mapk-like是一种信号转导相关促有丝分裂激活蛋白激酶家族基因,与线虫(Caenorhabditis elegans)中参与抵御Cry毒素的p38 MAPK含有类似的模序,其是否参与埃及伊蚊(Aedes aegypti)抵御苏云金芽胞杆菌(Bacillus thuringiensis,Bt)过程有待验证。为简便并大量获得RNAi验证所需的ds RNA,本研究利用RNaseⅢ缺陷型大肠杆菌(Escherichia coli)HT115高效和廉价的优点,根据已测序的促有丝分裂激活蛋白激酶家族基因mapk-like基因序列,设计特异性引物,PCR扩增大小为361 bp的目的片段mapk-like(Gen Bank登录号:AAEL003728-RA),将其连接到克隆载体p MD18-T上,利用限制性内切酶XbaⅠ和XhoⅠ将其插入到原核表达载体p Litmus28i的2个T7启动子之间,成功构建可诱导形成目的双链RNA(ds RNA)的原核表达重组载体p Litmus28i-mapk-like,并转化大肠杆菌HT115,经IPTG诱导,1 m L菌液的ds RNA产量可达1.18μg,电泳检测条带清晰,质量良好。此外,利用壳聚糖包裹ds RNA形成纳米微粒,上清经电泳检测表明,壳聚糖的聚沉效率良好,纳米微球可有效防止ds RNA从饲料琼脂块中游离出来,提高ds RNA的稳定性。埃及伊蚊幼虫摄取mapk-like基因的ds RNA后,相对转录表达水平为65.55%,表达量明显下调,饲喂效果良好。
Mapk-like, the signaling transduction related mitogen-activated protein kinase gene, shares aconserve motif with p38 MAPK that involves in cellular defense of Caenorhabditis elegans against Bacillus thuringiensis(Bt) Cry toxins. Little validation work, however, had been done in its important role for defense response of Aedes aegypti to Cry toxins. Therefore, RNaseⅢ-absent Escherichia coli HT115, with the high-efficiency and low-cost, was applied to establish a convenient and economical method for mass production of dsRNA for RNA silencing. Using specific primers, mapk-like gene (GenBank No. AAEL003728-RA) was amplified. Then 361 bp PCR products were cloned into the cloning vector pMD18-T and subcloned into the expression vector pLitmus28i, which contained 2 T7 promoters located in each side of multiple cloning sites with the digestion of restriction endonuclease XbaⅠ/XhoⅠ. The recombinant plasmid pLitmus28i-mapk-like was transformed into the HT115. dsRNAs of 1.18 μg/mL o
