本文關(guān)鍵詞： 褐飛虱 蛻皮激素合成 核受體 蛻皮激素信號(hào)轉(zhuǎn)運(yùn)通路 變態(tài)決定因子 表達(dá)模式 RNA干擾　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：褐飛虱Nilaparvata lugens(St?l)屬半翅目飛虱科,是我國(guó)和許多亞洲國(guó)家水稻生產(chǎn)上的重要害蟲(chóng)。調(diào)控昆蟲(chóng)蛻皮和變態(tài)的主要信號(hào)通路為蛻皮激素信號(hào)通路和保幼激素信號(hào)通路,本研究在基因組和轉(zhuǎn)錄組數(shù)據(jù)的基礎(chǔ)上,對(duì)褐飛虱核受體基因、蛻皮激素和保幼激素信號(hào)通路基因的保守結(jié)構(gòu)域、系統(tǒng)發(fā)育關(guān)系和基因表達(dá)模式等進(jìn)行了研究,并利用RNA干擾技術(shù)研究了褐飛虱蛻皮和變態(tài)信號(hào)通路上主要基因的功能,主要研究結(jié)果如下:1、本研究基于基因組和轉(zhuǎn)錄組數(shù)據(jù)搜索得到了褐飛虱蛻皮與變態(tài)信號(hào)途徑相關(guān)基因,包括蛻皮激素合成基因、蛻皮激素受體基因、受體轉(zhuǎn)運(yùn)蛋白基因、變態(tài)決定因子、保幼激素合成基因、保幼激素早期誘導(dǎo)基因和保幼激素信號(hào)通路下游基因。并搜索得到了褐飛虱體內(nèi)具有20個(gè)核受體家族基因(包括蛻皮激素響應(yīng)基因Nl E75、Nl HR3和Nl FTZ-F1)。2、本研究克隆得到了5個(gè)蛻皮激素合成的Halloween基因(Nl Cyp307、Nl Cyp306a1、Nl Cyp302a1、Nl Cyp315a1、Nl Cyp314a1)、蛻皮激素受體基因Nl USP的2個(gè)轉(zhuǎn)錄本、1個(gè)蛻皮激素受體轉(zhuǎn)運(yùn)蛋白基因Nl Ran、蛻皮激素前期響應(yīng)基因Nl E75的5個(gè)轉(zhuǎn)錄本、蛻皮激素中期響應(yīng)基因Nl HR3的2個(gè)轉(zhuǎn)錄本、蛻皮激素中后期響應(yīng)基因Nl FTZ-F1的2個(gè)轉(zhuǎn)錄本、2個(gè)保幼激素合成基因(Nl JHAMT和Nl FAMe T)、2個(gè)保幼激素信號(hào)通路基因(Nl Broad-C和Nl Kr-h1)、1個(gè)變態(tài)決定因子Nl E93。分析了各個(gè)基因的開(kāi)放閱讀框、預(yù)測(cè)氨基酸序列、理化特性、跨膜區(qū)域、保守結(jié)構(gòu)域和基因結(jié)構(gòu),初步預(yù)測(cè)了其功能地位。3、實(shí)時(shí)熒光定量PCR檢測(cè)這些基因的時(shí)空表達(dá)模式結(jié)果顯示,5個(gè)蛻皮激素合成基因在5齡蛻皮后的24h和60小時(shí)出現(xiàn)兩個(gè)表達(dá)峰值從而控制蛻皮激素滴度的波動(dòng),進(jìn)而完成對(duì)褐飛虱生長(zhǎng)變態(tài)的調(diào)節(jié)作用;蛻皮激素受體、Ec R核轉(zhuǎn)運(yùn)基因、蛻皮激素信號(hào)轉(zhuǎn)導(dǎo)基因均在蛻皮期間高表達(dá),協(xié)調(diào)褐飛虱的若蟲(chóng)-若蟲(chóng)和若蟲(chóng)-成蟲(chóng)的蛻皮過(guò)程,是褐飛虱蛻皮和變態(tài)的必需條件;Nl E93和Nl Kr-h1在不同時(shí)間段呈反向表達(dá)動(dòng)態(tài),這種表達(dá)平衡反映它們之間的相互抑制作用并保證了褐飛虱在正確時(shí)間點(diǎn)發(fā)生變態(tài)過(guò)程。4、本研究通過(guò)RNAi驗(yàn)證了這些基因的生物學(xué)功能,結(jié)果表明Nl Cyp314a1、Nl USP、Nl Ran、Nl E75、Nl HR3和Nl FTZ-F1對(duì)褐飛虱完成蛻皮過(guò)程是不可或缺的。分別將這6個(gè)基因干擾后,褐飛虱若蟲(chóng)都會(huì)出現(xiàn)蛻皮困難的致死表型:舊表皮自胸背板處開(kāi)裂但并未全部褪去,舊表皮蛻不下來(lái)在尾部形成拖尾而出現(xiàn)雙層表皮結(jié)構(gòu),或舊表皮并未開(kāi)裂但蟲(chóng)體變細(xì)長(zhǎng),并最終蛻皮失敗死亡。此外我們還發(fā)現(xiàn),Nl Cyp314a1基因干擾后褐飛虱卵母細(xì)胞的發(fā)育畸形、卵黃原蛋白的產(chǎn)生或填充受阻;Nl Ran基因干擾后褐飛虱卵巢發(fā)育畸形,無(wú)正常的卵和卵殼形成,無(wú)后代孵化;Nl HR3被干擾后也會(huì)導(dǎo)致褐飛虱卵巢發(fā)育畸形,卵巢中的卵為橢圓形小卵。Nl E93和Nl Kr-h1對(duì)褐飛虱完成正常的變態(tài)過(guò)程具有決定作用。干擾Nl Kr-h1后會(huì)有早熟畸形蟲(chóng)(若-成中間體)褐飛虱的出現(xiàn),而干擾Nl E93基因后會(huì)有超級(jí)若蟲(chóng)6齡褐飛虱的出現(xiàn)。再結(jié)合兩者的齡期定量結(jié)果,可以發(fā)現(xiàn)Nl Kr-h1能抑制變態(tài)的發(fā)生,而Nl E93的表達(dá)是褐飛虱變態(tài)起始地決定因子,兩者的平衡在褐飛虱變態(tài)中起到關(guān)鍵作用并決定褐飛虱在適當(dāng)?shù)臅r(shí)間點(diǎn)進(jìn)行變態(tài)。同時(shí)我們對(duì)干擾后各個(gè)基因及其相關(guān)基因的表達(dá)量進(jìn)行了檢測(cè),初步分析了蛻皮激素和保幼激素級(jí)聯(lián)反應(yīng)中各個(gè)基因的作用關(guān)系�；谏鲜鲅芯拷Y(jié)果,我們構(gòu)建了褐飛虱體內(nèi)蛻皮和變態(tài)信號(hào)轉(zhuǎn)導(dǎo)通路模式圖,包括蛻皮激素的合成、蛻皮激素受體、蛻皮激素響應(yīng)基因的信號(hào)轉(zhuǎn)導(dǎo)的通路以及Nl E93和Nl Kr-h1的互作調(diào)控變態(tài)的信號(hào)轉(zhuǎn)導(dǎo)通路。為進(jìn)一步了解蛻皮和變態(tài)信號(hào)轉(zhuǎn)導(dǎo)通路在褐飛虱生長(zhǎng)發(fā)育中的功能奠定了基礎(chǔ),為在基于RNAi的褐飛虱的防治中篩選合適靶標(biāo)提供了重要的參考信息。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S435.112.3
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本文編號(hào)：1492096