褐飞虱精液蛋白鉴定及离子转运肽基因功能分析

发布时间：2018-01-19 03:03

本文关键词： 褐飞虱精液蛋白 UPLC/MS/MS 蛋白组离子转运肽　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：精液蛋白(SFP)主要由雄性附腺产生并在交配中传递给雌虫,能够引起雌虫交配后的多种生理及行为变化；目前,对半翅目昆虫尚没有进行过精液蛋白组研究。褐飞虱是亚洲一种重要的水稻害虫,并且其雌虫在交配后产卵量增加且长时间拒绝再次交配。建立了雄性生殖系统转录组、雄性附腺表达谱,使用UPLC/MS/MS蛋白组对褐飞虱精液蛋白组成分进行了研究。通过蛋白组分析,鉴定得到94个可信度高的SFP,其中84个蛋白含有分泌信号肽。将褐飞虱的SFP同其他昆虫的精液蛋白组进行比较,发现褐飞虱精液胰蛋白酶及羧酸酯酶发生了明显扩张。褐飞虱精液胰蛋白酶的数量(n=20)是其他昆虫中发现的胰蛋白酶数量的两倍或以上。褐飞虱精液中发现了7个羧酸酯酶,而羧酸酯酶在其他昆虫精液中很少被发现。另外发现了新的昆虫SFP,包括：中脑星细胞来源的神经营养因子mesencephalic astrocyte-derived neurotrophic factor(MANF)、硒蛋白selenoprotein.含有EGF结构域蛋白以及一种神经肽离子转运肽的类似物ion transport peptide-like(ITPL).本研究是第一个报道的半翅目昆虫精液蛋白组,为今后褐飞虱精液蛋白的功能研究奠定了基础,并且为昆虫精液蛋白组提供了新知。对16个褐飞虱精液胰蛋白酶的RNAi研究,发现这16个精液胰蛋白酶并未参与雄虫对雌虫产卵的刺激。结合果蝇sex peptide通路中关键基因在褐飞虱精液蛋白中的缺失,暗示了褐飞虱存在与果蝇不同的交配后生理及行为改变的分子机制。神经肽离子转运肽ion transport peptide(ITP)及其可变剪切体ITPL在昆虫中非常保守并在昆虫发育中起到重要作用。在转录组中鉴定了褐飞虱ITP/ITPL的转录本,使用RT-qPCR研究了转录本的表达模式并采用RNAi研究了其功能。在褐飞虱精液中发现了ITPL后,对褐飞虱ITP及其可变剪切本ITPL进行了深入研究。首次发现褐飞虱ITPL(NLITPL)含有4种可变5’非翻译区(5’UTR),并分别命名为NLITPL-1、NL1TPL-2、NLITPL-3及NLITPL-4。通过RT-qPCR,发现NLITPL通过可变5’UTR控制其时空特异性表达。首先,NLITPL-1在雄性生殖系统中专性表达,并控制精液ITPL的合成；其次,首次报道了NLITPL在体壁中的专性表达(NLITPL-2及NLITPL-3),暗示了NLITPL在蜕皮中特化的功能。通过RNA干扰研究了NLITP/NLITPL的功能。当NLITPL的几个转录本被干扰后,没有观察到明显的表型。然而,当NLITP的转录本被干扰后,长翅成虫在羽化后无法伸展翅膀——证实ITP是鞣化激素bursicon之外另外一种控制昆虫翅膀伸展的神经肽。另外,同鞣化激素RNAi的效果相反,NLITP的RNAi加强了褐飞虱表皮的黑化现象。在褐飞虱中,NLITP并不影响羽化的成功,而对羽化后生理现象非常重要,这与在其他物种中ITP/ITPL的研究结果不同。ITP/ITPL在昆虫中的功能可能比之前认为的更加复杂。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.112.3

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