CAJ | 학술논문

目的:从苦瓜中克隆MAYO全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法.方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异件的引物,通过PCR技术扩增出全长861bp的MAP30基因.该基因经Xho Ⅰ和EcoR Ⅰ双酶切,连接至毕赤酵母表达载体pPIC9中.重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株.结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核骨酸同源性达99.9%,氨基酸同源性达100%.菌落PCR显示外源基因已整合人酵母GS115菌株中.结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础.
목적:종고과중극륭MAYO전장기인,병장해기인련접지표체재체pPIC9중,건립효모균락PCR사선방법.방법채용개량SDS법종고과표피중제취기인조DNA,설계특이건적인물,통과PCR기술확증출전장861bp적MAP30기인.해기인경Xho Ⅰ화EcoR Ⅰ쌍매절,련접지필적효모표체재체pPIC9중.중조재체전화GS115균주,운용균락PCR감정중조균주.결과:기인측서표명,해기인이성공삽입효모표체재체pPIC9α-factor분비신호하유,동원성분석표명해기인여GeneBank(AF284811)적핵골산동원성체99.9%,안기산동원성체100%.균락PCR현시외원기인이정합인효모GS115균주중.결론:성공지극륭료MAP30전장기인,병구건료함MAP30기인적중조필적효모표체재체,병획득료정합균주,위하일보연구전정료기출.
Objective:MAP30 gene cloning by PCR from total DNA of Momordica charantia L, the construction of recombinant vector Ppic9 - MAP30, and establishing the rapid screening method of recombinant GS115 strain.Method :The total genome DNA was extracted by modified SDS method from Momordica charantia L With designed specific primers, MAP30(Momordica anti - HIV protein of 30 Kd) gene was amplified by PCR.The PCR product was digested by Xho Ⅰ and EcoR Ⅰ ,then ligated to Ppic9 vector.Then the Ppic9 - MAP30 vector was transformed into GS115 strain, and the positive elones were screened by colony PCR.Result:Mter DNA sequencing, MAP30 gene was inserted into Ppic9, Nueleotide acid sequence and amino acid sequence showed 99.88％ and 100％ homology to GeneBand (AF284811) respectively.Four positive strains were identified by colony PCR.Condusion: MAP30 gene was suecessfuUy cloned into Ppic9 vector, and target gene was also integrated into chromosome of GS115 strain.