棉大卷叶螟丝素重链基因的克

发布时间：2018-06-11 10:00

本文选题：棉大卷叶螟 + 丝素重链基因　；参考：《扬州大学》2017年硕士论文

【摘要】：棉大卷叶螟(Sylepta derogataFabricius)是鳞翅目螟蛾科昆虫,分布范围广、寄主种类多,能对多种经济作物产生较大危害。为了进一步研究棉大卷叶螟的吐丝危害机理,本文以棉大卷叶螟的丝素重链基因为研究对象,克隆得到其cDNA全长序列,测定了不同发育阶段棉大卷叶螟吐丝基因的表达量,以及取食不同寄主后棉大卷叶螟吐丝基因的表达量。在此基础上,运用饲喂法干扰棉大卷叶螟丝素重基因的表达,进而影响卷叶螟的吐丝卷叶,并观察干扰表达后对棉大卷叶螟种群发育与蛹重的影响。结果如下:1.根据文献报导的其他昆虫的丝素重链基因,通过RACE法克隆得到棉大卷叶螟丝素重链基因cDNA全长。棉大卷叶螟丝素重链基因cDNA全长为1465bp,起始密码子为ATG,终止密码子为TAG,开放阅读框全长为1323bp。依据棉大卷叶螟丝素重链基因的核苷酸序列推导其氨基酸序列,估算该蛋白分子量为43.553kDa,等电点pI为6.26。将所获得的基因全长同其他已经登录到GenBank的昆虫丝素重链基因序列进行比对,发现与棉大卷叶螟丝素重链基因序列一致性最高的是外米缀蛾(Corcyra cephalonica),达到65%,其次为大蜡螟(Galleria mellonella)与琥珀蚕(Antheraea assama),一致性皆为 59%。2.通过实时定量PCR技术,分析了不同发育阶段棉大卷叶螟丝素重链基因的表达量,以及取食不同寄主后棉大卷叶螟丝素重链基因的表达量。结果表明,丝素重链基因表达量随虫态、虫龄的不同而不同:2龄、4龄幼虫以及预蛹期丝素重链基因表达量相对较高,其中2龄虫丝素重链基因表达量最高;3龄、5龄幼虫丝素重链基因表达量相对较低,蛹期及羽化后丝素重链基因的表达量更低,甚至近于零;取食不同寄主后棉大卷叶螟丝素重链基因的表达量不同,饲喂苘麻叶片的棉大卷叶螟丝素重链基因的相对表达量最高,棉花次之,木槿最低,且它们之间差异显著。3.通过饲喂法干扰棉大卷叶螟幼虫丝素重链基因的表达,并从菌液浓度、饲喂时间、饲喂的幼虫虫龄等方面研究其对棉大卷叶螟丝素重链基因表达量的影响。菌液浓度试验结果表明,随着饲喂棉大卷叶螟幼虫菌液浓度的增加,丝素重链基因相对表达量下降。喂食不同虫龄棉大卷叶螟幼虫不同天数菌液的试验结果表明,2L、3L、4L幼虫喂食表达dsRNA的菌液后其重链基因表达量均下降,且喂食表达dsRNA的菌液3天、5天后的丝素重链基因表达量比喂食1天的表达量显著下降。同时,2L、4L干扰效果比3L的好。试验结果说明,菌液浓度、饲喂时间、饲喂的幼虫虫龄均会对卷叶螟丝素重链基因的干扰效率产生影响。
[Abstract]:Sylepta derogata Fabriciusis is an insect of Lepidoptera, which has a wide range of distribution and many host species, which can cause great harm to many kinds of cash crops. In order to further study the mechanism of filamentous damage of cotton leaf roller, the full-length cDNA sequence was cloned from silk heavy chain gene of cotton leaf roller, and the expression amount of filamentous gene in different development stages was determined. And the expression amount of filamentous gene in cotton leaf roller after feeding on different hosts. On this basis, the feeding method was used to interfere with the expression of fibroin weight gene in the cotton leaf roller, and then to influence the filamentous leaf winding of the leaf roller, and to observe the effect of the interference on the population development and pupae weight of the cotton leaf roller. The result is as follows: 1. The full-length cDNA of silk fibroin heavy chain gene from other insects was cloned by race method. The full-length cDNA of silk fibroin heavy chain gene was 1465 BP, the start codon was ATG, the termination codon was tag, and the open reading frame was 1323 BP. According to the nucleotide sequence of the silk fibroin heavy chain gene, the amino acid sequence of the protein was deduced. The molecular weight of the protein was estimated to be 43.553 kDa, and the isoelectric point Pi was 6.26. The whole length of the obtained gene was compared with the sequence of other insect silk fibroin heavy chain genes that had been entered into GenBank. It was found that Corcyra cephalonica was the most consistent with the sequence of silk fibroin heavy chain gene, which was 65%, followed by Galleria mellonella) and Antheraea assamaa (59 路2). The expression of fibroin heavy chain gene in different developmental stages of cotton leaf roller borer and the expression amount of silk fibroin heavy chain gene of cotton leaf roller borer were analyzed by real-time quantitative PCR. The results showed that the expression of silk fibroin heavy chain gene was relatively higher in the 4th instar larvae of the 2nd instar and prepupae stage. The expression of silk fibroin heavy chain gene in the 2nd instar larvae was the highest, and the expression of silk fibroin heavy chain gene in the 3rd and 5th instar larvae was relatively low, and the expression of silk fibroin heavy chain gene in pupal stage and after emergence was even lower, even near zero. The relative expression of fibroin heavy chain gene was the highest in the leaves of Abutilon theophrasti, the second was in cotton, and the lowest in hibiscus, and the difference between them was significant (.3). The expression of silk fibroin heavy chain gene in cotton leaf roller larva was interfered with by feeding method, and the effects of it on the expression of silk fibroin heavy chain gene in cotton leaf roller moth were studied from the aspects of bacterial concentration, feeding time and larva age. The results showed that the relative expression of silk fibroin heavy chain gene decreased with the increase of the liquid concentration of the larva of cotton leaf roller. The results showed that the expression of heavy chain genes decreased after feeding the dsRNA-expressing bacteria solution to the larvae of different larvae of cotton leaf roller, the results showed that the expression of heavy chain genes in the larva was decreased after feeding the bacteria solution expressing dsRNA, and the results showed that the heavy chain gene expression of the larva was decreased after feeding the dsRNA. The expression of silk fibroin heavy chain gene in the medium fed with dsRNA for 3 days and 5 days was significantly lower than that in the first day of feeding. At the same time, the interference effect of 2L 4L is better than that of 3L. The results showed that the interference efficiency of silk fibroin heavy chain gene of leaf roller was affected by the concentration of bacteria liquid, feeding time and larva age.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.4

【参考文献】