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《Journal of Northwest A & F University(Natural Science Edition)》 2011-01
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Overlap extension PCR based site-directed mutant of PlxyCSP1 gene in Plutella xylostella

REN Zhen-zhen,LIU Jing-jing,LIU Xiao-lei,CHEN Yong,LI Xun,HU Mei-ying (Laboratory of Insect Toxicology,South China Agricultural University,Guangzhou,Guangdong 510642,China)  
【Objective】 This study explored overlap extension PCR based site-directed mutant of insects chemosensory and studied the effects of the cysteine(Cys32) in binding characteristics of PlxyCSP1.【Method】 The tertiary structure and binding characteristics of PlxyCSP1 and PlxyCSP1-M were simulated by computer software in order to confirm the mutation site.Overlapping primes were designed according to the sequence of PlxyCSP1 to obtain PlxyCSP1-M which the Cys32 was mutated to the Trp32.PlxyCSP1-M was linked with pET-32a(+) and transformed into E.coli BL21(DE3).【Result】 The result of molecular docking displayed that ligand compounds could easily enter binding pocket of PlxyCSP1 but could not enter the binding pocket of PlxyCSP1-M.The binding energies of ligand compounds and PlxyCSP1 were all positive while the binding energies of ligand compounds and PlxyCSP1-M were zero.Recombinant plasmid sequencing showed that the Cys32 was mutated to Trp32 in PlxyCSP1-M and the recombinant strain could express fusion protein which was 36 ku by IPTG induction.【Conclusion】 The Cys32 is important to the structure and function of PlxyCSP1.Binding characteristics of PlxyCSP1-M were changed and could not bind with ligand compounds.
【Fund】: 国家自然科学基金项目(31071713);; 教育部高等学校博士学科点专项科研基金项目(20094404110019)
【CateGory Index】: Q78
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