基于转录组数据库的桔小实蝇sHSP基因的挖掘及功能分析

发布时间：2018-03-14 03:04

本文选题：桔小实蝇　切入点：小分子热激蛋白　出处：《西南大学》2015年硕士论文　论文类型：学位论文

【摘要】：桔小实蝇Bactrocera dorsalis (Hendel)隶属双翅目Diptera、实蝇科Tephritidae,广泛分布于热带和亚热带地区。桔小实蝇寄主范围广、适应性、繁殖力以及扩散能力强,是一种重要的农业害虫。桔小实蝇的防治目前主要是依靠化学药剂,由于大量不合理地使用化学杀虫剂,造成了桔小实蝇对多种常用杀虫剂产生了严重的抗药性。因此,寻找新的防治突破点显得尤为重要。昆虫小分子热激蛋白可以参与调节机体的生长发育、抗逆、生殖以及细胞分化等重要生理过程。因此,阐明桔小实蝇小分子热激蛋白调控其变态发育和抗逆过程中的分子机制,可为寻找杀虫剂新的作用靶标提供理论基础。本学位论文基于桔小实蝇转录组数据库信息,运用RT-PCR技术,从桔小实蝇体内克隆获得了9个小分子热激蛋白(small heat shock protein, sHSP)基因的cDNA序列,并对其特征序列进行注释；利用qPCR技术解析了这9个基因在桔小实蝇不同发育阶段和成虫不同组织的表达特性,以及外源20E和热胁迫后的应激表达模式；最后利用RNAi技术探索了小分子热激蛋白基因Bdhsp23.8和3315在桔小实蝇卵巢发育中的作用,验证了小分子热激蛋白基因Bdhsp23.8和Bdhsp20.6在桔小实蝇抗热胁迫中的作用。研究结果将有助于解析桔小实蝇小分子热激蛋白基因的功能及其调控机制,为桔小实蝇的持续防控提供新的思路和方法。主要研究结果如下：1桔小实蝇9个sHSP基因的克隆与序列分析根据从桔小实蝇转录组数据库筛选鉴定的unigene序列,克隆获得了桔小实蝇9个sHSP基因的cDNA序列,这9个基因分别为Bdhsp20.4、Bdhsp21.6、 Bdhsp23.8、Bdhsp20.6、Bdhsp23.0、Bdhsp17.6、Bdhsp18.4、Bdhsp11.1和3315。通过序列分析,明确了其中8个基因的开放阅读框,并推导了其编码的氨基酸序列。2桔小实蝇9个sHSP基因的表达模式分析2.1桔小实蝇9个sHSP基因在不同发育阶段的表达模式利用qPCR技术分析了桔小实蝇9个sHSP基因在卵、幼虫、蛹和成虫期的mRNA表达模式。结果表明,这9个基因在桔小实蝇不同发育阶段的表达量差异明显,其中Bdhsp20.4和Bdhsp18.4在卵、幼虫和蛹前期的相对表达非常低,在蛹的后期和成虫期的相对表达量激增；Bdhsp23.8和Bdhsp23.0在卵、幼虫和蛹前期的相对表达量明显高于幼虫后期和蛹后期；Bdhsp21.6在卵期几乎不表达,在幼虫和蛹期的相对表达变化非常明显；Bdhsp17.6在卵中几乎不表达,在幼虫中的表达非常高,并且前期高于后期,蛹中则相反,随着日龄的增加,表达量逐渐升高。2.2桔小实蝇9个sHSP基因在成虫不同组织中的表达模式利用qPCR技术解析了桔小实蝇9个sHSP基因在成虫头、胸、中肠、马氏管、脂肪体、精巢和卵巢中的相对表达量。结果表明,Bdhsp23.8和3315在卵巢中的相对表达量显著高于其他组织,由此可推测Bdhsp23.8和3315在桔小实蝇的卵巢发育中起到了不可缺少的作用；Bdhsp21.6、Bdhsp23.0、Bdhsp20.6和Bdhsp11.1在各组织中的相对表达量也有显著性差异；Bdhsp20.4在脂肪体中的表达量显著高于其他组织,由此可初步推断该基因和桔小实蝇的能量代谢相关；Bdhsp17.6在头、胸和脂肪体中相对表达量显著高于其他组织,在精巢和卵巢中不表达；Bdhsp18.4仅在桔小实蝇的胸部高表达,其他组织中几乎不表达。3外源20E和热胁迫对9个sHSP基因表达的影响3.1 20E对9个sHSP基因表达的影响用不同剂量的20E处理桔小实蝇5日龄幼虫,结果表明,注射1000ng的20E后12h,Bdhsp20.4、Bdhsp21.6、Bdhsp23.8、Bdhsp23.0、Bdhsp20.6、Bdhsp18.4和3315的相对表达量均显著上调,Bdhsp11.1明显下调,Bdhsp17.6则没有显著变化。3.2热胁迫对9个sHSP基因表达的影响用不同温度处理桔小实蝇7日龄成虫,结果表明,Bdhsp20.4和Bdhsp21.6的相对表达量仅在-5℃胁迫下显著上调；Bdhsp23.8、Bdhsp20.6和Bdhsp18.4在-5℃和40℃胁迫下均显著上调；而Bdhsp17.6的相对表达量不会受温度的影响；Bdhsp11.1的相对表达量经-5℃和0℃胁迫后显著下调。4桔小实蝇Bdhsp23.8、Bdhsp20.6和3315基因的功能研究将体外合成的Bdhsp23.8和3315基因的dsRNA注射入未交配的桔小实蝇4日龄雌成虫体内,并在72h后解剖其卵巢,在体视镜下观察卵巢的发育情况,同时利用qPCR技术检测目的基因的沉默效率。结果显示,其mRNA的相对表达明显下调,且卵巢的发育较对照明显滞后。将体外合成的Bdhsp23.8和Bdhsp20.6基因的dsRNA注射入桔小实蝇7日龄成虫体内,经-5℃和40℃胁迫,观察桔小实蝇的死亡率,并利用qPCR技术检测目的基因的沉默效率。结果表明,干扰Bdhsp20.6基因12h后经-5℃和40℃胁迫,桔小实蝇死亡率显著高于对照组,mRNA的相对表达明显下调；然而48h后,桔小实蝇的死亡率和mRNA的沉默效率都出现大幅度降低。注射dsBdhsp23.8后,桔小实蝇几乎没有死亡,并且mRNA的相对表达也无明显下调,检测Bdhsp20.6基因的相对表达量,结果显示也没有明显下调,对Bdhsp23.8温度相关功能的RNAi仍需进一步探索。
[Abstract]:Bactrocera dorsalis (Hendel) Diptera belongs to Diptera, Tephritidae Tephritidae, widely distributed in tropical and subtropical regions. Its wide host range, adaptability, fecundity and diffusion ability, is an important agricultural pest control. Its currently relies mainly on chemical agents, due to the large number of unreasonable use of chemical pesticides. Which fruit developed serious resistance to various insecticides. Therefore, to find a new breakthrough point of prevention is particularly important. The insect sHSP can participate and regulate the body's growth and development, reproduction and resistance, cell differentiation and other physiological processes. Therefore, to clarify the B.dorsalis small heat shock protein regulates the metamorphosis and molecular mechanisms of stress resistance in the process, can provide a theoretical basis for finding new targets of insecticides. This paper base In the Bactrocera dorsalis transcriptome database, using RT-PCR technology, 9 small heat shock protein obtained from in vivo cloning (small heat shock on protein, sHSP) cDNA gene sequences, and comments on its characteristic sequence; using qPCR technology to analysis the 9 gene expression in different developmental characteristics of oriental fruit fly the stage and adult tissues, and exogenous 20E and heat stress after the stress expression pattern; finally explores the small heat shock protein gene Bdhsp23.8 and 3315 in the role of B.dorsalis ovarian development by using RNAi technology, verifies the effect of small heat shock protein genes Bdhsp23.8 and Bdhsp20.6 in its thermal stress. The results will be in have the function and regulatory mechanism contributes to the analysis of Bactrocera dorsalis small heat shock protein gene, provide new ideas and methods for the prevention and control of continuous. The main research fruit The results are as follows: 9 cloning and sequence analysis of sHSP gene according to the sequence of UniGene 1 Bactrocera dorsalis from the screening and identification of transcriptome database, cloned the cDNA sequence of 9 sHSP genes on, these 9 genes were Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp20.6, Bdhsp23.0, Bdhsp17.6, Bdhsp18.4, Bdhsp11.1 and 3315. by sequence analysis clearly, the 8 gene open reading frame, and deduced 2.1 analysis of 9 sHSP genes of Bactrocera dorsalis larvae of 9 sHSP genes in oocytes at different developmental stages, the expression patterns by qPCR expression patterns of 9 sHSP genes encoding the amino acid sequence of.2 dorsalis, pupa and adult stage the expression pattern of mRNA. The results showed that the expression of these 9 genes in different developmental stages of B.dorsalis was different, in which Bdhsp20.4 and Bdhsp18.4 in eggs, larvae and pupae before The relative expression is very low, the relative expression of late pupal and adult stages of the surge; Bdhsp23.8 and Bdhsp23.0 in eggs, larvae and pupae of the relative expression was significantly higher than that of larvae and pupae of the late late; Bdhsp21.6 almost no expression in the egg stage, very obvious changes in the relative scale of larval and pupal stages; Bdhsp17.6 almost no expression in eggs, expressed in larvae is very high, and higher than the pre pupa stage, on the contrary, with the increase of age, the expression of.2.2 gradually increased 9 Bactrocera sHSP gene in different tissues of adult expression pattern in the use of qPCR to analysis of fruit 9 sHSP genes in the adult head. The chest, midgut, Malpighian tubules, fat body, the relative expression in the testis and ovary. The results showed that Bdhsp23.8 and 3315 in the ovary of the relative expression was significantly higher than that in other tissues, which can be speculated that Bdhsp23.8 and 3315 in small orange Tephritidae ovarian development plays an indispensable role; Bdhsp21.6, Bdhsp23.0, Bdhsp20.6 and Bdhsp11.1 in tissues of relative expression also have significant differences; the expression of Bdhsp20.4 in fat body weight was significantly higher than that in other tissues, thus it is inferred that the energy metabolism related genes and Bactrocera Bdhsp17.6 in head,; the chest fat body and the relative expression level was significantly higher than that in other tissues, no expression in testis and ovary; Bdhsp18.4 high expression only in on chest, affect the other tissues almost does not affect the expression of.3 of exogenous 20E and heat stress on the expression of 9 sHSP genes of 3.1 20E on the expression of 9 sHSP genes by 20E treatment of Bactrocera dorsalis different doses of 5 day old larvae, the results show that the injection of 1000ng 20E 12h, Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp23.0, Bdhsp20.6, Bdhsp18.4 and 3315 relative expression were significantly. Bdhsp11.1, the down-regulation of Bdhsp17.6 is not significantly affected the change of.3.2 heat stress on the expression of 9 sHSP genes with different temperature of Bactrocera dorsalis 7 day old adults. The results showed that the relative expression of Bdhsp20.4 and Bdhsp21.6 only at -5 DEG C stress significantly increased; Bdhsp23.8, Bdhsp20.6 and Bdhsp18.4 at -5 DEG C and 40 DEG C under stress windgroup; Bdhsp17.6 relative expression quantity is not affected by the temperature effect; relative expression of Bdhsp11.1 by -5 stress degrees and 0 degrees after significant downregulation of.4 Bdhsp23.8, function of Bdhsp20.6 and Bdhsp23.8 3315 gene synthesis in vitro and 3315 gene dsRNA was injected into virgin fly 4 day old female adults in vivo, and the ovarian anatomy in 72h after ovarian development was observed under optical microscopy, and using qPCR technology to detect gene silencing efficiency. The results showed that the mRNA phase The expression was downregulated, and ovarian development is lagging behind. The lighting of Bdhsp23.8 and Bdhsp20.6 gene in vitro synthesis of dsRNA injected into the fly 7 day old adults in the -5 degrees and 40 degrees of stress, the observation of B.dorsalis mortality, and the use of qPCR technology to examine the gene silencing efficiency. The results show that the interference of Bdhsp20.6 gene 12h after -5 degrees and 40 degrees of stress, its mortality rate was significantly higher than the control group, the relative expression of mRNA was significantly reduced after 48h; however, its mortality and silencing efficiency of mRNA are greatly reduced. After injection of dsBdhsp23.8, mRNA and Bactrocera almost no death, the relative expression was significantly reduced without relative expression Bdhsp20.6 gene, showed no obvious reduction, the temperature of the Bdhsp23.8 function of RNAi still need further exploration.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433;Q78

【参考文献】