Identification and Gene Function Analysis of StCks1,a Cyclin-Dependent Kinase Subunit of Setosphaeria turcica
[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 SS.turcica,which regulates the development of appressorium through interaction with the cyclin-dependent kinase CDK1.
Setosphaeria turcicacell cycle regulationStCks1prokaryotic induced expression and purificationGST pull-down
万方数据
维普
