CAJ | 학술논문

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dsRNA，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 (GenBank登录号：AAEL003728-RA)，将其连接到克隆载体pMD18-T上，利用限制性内切酶XbaⅠ和XhoⅠ将其插入到原核表达载体pLitmus28i的2个T7启动子之间，成功构建可诱导形成目的双链RNA (dsRNA)的原核表达重组载体pLitmus28i-mapk-like，并转化大肠杆菌HT115，经IPTG诱导，1 mL菌液的dsRNA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dsRNA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dsRNA从饲料琼脂块中游离出来，提高dsRNA的稳定性。埃及伊蚊幼虫摄取mapk-like基因的dsRNA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。研究结果将有利于进一步筛选获得相关抵御基因，为建立一个基于RNA沉默防治蚊媒病的研究体系提供了基础资料。
mapk-like시일충신호전도상관촉유사분렬격활단백격매가족기인，여선충(Caenorhabditis elegans)중삼여저어Cry독소적p38 MAPK함유유사적모서，기시부삼여애급이문(Aedes aegypti)저어소운금아포간균(Bacillus thuringiensis, Bt)과정유대험증。위간편병대량획득RNAi험증소수적dsRNA，본연구이용RNaseⅢ결함형대장간균(Escherichia coli) HT115고효화렴개적우점，근거이측서적촉유사분렬격활단백격매가족기인mapk-like기인서렬，설계특이성인물，PCR확증대소위361 bp적목적편단mapk-like (GenBank등록호：AAEL003728-RA)，장기련접도극륭재체pMD18-T상，이용한제성내절매XbaⅠ화XhoⅠ장기삽입도원핵표체재체pLitmus28i적2개T7계동자지간，성공구건가유도형성목적쌍련RNA (dsRNA)적원핵표체중조재체pLitmus28i-mapk-like，병전화대장간균HT115，경IPTG유도，1 mL균액적dsRNA산량가체1.18μg，전영검측조대청석，질량량호。차외，이용각취당포과dsRNA형성납미미립，상청경전영검측표명，각취당적취침효솔량호，납미미구가유효방지dsRNA종사료경지괴중유리출래，제고dsRNA적은정성。애급이문유충섭취mapk-like기인적dsRNA후，상대전록표체수평위65.55%，표체량명현하조，사위효과량호。연구결과장유리우진일보사선획득상관저어기인，위건립일개기우RNA침묵방치문매병적연구체계제공료기출자료。
Mapk-like, the signaling transduction related mitogen-activated protein kinase gene, shares a conserve 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 of bacteria liquid with high quality were obtained after 0.5 mmol/L IPTG induction. In addition, nanoparticles were prepared by mixing resulted dsRNA and chitosan and the supernatant was examined by agarose gel electrophoresis. These results indicated a good coagulation effect for chitosan, by which nanoparticles could protect dsRNA from dissociation from the mixture of feed andagarose and the stability was improved. Finally, the relative expression levels of mapk-like gene after the effective dsRNA feeding decreased to 65%. These results provide potential for use in RNAi, screening for more defense related gene, and basic research for RNA silencing to control mosquito transmitted diseases.