本文關(guān)鍵詞： 褐飛虱 精液蛋白 UPLC/MS/MS 蛋白組 離子轉(zhuǎn)運肽　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：精液蛋白(SFP)主要由雄性附腺產(chǎn)生并在交配中傳遞給雌蟲,能夠引起雌蟲交配后的多種生理及行為變化；目前,對半翅目昆蟲尚沒有進行過精液蛋白組研究。褐飛虱是亞洲一種重要的水稻害蟲,并且其雌蟲在交配后產(chǎn)卵量增加且長時間拒絕再次交配。建立了雄性生殖系統(tǒng)轉(zhuǎn)錄組、雄性附腺表達譜,使用UPLC/MS/MS蛋白組對褐飛虱精液蛋白組成分進行了研究。通過蛋白組分析,鑒定得到94個可信度高的SFP,其中84個蛋白含有分泌信號肽。將褐飛虱的SFP同其他昆蟲的精液蛋白組進行比較,發(fā)現(xiàn)褐飛虱精液胰蛋白酶及羧酸酯酶發(fā)生了明顯擴張。褐飛虱精液胰蛋白酶的數(shù)量(n=20)是其他昆蟲中發(fā)現(xiàn)的胰蛋白酶數(shù)量的兩倍或以上。褐飛虱精液中發(fā)現(xiàn)了7個羧酸酯酶,而羧酸酯酶在其他昆蟲精液中很少被發(fā)現(xiàn)。另外發(fā)現(xiàn)了新的昆蟲SFP,包括：中腦星細胞來源的神經(jīng)營養(yǎng)因子mesencephalic astrocyte-derived neurotrophic factor(MANF)、硒蛋白selenoprotein.含有EGF結(jié)構(gòu)域蛋白以及一種神經(jīng)肽離子轉(zhuǎn)運肽的類似物ion transport peptide-like(ITPL).本研究是第一個報道的半翅目昆蟲精液蛋白組,為今后褐飛虱精液蛋白的功能研究奠定了基礎(chǔ),并且為昆蟲精液蛋白組提供了新知。對16個褐飛虱精液胰蛋白酶的RNAi研究,發(fā)現(xiàn)這16個精液胰蛋白酶并未參與雄蟲對雌蟲產(chǎn)卵的刺激。結(jié)合果蠅sex peptide通路中關(guān)鍵基因在褐飛虱精液蛋白中的缺失,暗示了褐飛虱存在與果蠅不同的交配后生理及行為改變的分子機制。神經(jīng)肽離子轉(zhuǎn)運肽ion transport peptide(ITP)及其可變剪切體ITPL在昆蟲中非常保守并在昆蟲發(fā)育中起到重要作用。在轉(zhuǎn)錄組中鑒定了褐飛虱ITP/ITPL的轉(zhuǎn)錄本,使用RT-qPCR研究了轉(zhuǎn)錄本的表達模式并采用RNAi研究了其功能。在褐飛虱精液中發(fā)現(xiàn)了ITPL后,對褐飛虱ITP及其可變剪切本ITPL進行了深入研究。首次發(fā)現(xiàn)褐飛虱ITPL(NLITPL)含有4種可變5’非翻譯區(qū)(5’UTR),并分別命名為NLITPL-1、NL1TPL-2、NLITPL-3及NLITPL-4。通過RT-qPCR,發(fā)現(xiàn)NLITPL通過可變5’UTR控制其時空特異性表達。首先,NLITPL-1在雄性生殖系統(tǒng)中專性表達,并控制精液ITPL的合成；其次,首次報道了NLITPL在體壁中的專性表達(NLITPL-2及NLITPL-3),暗示了NLITPL在蛻皮中特化的功能。通過RNA干擾研究了NLITP/NLITPL的功能。當(dāng)NLITPL的幾個轉(zhuǎn)錄本被干擾后,沒有觀察到明顯的表型。然而,當(dāng)NLITP的轉(zhuǎn)錄本被干擾后,長翅成蟲在羽化后無法伸展翅膀——證實ITP是鞣化激素bursicon之外另外一種控制昆蟲翅膀伸展的神經(jīng)肽。另外,同鞣化激素RNAi的效果相反,NLITP的RNAi加強了褐飛虱表皮的黑化現(xiàn)象。在褐飛虱中,NLITP并不影響羽化的成功,而對羽化后生理現(xiàn)象非常重要,這與在其他物種中ITP/ITPL的研究結(jié)果不同。ITP/ITPL在昆蟲中的功能可能比之前認(rèn)為的更加復(fù)雜。
[Abstract]:Semen protein SFPs are mainly produced by male accessory glands and transmitted to females during mating, which can cause various physiological and behavioral changes after mating. At present, seminal proteome has not been studied on Hemiptera insects. Brown planthopper is an important rice pest in Asia. The female spawning increased after mating and refused to mate again for a long time. The male reproductive system transcriptional group and the male accessory gland expression profile were established. The UPLC/MS/MS protein group was used to study the protein composition of brown planthopper semen. 94 SFP with high reliability were identified by proteome analysis. Eighty-four of them contained secretory signal peptides. The SFP of brown planthopper was compared with the semen protein group of other insects. It was found that the semen trypsin and carboxylesterase of brown planthopper expanded obviously. The quantity of trypsin in semen of brown planthopper. This is more than twice the amount of trypsin found in other insects. Seven carboxylic esterases were found in brown planthopper semen. Carboxylesterase was rarely found in other insect semen and a new insect SFP was found. Including: mesencephalic astrocyte-derived neurotrophic factor, a neurotrophic factor derived from midbrain stellate cells. MANF. Selenoprotein. Ion transport peptide-like containing EGF domain protein and a neuropeptide ion transporter peptide. The present study is the first reported seminal protein group of Hemiptera insects. It provides a basis for the study of protein function of brown planthopper semen, and provides a new knowledge for the protein group of insect semen. The RNAi study on trypsin in semen of 16 brown planthopper was carried out. It was found that these 16 semen trypsin were not involved in the male stimulation of female oviposition. The deletion of the key gene in the sex peptide pathway in brown planthopper semen protein was found. The results suggest that there is a molecular mechanism of physiological and behavioral changes in brown planthopper (ion transport peptidea) after mating with Drosophila melanogaster. And its variable shearing ITPL is very conserved in insects and plays an important role in insect development. The ITP/ITPL transcripts of brown planthopper have been identified in the transcriptome. RT-qPCR was used to study the expression pattern of transcripts and RNAi was used to study its function. ITPL was found in the semen of brown planthopper. In this paper, ITP and its variable shear ITPL were studied. It was found for the first time that ITP contained four variable 5 'untranslated region (5' UTRs). They were named NLITPL-1NL1TPL-2NLITPL-3 and NLITPL-4respectively. It was found that NLITPL controlled its space-time specific expression by variable 5UTR. Firstly, NLITPL-1 was specifically expressed in male reproductive system, and the synthesis of ITPL in semen was controlled. Secondly, the specific expression of NLITPL in the body wall, NLITPL-2 and NLITPL-3, was reported for the first time. It is suggested that NLITPL is specialized in molting. The function of NLITP/NLITPL is studied by RNA interference. When several transcripts of NLITPL are interfered with. No obvious phenotype was observed. However, when the transcripts of NLITP were interfered with. Adults cannot stretch their wings after emergence-proving that ITP is a neuropeptide that controls the spread of insect wings in addition to the tanning hormone bursicon. In contrast to the effect of tanning hormone RNAi, the RNAi of NLITP enhanced the melanization of brown planthopper epidermis, but in the brown planthopper, NLITP did not affect the success of Eclosion. But it is very important for post-emergence physiological phenomena, which is different from the results of ITP/ITPL in other species. The function of ITP / ITPL in insects may be more complex than previously thought.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.112.3

【參考文獻】

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