亚洲玉米螟丝素轻链基因的克

发布时间：2018-06-24 13:19

  本文选题：亚洲玉米螟 + 丝素轻链基因　； 参考：《扬州大学》2015年硕士论文

【摘要】：亚洲玉米螟(Ostrinia furnacalis)是一种分布广泛且多食性的害虫,对我国的玉米、棉花危害较大。为研究亚洲玉米螟的吐丝机理,本文克隆了亚洲玉米螟的丝素轻链基因片段且进行了全长拼接,并研究了在其不同发育阶段、不同温度、不同寄主条件下该基因的相对表达量；同时,通过饲喂法对亚洲玉米螟丝素轻链基因进行了RNA干扰。现将研究结果报告如下：1、采用RACE技术克隆到亚洲玉米螟丝素轻链基因的cDNA全长(已提交GeneBank: KF669645)。该基因全长1098bp,自39bp至830bp为其开放阅读框,起始密码子ATG,终止密码子TAA,一共编码263个氨基酸,相对分子量为92.76kDa,等电点pI为4.99。通过同源性比对,亚洲玉米螟丝素轻链基因与棉大卷叶螟(Sylepta derogata Fabricius)同源性最高,为75%,与外米缀蛾(Corcyra cephalonica)的同源性为69%；与大蜡螟(Galleria mellonella)的同源性为68%。2、运用实时荧光定量PCR,检测了在不同发育阶段、不同温度条件、喂食不同寄主后亚洲玉米螟丝素轻链基因的相对表达量。结果表明,丝素轻链基因在5龄幼虫时相对表达量最高,其次是2龄幼虫与3龄幼虫；当幼虫进入预蛹后,该基因相对表达量开始降低,至成虫时,相对表达量几乎为零；研究了22℃,25℃,28℃,31℃,34℃条件下亚洲玉米螟丝素轻链基因的相对表达量,发现34℃时相对表达量最高；在棉花、玉米、酸模叶蓼三种寄主间,取食棉花的亚洲玉米螟丝素轻链基因的表达量最高,其次分别是玉米、酸模叶蓼。文章对处于逆境条件下玉米螟丝素轻链基因的相对表达量较高进行了讨论。3、通过饲喂法,将表达目标基因dsRNA的菌液饲喂亚洲玉米螟幼虫,检测沉默效果。结果发现,相对于喂食添加了清水的常规饲料的亚洲玉米螟幼虫(CK),喂食添加了表达dsRNA菌液饲料的亚洲玉米螟的丝素轻链基因表达量分别下调了34.81%、35.05%。经过RNA干扰后,相对于对照,亚洲玉米螟的幼虫发育历期缩短0.99d、蛹重下降8.53%、单雌产卵量减少34.86%。这表明丝素轻链基因表达量的降低与亚洲玉米螟的生长发育是有关联的。
[Abstract]:Asian corn borer (Ostrinia furnacalis) is a widely distributed and multifood pest, which is harmful to maize and cotton in China. In order to study the silking mechanism of Asian corn borer, the silk light chain gene fragment of Asian corn borer was cloned and spliced, and the relative expression amount of the gene was studied at different development stages, different temperatures and different host conditions. At the same time, RNA interference of Asian corn borer fibroin light chain gene was carried out by feeding method. The results are reported as follows: 1. The full-length cDNA of Asian corn borer fibroin light chain gene was cloned by race technique (GeneBank: KF669645). The total length of the gene is 1098bp. the open reading frame is from 39bp to 830bp, the start codon is ATG, and the stop codon is TAA. It encodes 263 amino acids, the relative molecular weight is 92.76 kDaand the isoelectric point Pi is 4.99. By homology comparison, Asian corn borer silk fibroin light chain gene has the highest homology with Sylepta derogata Fabricius, which is 75% and 69% with Corcyra cephalonica. The homology with Galleria mellonella was 68.2. The relative expression of silk fibroin light chain gene of Asian corn borer at different development stage and different temperature was detected by real-time fluorescence quantitative PCR. The results showed that the relative expression of silk fibroin light chain gene was the highest in the 5th instar larva, followed by the 2nd instar larva and the 3rd instar larva, and the relative expression level of the fibroin light chain gene began to decrease when the larva entered the prepupae, and almost zero at the adult stage. The relative expression of fibroin light chain gene of Asian corn borer was studied at 22 鈩，

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