Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (5): 900-908.doi: 10.3864/j.issn.0578-1752.2024.05.006

• PLANT PROTECTION • Previous Articles     Next Articles

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. 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 Online:2024-03-01 Published:2024-03-06
  • Contact: MENG YaNan, DONG JinGao

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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

Table 1

Primers used in this study"

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

Fig. 1

StCks1 structure analysis"

Fig. 2

Phylogenetic tree construction and conserved domain analysis of StCks1 protein"

Fig. 3

Amino acid sequence alignment and protein structure model of StCks1"

Fig. 4

Development morphology and expression level of StCks1 at different appressorium stages"

Fig. 5

StCks1 digestion validation and plasmid map"

Fig. 6

Induced expression of StCks1 protein"

Fig. 7

Purification of StCks1 protein"

Fig. 8

The interaction analysis between StCks1 and CDK1"

Fig. 9

Yeast two-hybrid verification of the interaction between CDK1 and StCks1"

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