国家转基因生物新品种培育科技重大专项(2008ZX08002-001)
为了明确人工合成的萝卜抗菌肽Rs-AFP2基因编码产物是否对一些重要农作物病原真菌有抑制作用,本文将该基因编码序列亚克隆到原核表达载体pGEX-4T-1上,构建成GST-Rs-AFP2融合蛋白表达载体。经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得了以包涵体形式表达的GST-Rs-AFP2重组蛋白。经过裂解、洗涤、溶解、复性、离心等处理,获得了纯化的GST-Rs-AFP2融合蛋白。对重要作物病原真菌进行体外抑菌分析的结果表明,Rs-AFP2可以显著抑制水稻纹枯病菌、小麦纹枯病菌、小麦根腐病菌、棉花黄萎病菌、小麦赤霉病菌的菌丝生长,说明RsAFP2可作为上述植物病害抗性基因育种的潜在基因。
Rs-AFP2 is an antifungal peptide from radish(Raphanus sativus).In this study,the full cDNA sequence of Rs-AFP2 gene was synthesized,and was sub-cloned in-frame to the 3′-terminus of the ORF of Glutathione S-transferase(GST)in the prokaryotic expression vector pGEX-4T-1.This allowed the recombinant protein GST-Rs-AFP2 to be highly expressed in cells of Escherichia coli.The recombinant protein GST-Rs-AFP2 could be expressed in the form of inclusion bodies after induction by isopropyl-β-D-thiogalactoside.Then,the GST-Rs-AFP2 protein was solubilized,denatured,refolded,and purified.The results of antifungal assay in vitro showed that the Rs-AFP2 protein exerted obvious inhibition against the growth of phytopathogenic fungi,such as Rhizoctonia solani,R.cerealis,Bipolaris sorokiniana,VerticilliuIll dahliae, and Fusarium graminearum.Therefore,Rs-AFP2 gene may be useful for improving the crop resistance against the above pathogenic fungi.
路妍,刘宝业,井金学,等.抗菌肽Rs-AFP2的原核表达及抑菌活性[J].植物遗传资源学报,2010,11(1):84-88.
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