【摘要】：幾丁質(zhì)脫乙酰酶(chitin deacetylase)是一種昆蟲體內(nèi)幾丁質(zhì)降解途徑中的酶,它能降解幾丁質(zhì)生成殼聚糖。斜紋夜蛾(Spodoptera litura)是一種世界性分布廣泛的害蟲,發(fā)生蟲害時給農(nóng)業(yè)生產(chǎn)造成巨大的經(jīng)濟損失。深入研究斜紋夜蛾的生理生化機制,并在此基礎上探尋有效的防治方法已勢在必行。本論文以斜紋夜蛾為實驗對象,研究斜紋夜蛾幾丁質(zhì)脫乙酰酶功能為斜紋夜蛾綠色防控提出新的思路與方法。主要結果如下:從斜紋夜蛾轉錄組數(shù)據(jù)中篩選并克隆6個斜紋夜蛾幾丁質(zhì)脫乙酰酶基因(SlCDA),分別命名為SlCDA1、SlCDA2、SlCDA3、SlCDA4、SlCDA5、SlCDA6,開放閱讀框分別包含1620、1446、7224、1131、1167、1125個堿基,分別編碼540、482、2408、377、389、375個氨基酸。系統(tǒng)發(fā)育分析結果表明:SlCDA1屬于第一組(GroupⅠ),SlCDA2屬于第三組(GroupⅢ),SlCDA3屬于第四組(GroupⅣ),SlCDA4、SlCDA5、SlCDA6均屬于第五組(GroupⅤ)。氨基酸序列同源性比對結果顯示,SlCDA與家蠶、棉鈴蟲等昆蟲的幾丁質(zhì)脫乙酰酶氨基酸序列相似度極高。通過qRT-PCR確定了6個Sl CDA基因表達譜,結果表明:SlCDA1、SlCDA2在表皮中周期性表達,蛻皮后表達量急劇上升,在預蛹期第一天達到最高;SlCDA3在表皮及脂肪體中均有一定表達,但在檢測時期內(nèi)無特異性;SlCDA4、SlCDA6在6齡第2天的中腸中表達量最高;SlCDA5在檢測時期內(nèi)表皮、脂肪體和中腸中表達水平均偏低。表明SlCDA1、SlCDA2可能與斜紋夜蛾蛻皮生理過程相關,SlCDA4、SlCDA6可能與中腸生長發(fā)育相關,SlCDA3、SlCDA5可能不參與斜紋夜蛾的蛻皮等過程。選擇斜紋夜蛾表皮特異性基因SlCDA1、SlCDA2對斜紋夜蛾進行RNA干擾并檢測干擾效率,結果顯示目的基因表達量下調(diào)但并沒有對當代斜紋夜蛾致畸或致死。采用昆蟲桿狀病毒表達系統(tǒng)獲得重組蛋白SlCDA1、SlCDA2,經(jīng)SDS-PAGE、Western Blotting鑒定其分子量分別為62 kDa、55 kDa,并能與幾丁質(zhì)結合。
[Abstract]:Chitin deacetylase (chitin deacetylase) is an enzyme in chitin degradation pathway in insects, which can degrade chitin to produce chitosan. Spodoptera litura is a widely distributed pest in the world. It is imperative to study the physiological and biochemical mechanism of Spodoptera litura and explore effective control methods. In this paper, chitin deacetylase function of Spodoptera litura was studied, and a new idea and method for green control of Spodoptera litura was put forward. The main results were as follows: six chitinase genes (SlCDA) were screened and cloned from the transcriptional data of Spodoptera litura, named SlCDA1, SlCDA2, SlCDA3, SlCDA5, SlCDA6, respectively, and the open reading frame contained 1620144672224A1131113116H1 125 bases, encoding 540482,240837389375 amino acids, respectively. The results of phylogenetic analysis showed that the first group (Group 鈪，

本文編號：2135805