Abstract:

To further clarify pathogenic factor of potato late blight, we cloned the cysteine protease family gene in the secreted protein of Phytophthora infestans causing potato late blight and express in E. coli BL21. In this study, TA cloning method was used to extract total RNA of P. infestans XH05-5-4 of Yunnan, synthesize cDNA by reverse transcription and connect gene fragment to the vector after PCR amplification. Then we induced the expression of it in E. coli BL21. The full length of cysteine protease family gene is 1176 bp, containing 1077 bp open reading frame (ORF) encoding 387 amino acids. Predicted by bioinformatics analysis, its protein molecular weight is 41.671 KDa, isoelectric point is 4.88. After IPTG induced expression and SDS-PAGE identification, the protein expressed in the constructed prokaryotic expression vector is consistent with the expected protein. The results of biological activity examination show that potato leaves have necrosis and this symptom is similar to potato late blight. A cysteine protease family gene (GenBank Accession: KP938956, name: C1A-PH-SCH1) was cloned from P. infestans XH05-5-4 by TA cloning method. It is a papain enzyme subfamily gene by homology comparison, and the prokaryotic expression vector is successfully constructed and expressed in E. coli BL21. The symptom is similar to watery necrosis of potato late blight, therefore, it is speculated that C1A-PH-SCH1 is involved in the pathogenic process of potato late blight.

Key words: potato, Phytophthora infestans, papain-like cysteine proteases, C1A-PH-SCH1 gene, clone