白蜡虫蜡酯合酶基因表达动态和初步功能研究

发布时间：2018-06-19 03:42

本文选题：白蜡虫 + WS　；参考：《中国林业科学研究院》2017年硕士论文

【摘要】：白蜡虫(Ericerus pela Chavannes)是我国具有重要经济价值的资源昆虫,其2龄雄幼虫所分泌的白蜡是一种天然高分子化合物,被广泛应用于机械、医疗、食品、化妆品等诸多领域。研究表明,不同生物体内存在保守的蜡酯合成途径,在不同生物中,蜡酯最终都是在蜡酯合酶(wax synthase,WS)的催化下由长链脂肪酸和长链脂肪醇通过酯化反应形成的,因此,WS是催化蜡酯合成最关键的酶。本研究在前期鉴定到白蜡虫ws基因的基础上,围绕基因表达动态、基因体内功能和体外活性,采用实时荧光定量PCR技术(Real-time Quantitative PCR,RT-qPCR)、昆虫细胞表达、RNA干扰(RNA interference,RNAi)等方法,分析了白蜡虫ws在不同虫态、不同组织的表达情况,验证了白蜡虫ws基因表达动态与白蜡虫泌蜡特点一致,ws基因沉默导致白蜡虫泌蜡量显著降低,WS体外表达具有生成蜡酯的活性,主要结果如下:(1)对白蜡虫雌雄虫不同时期ws基因进行检测发现,ws基因在整个2龄雄幼虫都上调表达,尤其是在2龄雄幼虫前期;对不同组织进行检测发现,ws基因在雄虫表皮的表达量明显高于其他组织器官,与白蜡虫泌蜡特点一致;(2)体外合成了ws dsRNA进行RNA干扰,经RT-qPCR检测,相比于空白对照和gfp(Green Fluorescent Protein)dsRNA对照,RNA干扰后白蜡虫雄幼虫体内ws表达下调,RNA干扰后发现ws实验组相对于空白对照组和gfp dsRNA对照组单头泌蜡量降低;(3)利用昆虫-杆状病毒表达系统将白蜡虫ws基因在BmN家蚕细胞中表达,Western Blot表明,WS表达成功,且表达量在转染36小时时较高;(4)加入底物24酯酰辅酶A和24C脂肪醇,利用表达产物进行体外酶活反应,HPLC检测产物有酯的生成。以上研究表明,鉴定到的白蜡虫ws基因是白蜡虫泌蜡的关键酶基因,对于研究白蜡虫泌蜡机理具有重要意义。
[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.
【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S899.1

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