【摘要】 利用基因克隆技术探究了松材线虫携带的荧光假单胞菌(Pseudomonas fluorescens)GcM5-1A是否含有溶血素基因以及溶血素活性。根据荧光假单胞菌疑似溶血素的基因序列设计一对引物,通过PCR技术扩增得到疑似溶血素基因Hly。将该基因克隆到表达载体pET-15b上,构建了pET-15b-Hly。重组质粒pET-15b-Hly转化E.coli BL21(DE3)构建了工程菌,工程菌经过异丙基-β-D硫代半乳糖苷(IPTG)诱导后,SDS-PAGE分析表明,疑似溶血素在大肠杆菌中得到了表达。利用Ni-Sepharose 6(FF)亲和树脂纯化了重组蛋白,溶血实验显示,纯化的重组蛋白可使鸡的红细胞发生溶血,证实本研究所克隆基因确实编码溶血素,利用黑松切根苗的生测表明,重组荧光假单胞菌溶血素对黑松幼苗有致萎活性,这为深入研究松材线虫携带细菌在松材线虫病病理进程中的作用打下了基础。更多还原

【Abstract】 A pair of special primers were designed according to the gene encoding hypothetical hemolysin of Pseudomonas fluorescens GcM5-1Acarried by Busaphelenchus xylophilus.This gene was amplified by PCR using the designed primers and the genomic DNA of P.fluorescens GcM5-1Aas the template.The PCR product was cloned into expression vector pET-15 bto construct pET-15b-Hly,followed by transforming this recombinant plasmid to E.coli BL21(DE3)to construct engineering bacteria.SDS-PAGE analysis showed that the expression of hypothetical hemolysin in engineering bacteria was achieved after IPTG induction was applied.The recombinant protein was purified by Ni-Sepharose 6(FF)affinity chromatography.Red cell lysis test indicated that the recombinant protein could lyse chicken erythrocytes,which illustrated that the gene cloned in this paper indeed coded hemolysin.The result of bioassay with root-cutting seedlings of Pinus thunbergii showed that the recombinant hemolysin could cause seedlings wilting.This study offers some clues to further investigate the roles of bacteria carried by pine wood nematode in the pathogenesis of pine wilt disease.更多还原