Gene Clone and Bioinformatics Analysis of Subtilisin-Like Protease in Cucumis sativus

Abstract

Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants. Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay. Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion. Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain. Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.