褐飞虱蜕皮及变态信号途径相关基因的功能分析

发布时间：2018-02-05 04:17

  本文关键词： 褐飞虱 蜕皮激素合成 核受体 蜕皮激素信号转运通路 变态决定因子 表达模式 RNA干扰　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：褐飞虱Nilaparvata lugens(St?l)属半翅目飞虱科,是我国和许多亚洲国家水稻生产上的重要害虫。调控昆虫蜕皮和变态的主要信号通路为蜕皮激素信号通路和保幼激素信号通路,本研究在基因组和转录组数据的基础上,对褐飞虱核受体基因、蜕皮激素和保幼激素信号通路基因的保守结构域、系统发育关系和基因表达模式等进行了研究,并利用RNA干扰技术研究了褐飞虱蜕皮和变态信号通路上主要基因的功能,主要研究结果如下:1、本研究基于基因组和转录组数据搜索得到了褐飞虱蜕皮与变态信号途径相关基因,包括蜕皮激素合成基因、蜕皮激素受体基因、受体转运蛋白基因、变态决定因子、保幼激素合成基因、保幼激素早期诱导基因和保幼激素信号通路下游基因。并搜索得到了褐飞虱体内具有20个核受体家族基因(包括蜕皮激素响应基因Nl E75、Nl HR3和Nl FTZ-F1)。2、本研究克隆得到了5个蜕皮激素合成的Halloween基因(Nl Cyp307、Nl Cyp306a1、Nl Cyp302a1、Nl Cyp315a1、Nl Cyp314a1)、蜕皮激素受体基因Nl USP的2个转录本、1个蜕皮激素受体转运蛋白基因Nl Ran、蜕皮激素前期响应基因Nl E75的5个转录本、蜕皮激素中期响应基因Nl HR3的2个转录本、蜕皮激素中后期响应基因Nl FTZ-F1的2个转录本、2个保幼激素合成基因(Nl JHAMT和Nl FAMe T)、2个保幼激素信号通路基因(Nl Broad-C和Nl Kr-h1)、1个变态决定因子Nl E93。分析了各个基因的开放阅读框、预测氨基酸序列、理化特性、跨膜区域、保守结构域和基因结构,初步预测了其功能地位。3、实时荧光定量PCR检测这些基因的时空表达模式结果显示,5个蜕皮激素合成基因在5龄蜕皮后的24h和60小时出现两个表达峰值从而控制蜕皮激素滴度的波动,进而完成对褐飞虱生长变态的调节作用;蜕皮激素受体、Ec R核转运基因、蜕皮激素信号转导基因均在蜕皮期间高表达,协调褐飞虱的若虫-若虫和若虫-成虫的蜕皮过程,是褐飞虱蜕皮和变态的必需条件;Nl E93和Nl Kr-h1在不同时间段呈反向表达动态,这种表达平衡反映它们之间的相互抑制作用并保证了褐飞虱在正确时间点发生变态过程。4、本研究通过RNAi验证了这些基因的生物学功能,结果表明Nl Cyp314a1、Nl USP、Nl Ran、Nl E75、Nl HR3和Nl FTZ-F1对褐飞虱完成蜕皮过程是不可或缺的。分别将这6个基因干扰后,褐飞虱若虫都会出现蜕皮困难的致死表型:旧表皮自胸背板处开裂但并未全部褪去,旧表皮蜕不下来在尾部形成拖尾而出现双层表皮结构,或旧表皮并未开裂但虫体变细长,并最终蜕皮失败死亡。此外我们还发现,Nl Cyp314a1基因干扰后褐飞虱卵母细胞的发育畸形、卵黄原蛋白的产生或填充受阻;Nl Ran基因干扰后褐飞虱卵巢发育畸形,无正常的卵和卵壳形成,无后代孵化;Nl HR3被干扰后也会导致褐飞虱卵巢发育畸形,卵巢中的卵为椭圆形小卵。Nl E93和Nl Kr-h1对褐飞虱完成正常的变态过程具有决定作用。干扰Nl Kr-h1后会有早熟畸形虫(若-成中间体)褐飞虱的出现,而干扰Nl E93基因后会有超级若虫6龄褐飞虱的出现。再结合两者的龄期定量结果,可以发现Nl Kr-h1能抑制变态的发生,而Nl E93的表达是褐飞虱变态起始地决定因子,两者的平衡在褐飞虱变态中起到关键作用并决定褐飞虱在适当的时间点进行变态。同时我们对干扰后各个基因及其相关基因的表达量进行了检测,初步分析了蜕皮激素和保幼激素级联反应中各个基因的作用关系。基于上述研究结果,我们构建了褐飞虱体内蜕皮和变态信号转导通路模式图,包括蜕皮激素的合成、蜕皮激素受体、蜕皮激素响应基因的信号转导的通路以及Nl E93和Nl Kr-h1的互作调控变态的信号转导通路。为进一步了解蜕皮和变态信号转导通路在褐飞虱生长发育中的功能奠定了基础,为在基于RNAi的褐飞虱的防治中筛选合适靶标提供了重要的参考信息。
[Abstract]:The brown planthopper Nilaparvata lugens (St? L) belong to Hemiptera Delphacidae, is an important pest in China and many Asian countries on rice production. The main signal pathway regulation of insect molting and metamorphosis for ecdysone and juvenile hormone signaling pathway, based on the research of genome and transcriptome data, the nucleus of brown planthopper receptor gene, conserved domain of ecdysone and juvenile hormone signaling gene, and analyze the relationship between the gene expression pattern and system development, using RNA interference technology of brown planthopper molting and metamorphosis on the main pathway of gene function, the main results are as follows: 1. The study of genome and transcriptome data search based on the brown planthopper and abnormal molting related signaling pathway genes, including the ecdysone synthesis genes, ecdysone receptor gene, receptor transporter gene, abnormal factors, The gene of juvenile hormone synthesis, juvenile hormone induced gene and early juvenile hormone signaling downstream genes. And the search was obtained with 20 nuclear receptor gene family in BPH (including ecdysone response genes Nl E75, Nl HR3 and Nl FTZ-F1).2, we cloned 5 Halloween genes (ecdysone synthesis Nl Cyp307, Nl Cyp306a1, Nl Cyp302a1, Nl Cyp315a1, Nl Cyp314a1), 2 transcripts of ecdysone receptor gene Nl USP, 1 ecdysone receptor transporter gene Nl Ran, ecdysone response early 5 transcripts of Nl gene E75, ecdysone response mid 2 transcripts of Nl gene HR3, 2 transcription the gene Nl FTZ-F1 in late 2 in response to ecdysone and juvenile hormone biosynthesis genes (Nl JHAMT and Nl FAMe T), 2 juvenile hormone signaling genes (Nl Broad-C and Nl Kr-h1), Nl E93. factor analysis 1 metamorphosis Each gene open reading frame predicted amino acid sequence, physicochemical properties, transmembrane domain, conserved domain and gene structure, preliminary forecast of its functional status of.3, real time fluorescence quantitative PCR detection of spatio-temporal patterns of expression of these genes showed that 5 genes appeared ecdysone synthesis two expression peak fluctuations so as to control the ecdysteroid titer in the 5 instar molting after 24h and 60 hours, and then complete metamorphosis of brown planthopper on the regulating effect of ecdysone receptor, Ec; nuclear translocation of R gene, gene ecdysone signal transduction were highly expressed during the molting molting process, coordination of brown planthopper nymphs and adult nymph - nymphs, is a necessary condition for Brown Planthopper molting and the abnormal Nl E93 and Nl Kr-h1; the inverse dynamic expression at different time, the expression of the balance between them reflects the mutual inhibition and ensure Brown lice at the right time to fly Point.4 metamorphosis, this study tested the biological functions of these genes by RNAi, the results showed that Nl Cyp314a1, Nl USP, Nl Ran, Nl E75, Nl HR3 and Nl FTZ-F1 of brown planthopper in the molting process is indispensable. Which of these 6 genes after interference, BPH will appear if insect molting difficult: the old cuticle from the lethal phenotype mesonotum cracked but did not completely faded, the old cuticle shedding down to form a tail in the tail and double skin structure, or the old cuticle did not crack but the body became slender, and ultimately defeat death. In addition I molt have also found that Nl Cyp314a1 gene interference after BPH oocytes cell developmental abnormalities, or filling up vitellogenin; Nl Ran gene interference after BPH ovarian development deformity, no normal egg and eggshell formation, no offspring hatch; HR3 is Nl after interference will lead to growth of brown planthopper in ovary Deformity in ovarian eggs has decisive effect for.Nl E93 and Nl small oval egg Kr-h1 of brown planthopper complete metamorphosis normal. There will be early after Kr-h1 interference of Nl worm deformity (if - Intermediate) appear brown planthopper, and Nl interference E93 gene after a super 6 instar BPH nymphs. Combined with the results of quantitative age two, it was found that the Nl Kr-h1 can inhibit the expression of Nl E93 and metamorphosis, the brown planthopper determines the onset of metamorphosis factor, balance plays a key role and decided to brown planthopper metamorphosis at the appropriate time in Nilaparvatalugens metamorphosis. At the same time we express the interference after all genes and related genes were detected, interactions between each ecdysone and juvenile hormone cascade genes analyzed. Based on the above results, we constructed in n.lugens molting and metamorphosis signal Pathway pattern, including ecdysteroid synthesis, ecdysone receptor, ecdysone response signal transduction pathway of signal transduction pathway and Nl gene E93 and Nl Kr-h1 interaction. To further understand the abnormal regulation of signal transduction pathway in the molting and metamorphosis of brown planthopper in the growth and development of the function of the foundation, in order to provide an important the reference information to select the appropriate target for treatment of BPH in RNAi based on.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.112.3
，

本文编号：1492096