Cloning and overexpression of Zea mays cystatin 2 (ccii) gene in Bacillus subtilis to reduce root-knot nematode infection in cucumber plants in Iraq

Root-knot nematodes significantly reduce cucumber yields, prompting the development of a genetic engineering strategy as an alternative to chemical nematicides. In this study, the maize CCII gene was cloned into the pHT1469 shuttle vector and transformed into the Bacillus subtilis expression system to produce a cystatin that interferes with nematode digestive proteins. The full-length CCII cDNA sequence was optimized for overexpression in B. subtilis, driven by the Pgrac promoter, resulting in a protein of 134 amino acids with a deduced molecular mass of 15.01 kDa. After optimization, the relative adaptiveness in the heterologous B. subtilis system improved by 6.72% compared to non-optimized codons. Purification results using immobilized metal affinity chromatography (IMAC) indicated that the third fraction exhibited the highest inhibition activity, and the purified cystatin was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 15 kDa. Treatments with nematode juveniles showed the highest mortality percentages at 30% and 40% concentrations of purified cystatin isolated from both competent and wild B. subtilis cells, yielding mortality rates of 65%, 73.7%, 75.3%, and 89% respectively after 72 h. Furthermore, the recombinant cystatin isolated from the first B. subtilis (wild) strain had a greater impact on juvenile mortality than the second strain. This study aimed to clone and express corn cystatin in heterologous B. subtilis and evaluate its effectiveness in reducing nematode populations, exploring its potential as a biocontrol agent.