, PENG Y, WANG Z. The cyclin dependent kinase subunit Cks1 is required for infection-associated development of the rice blast fungus Magnaporthe oryzae. Environmental Microbiology, 2017, 19: 3959-3981. doi: 10.1111/emi.2017.19.issue-10
[24]	HAYLES J, AVES S, NURSE P. Suc1 is an essential gene involved in both the cell cycle and growth in fission yeast. The EMBO Journal, 1986, 5(12): 3373-3379. doi: 10.1002/embj.1986.5.issue-12

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

RichHTML

PDF

赞

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 24

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	邹金鹏, 岳浩峰, 李海笑, 刘峥, 刘宁, 曹志艳, 董金皋. 基于非靶向代谢组学分析StLAC2和StLAC6差异影响玉米大斑病菌的机制[J]. 中国农业科学, 2023, 56(16): 3110-3223.
[2]	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.
[3]	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.
[4]	代玉立,甘林,滕振勇,杨静民,祁月月,石妞妞,陈福如,杨秀娟. 玉米大斑病菌和小斑病菌交配型多重PCR 检测方法的建立与应用[J]. 中国农业科学, 2020, 53(3): 527-538.
[5]	龙凤,王擎,朱行,王建霞,申珅,刘宁,郝志敏,董金皋. 玉米大斑病菌Septin基因家族的鉴定与表达模式分析[J]. 中国农业科学, 2020, 53(24): 5017-5026.
[6]	窦万福,祁静静,胡安华,陈善春,彭爱红,许兰珍,雷天刚,姚利晓,何永睿,李强. GST pull-down联合LC-MS/MS筛选柑橘抗溃疡病转录因子CsBZIP40的互作蛋白[J]. 中国农业科学, 2019, 52(13): 2243-2255.
[7]	冯胜泽，刘星晨，王海祥，赵洁，赵立卿，郑亚男，巩校东，韩建民，谷守芹，董金皋. 玉米大斑病菌分生孢子形成的影响因素及GATA 转录因子家族的表达分析[J]. 中国农业科学, 2017, 50(7): 1234-1241.
[8]	赵洁，赵立卿，巩校东，冯胜泽，刘星晨，郑亚男，李志勇，孙海月，王冬雪，韩建民，谷守芹，董金皋. 玉米大斑病菌Homeobox转录因子家族全基因组鉴定及其表达规律分析[J]. 中国农业科学, 2017, 50(4): 669-678.
[9]	申珅, 李贞杨, 赵玉兰, 李盼, 韩建民, 郝志敏, 董金皋. 玉米大斑病菌环腺苷酸磷酸二酯酶基因克隆及表达分析[J]. 中国农业科学, 2017, 50(16): 3135-3144.
[10]	张运峰，张淑红，武秋颖，范永山. STK1对玉米大斑病菌附着胞发育过程中糖原和脂肪积累的影响[J]. 中国农业科学, 2017, 50(15): 2928-2935.
[11]	巩校东，刘星晨，赵立卿，郑亚男，范永山，韩建民，谷守芹，董金皋. 高渗胁迫对玉米大斑病菌生长发育的影响及菌丝细胞中渗透调节物质的分析[J]. 中国农业科学, 2017, 50(10): 1922-1929.
[12]	马双新，刘宁，贾慧，戴冬青，许苗苗，曹志艳，董金皋. 玉米大斑病菌漆酶基因Stlac2结构分析及原核表达[J]. 中国农业科学, 2016, 49(21): 4130-4139.
[13]	贾慧，孟庆江，李志勇，巩校东，藏金萍，郝志敏，曹志艳，董金皋. 玉米大斑病菌黑色素合成酶基因的全基因组定位及表达模式分析[J]. 中国农业科学, 2015, 48(14): 2767-2776.
[14]	巩校东，王玥，张盼, 范永山, 谷守芹，韩建民，董金皋. 玉米大斑病菌MAPK基因StIME2的基因组定位蛋白质结构预测及表达分析[J]. 中国农业科学, 2015, 48(13): 2549-2558.
[15]	巩校东1, 张晓玉1, 田兰1, 王星懿1, 李坡2, 张盼1, 王玥1, 范永山3, 韩建民1, 谷守芹1, 董金皋1. 玉米大斑病菌MAPK超家族的全基因组鉴定及途径模型建立[J]. 中国农业科学, 2014, 47(9): 1715-1724.