黄曲条跳甲化学感受基因的鉴定与表达分析

发布时间：2018-06-06 07:36

本文选题：黄曲条跳甲 + 化学感受基因　；参考：《仲恺农业工程学院》2015年硕士论文

【摘要】：黄曲条跳甲Phyllotreta striolata(Fabricius)是十字花科蔬菜的主要害虫之一,在中国南方地区严重危害十字花科蔬菜的生产。目前黄曲条跳甲的防治主要依靠化学防治,但农药的过度使用导致抗药性产生和食品安全隐患。因此,需要发展新型的防治策略。应用引诱化合物或驱避化合物是害虫防治的发展方向之一,研究黄曲条跳甲的化学感受基因可以加深对化学感受基因的认知,同时也为将来对其功能研究奠定科学基础。本论文对本实验室前期构建的黄曲条跳甲转录组数据进行生物信息学分析,通过同源性分析和c DNA末端快速扩增技术(RACE)从黄曲条跳甲转录组数据库中鉴定化学感受相关基因并获得基因全长;应用生物信息学方法分析候选基因的结构和特征,并构建系统发育进化树;应用实时荧光定量PCR技术,研究化学感受基因在不同性别的嗅觉器官与非嗅觉器官的表达差异。通过以上研究,获得以下结果:1)黄曲条跳甲化学感受基因鉴定和生物信息学分析应用生物信息学方法和RACE技术共鉴定16个化学感受基因,其中气味结合蛋白(Odorant-binding proteins,OBPs)基因3个,化学感受蛋白(Chemosensory proteins,CSPs)基因2个,气味受体(Odorant receptors,ORs)基因1个,离子型受体(Ionotropic receptors,IRs)基因7个,感受神经膜蛋白(Sensory neuron membrane proteins,SNMPs)基因2个,味觉受体(Gustatory receptors,GRs)基因1个。通过选取部分昆虫的化学感受基因与鉴定所得的黄曲条跳甲化学感受基因构建系统发育进化树,分析显示黄曲条跳甲化学感受基因多与赤拟谷盗等鞘翅目昆虫的化学感受基因的进化关系较为接近。(1)Pstr OBP1、Pstr OBP2、Pstr OBP3分别与松墨天牛Monochamus alternatus的Malt OBP4、Malt OBP3和赤拟谷盗Tribolium castaneum的Tcas OBP19d同源性最高,分别为52%、41%、42%。(2)Pstr CSP1与Pstr CSP2分别与与黄粉甲Tenebrio molitor的Tmol CSP3和赤拟谷盗T.castaneum的Tcas CSP6同源性最高,分别为57%和37%。(3)Pstr SNMP1和Pstr SNMP2分别与松山甲虫Dendroctonus ponderosae的Dpon SNMP(AFI45066.1)和赤拟谷盗T.castaneum的Tcas SNMP2-like(XP_008198962.1)最高,分别为49%和46%;系统进化分析显示,2个SNMP分在SNMP1和SNMP2两类的分支上。(4)Pstr IR1、Pstr IR2、Pstr IR5、Pstr IR6、Pstr IR7均与glutamate receptor ionotropic,kainate类基因同源性较高,Pstr IR3与赤拟谷盗T.castaneum的glutamate receptor 2的同源性最高,同源性为59%,Pstr IR4与赤拟谷盗glutamate[NMDA]receptor subunit 1同源性最高,同源性达到87%。(5)Pstr OR2与赤拟谷盗T.castaneum的Tcas OR37同源性最高,同源性为34%。(6)Pstr GR1与Tcas GR10同源性最高同源性为69%,与Dmel GR21a为直系同源。2)嗅觉感受基因在黄曲条跳甲两性嗅觉和非嗅觉的组织的表达分析采用q RT-PCR方法,分别选取黄曲条跳甲不同性别的触角和腹端,研究了其雌雄虫Pstr OBP1、Pstr OBP2、Pstr OBP3、Pstr CSP1、Pstr IR1、Pstr IR2、Pstr IR3、Pstr SNMP1和Pstr SNMP2在这2个部位的表达情况。(1)OBPs的表达研究显示:Pstr OBP1与Pstr OBP2为触角特异表达,Pstr OBP1在雄虫触角表达量是雌虫触角表达量的2倍,Pstr OBP2在雄虫和雌虫触角均是特异表达,且在两性触角的表达量无明显差异,Pstr OBP3在雄虫腹部的表达量高于触角,同时雄虫触角与雌虫触角表达量相差不大,因此推测Pstr OBP1可能在雄虫感知异性性信息素的过程中起作用;推测Pstr OBP2可能参与感知普通气味;推测Pstr OBP3可能参与雄虫聚集信息素的释放和对聚集信息素的感知;(2)CSPs的表达研究显示:Pstr CSP1在触角和腹部均有表达,在雌虫腹部表达最低,其余部位表达差异不显著;(3)IRs表达研究显示:Pstr IR1、Pstr IR2、Pstr IR3在触角和腹部都有表达,总体上雄虫表达量高于雌虫;4)SNMPs表达研究显示:Pstr SNMP1与Pstr SNMP2在触角和腹部都有表达,但表达差异不明显。
[Abstract]:Phyllotreta striolata (Fabricius) is one of the main pests of cruciferous vegetables and seriously endangering the production of cruciferous vegetables in the south of China. At present, the prevention and control of the beetle is mainly based on chemical control, but the excessive use of pesticides leads to the potential of resistance and food safety. Therefore, a new type of food is needed to develop a new type. The application of attractant compounds or repellent compounds is one of the development directions of pest control. The study of the chemoreceptor genes of the Yellow curved strip can deepen the cognition of the chemoreceptor genes and lay a scientific foundation for the study of its function in the future. According to bioinformatics analysis, using homology analysis and C DNA terminal rapid amplification technique (RACE) to identify the chemoreceptor related genes and obtain the whole length of the gene from the database of the Yellow curved strip armor transcriptome, the structure and characteristics of the candidate genes were analyzed by bioinformatics, and the phylogenetic tree was constructed; real time fluorescence determination was applied. PCR technique was used to study the difference between the olfactory organ and the non olfactory organ in different sexes. Through the above study, the following results were obtained: 1) the identification of the chemoreceptor gene and the bioinformatics analysis, the application of bioinformatics and the RACE technology to identify 16 chemoreceptor genes. There are 3 Odorant-binding proteins (OBPs) genes, 2 Chemosensory proteins (CSPs) genes, 1 Odorant receptors (ORs) genes, 7 ionic receptors (Ionotropic receptors, IRs) genes, and 2 receptor genes. Ptors, GRs) gene 1. The phylogenetic tree was constructed by selecting the chemoreceptor genes of some insects and the identification of the chemoreceptor genes of the Yellow curved strip armor. The analysis showed that the evolution of chemoreceptor genes of Coleoptera and other Coleoptera insects, such as hellfish, was close to that of the chemoreceptor genes of the beetle. (1) Pstr OBP1, Pstr OBP2, P STR OBP3 is the highest homology with Malt OBP4, Malt OBP3 and Tribolium castaneum of Monochamus alternatus, respectively, 52%, 41%. 37%. (3) Pstr SNMP1 and Pstr SNMP2 are respectively the highest of Dpon SNMP (AFI45066.1) and the T.castaneum of Matsuyama Kousa Dendroctonus ponderosae, respectively, 49% and 46%, respectively. The homology of IR7 is higher than that of glutamate receptor ionotropic and kainate gene. Pstr IR3 has the highest homology with the glutamate receptor 2 of the ash T.castaneum, and the homology is 59%. The homology of OR37 is the highest, the homology is 34%. (6) Pstr GR1 and Tcas GR10 homology with the highest homology, and the expression of the olfactory receptor gene with the Dmel GR21a as the direct homologous.2) in the olfactory and non olfactory tissues of the stripe dive is analyzed by Q RT-PCR method, respectively, to select the antennae and the abdominal end of the different sex of the stripe diaphora. The expression of Pstr OBP1, Pstr OBP2, Pstr OBP3, Pstr CSP1, Pstr IR1, Pstr IR2, and Pstr IR2 are expressed as the specific expression of the antennae, and the expression of the male antennae is 2 times the female's antennae expression. The expression of Pstr OBP3 is higher than the antennae in the abdomen of the male, and the difference between the male antennae and the female antennae is not significant. Therefore, it is suggested that Pstr OBP1 may play a role in the process of male perception of heterosexual pheromone; Pstr OBP2 may be involved in the perception of common odor. It is speculated that Pstr OBP3 may be involved in the release of the aggregation pheromone of the male and the perception of the aggregation pheromone. (2) the expression of CSPs shows that Pstr CSP1 is expressed in the antennae and abdomen, and the expression in the abdomen is the lowest, and the expression of the rest is not significant. (3) the expression of IRs shows that Pstr IR1, Pstr IR2, Pstr IR3 have a table in the antennae and abdomen. In general, the expression level of male was higher than that of female. 4) SNMPs expression study showed that Pstr SNMP1 and Pstr SNMP2 were expressed in antennae and abdomen, but the difference was not obvious.
【学位授予单位】：仲恺农业工程学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.5

【引证文献】