CAJ | 학술논문

以连接在pMD-18T载体上的香蕉条斑病毒（BSV）全基因组为模板,PCR扩增得到了带有Gateway R重组反应接头序列的香蕉条斑病毒ORFⅠ及ORFⅡ基因片段,经过BP、LR重组后,构建成带有目的片段的酵母双杂诱饵质粒pDEST32-O1及pDEST32-O2。将质粒转化E.coli DH5α感受态细胞,筛选序列和编码框均正确的重组质粒。将以上2种正确的重组质粒分别与用于构建诱饵质粒的pDEST22空质粒共转化酵母MaV203感受态细胞,利用营养缺陷培养基（SC-Leu-Trp）筛选转化子,并对转化子进行毒性鉴定、自激活鉴定以及3-AT抑制浓度鉴定。结果显示,2种转化子均生长正常,说明所构建的2个重组质粒表达蛋白对酵母无毒性作用,其在特异营养缺陷培养基（SC-Leu-Trp-Ura）中无法生长,不能产生自激活现象,3-AT抑制浓度分别为50、75 mmol/L。以上结果说明所构建的2种诱饵质粒均可用于后续酵母双杂工作,为BSV与宿主的蛋白互作奠定了基础。
이련접재pMD-18T재체상적향초조반병독（BSV）전기인조위모판,PCR확증득도료대유Gateway R중조반응접두서렬적향초조반병독ORFⅠ급ORFⅡ기인편단,경과BP、LR중조후,구건성대유목적편단적효모쌍잡유이질립pDEST32-O1급pDEST32-O2。장질립전화E.coli DH5α감수태세포,사선서렬화편마광균정학적중조질립。장이상2충정학적중조질립분별여용우구건유이질립적pDEST22공질립공전화효모MaV203감수태세포,이용영양결함배양기（SC-Leu-Trp）사선전화자,병대전화자진행독성감정、자격활감정이급3-AT억제농도감정。결과현시,2충전화자균생장정상,설명소구건적2개중조질립표체단백대효모무독성작용,기재특이영양결함배양기（SC-Leu-Trp-Ura）중무법생장,불능산생자격활현상,3-AT억제농도분별위50、75 mmol/L。이상결과설명소구건적2충유이질립균가용우후속효모쌍잡공작,위BSV여숙주적단백호작전정료기출。
In the present study,the two target segments containing the Gateway recombinant sequences were firstly generated by two PCR amplifications,respectively,using the whole genome of BSV-Yunnan as the template.Then the two recombinant plasmids pDEST32-O1 and pDEST32-O2 were generated by two steps of Gateway recombination reactions,BP and LR respectively.The two plasmids were then transformed into the competent cells of E.coli DH5α,the plasmids with both accurate sequences and the reading-frames were selected by colony PCR amplification and sequencing.The two qualified plasmids were thereafter co-transformed with pDEST-22 empty plasmid respectively into the competent cells of MaV203 Yeast strains,and the tests of toxicity,self-activation and the inhibition concentration of 3-AT were performed by the utilization of selective plates.The results showed that there was no toxicity and self-activation among both of the recombinant plasmids,moreover,the inhibition concentration of 3-AT was 50 mmol/L and 75 mmol/L respectively for the two plasmids.These results suggested that both of the bait plasmids were qualified to be utilized in the subsequent yeast two-hybrid analysis and thus pave the way for the further investigation of interactions between proteins encoded by BSV and hosts.