發(fā)布時間：2018-06-11 10:00

  本文選題：棉大卷葉螟 + 絲素重鏈基因��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：棉大卷葉螟(Sylepta derogataFabricius)是鱗翅目螟蛾科昆蟲,分布范圍廣、寄主種類多,能對多種經(jīng)濟(jì)作物產(chǎn)生較大危害。為了進(jìn)一步研究棉大卷葉螟的吐絲危害機(jī)理,本文以棉大卷葉螟的絲素重鏈基因為研究對象,克隆得到其cDNA全長序列,測定了不同發(fā)育階段棉大卷葉螟吐絲基因的表達(dá)量,以及取食不同寄主后棉大卷葉螟吐絲基因的表達(dá)量。在此基礎(chǔ)上,運(yùn)用飼喂法干擾棉大卷葉螟絲素重基因的表達(dá),進(jìn)而影響卷葉螟的吐絲卷葉,并觀察干擾表達(dá)后對棉大卷葉螟種群發(fā)育與蛹重的影響。結(jié)果如下:1.根據(jù)文獻(xiàn)報導(dǎo)的其他昆蟲的絲素重鏈基因,通過RACE法克隆得到棉大卷葉螟絲素重鏈基因cDNA全長。棉大卷葉螟絲素重鏈基因cDNA全長為1465bp,起始密碼子為ATG,終止密碼子為TAG,開放閱讀框全長為1323bp。依據(jù)棉大卷葉螟絲素重鏈基因的核苷酸序列推導(dǎo)其氨基酸序列,估算該蛋白分子量為43.553kDa,等電點pI為6.26。將所獲得的基因全長同其他已經(jīng)登錄到GenBank的昆蟲絲素重鏈基因序列進(jìn)行比對,發(fā)現(xiàn)與棉大卷葉螟絲素重鏈基因序列一致性最高的是外米綴蛾(Corcyra cephalonica),達(dá)到65%,其次為大蠟螟(Galleria mellonella)與琥珀蠶(Antheraea assama),一致性皆為 59%。2.通過實時定量PCR技術(shù),分析了不同發(fā)育階段棉大卷葉螟絲素重鏈基因的表達(dá)量,以及取食不同寄主后棉大卷葉螟絲素重鏈基因的表達(dá)量。結(jié)果表明,絲素重鏈基因表達(dá)量隨蟲態(tài)、蟲齡的不同而不同:2齡、4齡幼蟲以及預(yù)蛹期絲素重鏈基因表達(dá)量相對較高,其中2齡蟲絲素重鏈基因表達(dá)量最高;3齡、5齡幼蟲絲素重鏈基因表達(dá)量相對較低,蛹期及羽化后絲素重鏈基因的表達(dá)量更低,甚至近于零;取食不同寄主后棉大卷葉螟絲素重鏈基因的表達(dá)量不同,飼喂苘麻葉片的棉大卷葉螟絲素重鏈基因的相對表達(dá)量最高,棉花次之,木槿最低,且它們之間差異顯著。3.通過飼喂法干擾棉大卷葉螟幼蟲絲素重鏈基因的表達(dá),并從菌液濃度、飼喂時間、飼喂的幼蟲蟲齡等方面研究其對棉大卷葉螟絲素重鏈基因表達(dá)量的影響。菌液濃度試驗結(jié)果表明,隨著飼喂棉大卷葉螟幼蟲菌液濃度的增加,絲素重鏈基因相對表達(dá)量下降。喂食不同蟲齡棉大卷葉螟幼蟲不同天數(shù)菌液的試驗結(jié)果表明,2L、3L、4L幼蟲喂食表達(dá)dsRNA的菌液后其重鏈基因表達(dá)量均下降,且喂食表達(dá)dsRNA的菌液3天、5天后的絲素重鏈基因表達(dá)量比喂食1天的表達(dá)量顯著下降。同時,2L、4L干擾效果比3L的好。試驗結(jié)果說明,菌液濃度、飼喂時間、飼喂的幼蟲蟲齡均會對卷葉螟絲素重鏈基因的干擾效率產(chǎn)生影響。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433.4

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本文編號：2004851