本文選題：白蠟蟲 + WS　； 參考：《中國林業(yè)科學研究院》2017年碩士論文

【摘要】：白蠟蟲(Ericerus pela Chavannes)是我國具有重要經(jīng)濟價值的資源昆蟲,其2齡雄幼蟲所分泌的白蠟是一種天然高分子化合物,被廣泛應用于機械、醫(yī)療、食品、化妝品等諸多領域。研究表明,不同生物體內(nèi)存在保守的蠟酯合成途徑,在不同生物中,蠟酯最終都是在蠟酯合酶(wax synthase,WS)的催化下由長鏈脂肪酸和長鏈脂肪醇通過酯化反應形成的,因此,WS是催化蠟酯合成最關鍵的酶。本研究在前期鑒定到白蠟蟲ws基因的基礎上,圍繞基因表達動態(tài)、基因體內(nèi)功能和體外活性,采用實時熒光定量PCR技術(Real-time Quantitative PCR,RT-qPCR)、昆蟲細胞表達、RNA干擾(RNA interference,RNAi)等方法,分析了白蠟蟲ws在不同蟲態(tài)、不同組織的表達情況,驗證了白蠟蟲ws基因表達動態(tài)與白蠟蟲泌蠟特點一致,ws基因沉默導致白蠟蟲泌蠟量顯著降低,WS體外表達具有生成蠟酯的活性,主要結果如下:(1)對白蠟蟲雌雄蟲不同時期ws基因進行檢測發(fā)現(xiàn),ws基因在整個2齡雄幼蟲都上調(diào)表達,尤其是在2齡雄幼蟲前期;對不同組織進行檢測發(fā)現(xiàn),ws基因在雄蟲表皮的表達量明顯高于其他組織器官,與白蠟蟲泌蠟特點一致;(2)體外合成了ws dsRNA進行RNA干擾,經(jīng)RT-qPCR檢測,相比于空白對照和gfp(Green Fluorescent Protein)dsRNA對照,RNA干擾后白蠟蟲雄幼蟲體內(nèi)ws表達下調(diào),RNA干擾后發(fā)現(xiàn)ws實驗組相對于空白對照組和gfp dsRNA對照組單頭泌蠟量降低;(3)利用昆蟲-桿狀病毒表達系統(tǒng)將白蠟蟲ws基因在BmN家蠶細胞中表達,Western Blot表明,WS表達成功,且表達量在轉(zhuǎn)染36小時時較高;(4)加入底物24酯酰輔酶A和24C脂肪醇,利用表達產(chǎn)物進行體外酶活反應,HPLC檢測產(chǎn)物有酯的生成。以上研究表明,鑒定到的白蠟蟲ws基因是白蠟蟲泌蠟的關鍵酶基因,對于研究白蠟蟲泌蠟機理具有重要意義。
[Abstract]:Ericerus PELA Chavannes is a resource insect with important economic value in China. The white wax secreted by the 2 instar male larvae is a natural polymer compound, which is widely used in many fields, such as mechanical, medical, food, cosmetics and so on. The wax ester was finally formed by the esterification of long chain fatty acids and long chain fatty alcohols under the catalysis of wax synthase (WS). Therefore, WS is the most important enzyme to catalyze the synthesis of wax ester. Based on the earlier identification of the WS gene of paraffin, this study was based on the gene expression, in vivo function and in vitro activity. The expression of Real-time Quantitative PCR (RT-qPCR), insect cell expression, RNA interference (RNA interference, RNAi) and other methods were used to analyze the expression of WS in different insect States and different tissues. It was proved that the expression of the WS gene expression was consistent with the wax secretion of paraffin, and the WS gene silencing resulted in the significant wax secretion. The results were as follows: (1) the main results were as follows: (1) the detection of WS gene in different stages of the female and male insect of white wax insect found that the WS gene was up-regulated in the whole 2 age male larvae, especially at the early stage of the male larvae of 2. The expression of the WS gene in the male insect epidermis was significantly higher than that of the other groups. The weave characteristics were the same as that of paraffin wax; (2) ws dsRNA was synthesized in vitro for RNA interference. Compared with the blank control and GFP (Green Fluorescent Protein) dsRNA control, the WS expression in the male larvae was down after RNA interference, and the experimental group was found to be single head compared to the blank control group and the control group after the RNA interference. The amount of wax secretion was reduced; (3) the WS gene was expressed in the BmN silkworm cells by the insect baculovirus expression system. Western Blot showed that the expression of WS was successful and the expression was higher at 36 hours. (4) the substrate 24 ester acyl coenzyme A and 24C fatty alcohol were added to the enzyme activity reaction in vitro, and the product of HPLC detection product was formed. The above studies indicated that the WS gene identified from the wax insect was the key enzyme gene of wax secretion, and it is important for studying the wax secretion mechanism of wax insect.
【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S899.1

【參考文獻】

相關期刊論文 前10條

1 孫濤;王雪慶;趙遵嶺;于淑慧;陳曉鳴;劉魏魏;亓倩;楊璞;;白蠟蟲熱激蛋白序列分析[J];林業(yè)科學研究;2016年05期

2 劉博文;王雪慶;孫濤;亓倩;于淑惠;楊璞;陳曉鳴;;白蠟蟲蠟酯合酶基因cDNA全長克隆及原核表達[J];林業(yè)科學研究;2016年04期

3 亓倩;于淑惠;孫濤;王雪慶;劉博文;楊璞;陳曉鳴;;白蠟蟲蠟酯合酶在昆蟲細胞Sf9中的表達[J];林業(yè)科學研究;2016年02期

4 楊璞;徐冬麗;陳曉鳴;劉魏魏;鞏中軍;胡艷紅;;蠟酯合成途徑及關鍵酶的研究進展[J];中國細胞生物學學報;2012年07期

5 何晨柳;孔任秋;胡晗華;;產(chǎn)蠟酯聚球藻PCC7002的構建[J];水生生物學報;2010年06期

6 盛春霞;孔任秋;;基因工程藍藻產(chǎn)蠟酯的研究[J];水生生物學報;2010年03期

7 楊廣;尤民生;趙伊英;劉春輝;;昆蟲的RNA干擾[J];昆蟲學報;2009年10期

8 倪郁;郭彥軍;;植物超長鏈脂肪酸及角質(zhì)層蠟質(zhì)生物合成相關酶基因研究現(xiàn)狀[J];遺傳;2008年05期

9 馬李一;王有瓊;張重權;鄭華;甘瑾;段瓊芬;;蟲白蠟制備高級烷醇混合物研究[J];林產(chǎn)化學與工業(yè);2007年S1期

10 陳曉鳴;王自力;陳勇;葉壽德;王紹云;馮穎;;影響白蠟蟲泌蠟主要氣候因子及白蠟蟲生態(tài)適應性分析[J];昆蟲學報;2007年02期

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