小菜蛾保幼激素受体基因的分子特性及生殖调控作用研究

发布时间：2018-01-14 16:15

本文关键词：小菜蛾保幼激素受体基因的分子特性及生殖调控作用研究　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：小菜蛾Plutella xyPostella(L.)(鳞翅目菜蛾科Lepidoptera,Plutellidae)是危害十字花科蔬菜的世界性害虫,生殖能力强是其成为全球分布最广的鳞翅目害虫的重要因素之一。保幼激素(juvenile hormone,JH)是参与昆虫生殖调控的重要物质,在昆虫卵子发生中起着至关重要的作用,但对其分子机制的了解仍然有限,尤其是在非模式昆虫中。保幼激素受体(methoprene-tolerant,Met)作为JH的关键受体之一,对JH发挥功能效应起着重要调节作用,但具有明显的物种特异性。本研究围绕生殖调控决定昆虫生殖潜能这一科学问题,以激素调控生殖为核心,采用生物化学与分子生物学、解剖形态学与实验生物学等方法,从JH的作用受体方面开展研究,重点研究:小菜蛾Met基因的分子结构特征与进化关系,小菜蛾Met基因在不同龄期与不同组织的表达模式,以及Met基因在小菜蛾生殖调控中的作用。主要结果如下:PxMet-1与PxMet-2开放阅读框(ORF)全长分别为1575和2100bp,预计编码524和699个氨基酸,理论分子质量为60.5和70.7 kDa,预测等电点为6.73和5.50。PxMet-1和PxMet-2都具有4个保守结构域,分别是Helix-Loop-Helix结构域(bHLH)以及两个PAS保守结构域及一个PAC保守基序。氨基酸序列同源比对分析结构显示,PxMet-1与PxMet-2与鳞翅目昆虫中的家蚕、棉铃虫、冬尺蠖蛾的相似性均大于50%。进化树分析结果显示,小菜蛾PxMet-1,PxMet-2聚为不同的两支,说明PxMet-1,PxMet-可能具有不同的生物学功能以及进化模式,但两者均与鳞翅目昆虫聚在一起,且分别与家蚕的PxMet-1与PxMet-2聚在一起,说明PxMet-与PxMet-2与鳞翅目昆虫Mets可能发挥相同作。PxMet-1与PxMet-2在蛹期(1-3d)与雌成虫期(0-72h)均有表达,PxMet-1在蛹期的表达量无明显差异,但均显著高于雌成虫期(0-48h);而PxMet-2在雌成虫期(0-48 h)的表达量呈先上升后下降的趋势,在羽化后12 h出现表达高峰,且成虫期(0-36h)的表达量显著高于蛹期;PxMet-1与PxMet-2均在雌虫羽化后72h出现一个明显的表达升高,推测可能与小菜蛾生殖与寿命维持的能量权衡相关。PxMet-1与PxMet-2在脂肪体的表达量显著高于其他组织(头、肠道、卵巢、残渣)。利用RNAi技术同时干扰PxMet-1和PxMet-2,结果显示,在注射Met-l+Met-2 dsRNA24 h后,PxMet-1和PxMet-2的表达量均受到显著抑制,但48 h与72 h时,干扰效应明显减弱,处理组与对照组表达量无显著差异。干扰PxMet-1和PxMet-2后,小菜蛾卵巢发育受到明显抑制,卵黄沉积减缓,成熟卵子数目减少,产卵量也随之下降。本研究明确了小菜蛾Met的分子结构特征、表达模式及基因功能,可为初步阐明JH调控小菜蛾生殖过程的分子信号途径奠定基础。在实践上有助于筛选控制小菜蛾的潜在靶标,为有效防治和持续控制小菜蛾种群暴发与危害提供创新思路和途径。
[Abstract]:Diamondback moth Plutella xyPostellaa L. (Lepidoptera, Lepidoptera). Plutellidaeis a worldwide pest that harms cruciferous vegetables. Strong reproductive ability is one of the most widely distributed Lepidoptera pests in the world, and juvenile hormone rejuvenile hormoney (JHH) is an important substance involved in insect reproductive regulation. It plays an important role in insect oogenesis, but its molecular mechanism is still limited. Especially in non-model insects, methoprene-tolerantantone is one of the key receptors of JH. JH plays an important role in regulating the function of JH, but it has obvious species-specificity. This study focuses on the scientific issue of reproductive regulation and determination of insect reproductive potential, with hormone regulation of reproduction as the core. By means of biochemical and molecular biology, anatomical morphology and experimental biology, the study was carried out from the aspect of JH acting receptor, with emphasis on the molecular structural characteristics and evolutionary relationship of the Met gene of Plutella xylostella (Plutella xylostella). Expression patterns of Met gene in different age and tissue of Plutella xylostella. And the role of Met gene in the reproductive regulation of Plutella xylostella. The main results were as follows: 1 575 BP and 2100bp of PxMet-2 open reading frame (ORF). It is predicted to encode 524 and 699 amino acids with theoretical molecular weights of 60.5 and 70.7 kDa. The predicted isoelectric points were 6.73 and 5.50. PxMet-1 and PxMet-2 had four conserved domains. Helix-Loop-Helix domain, two PAS conserved domains and one PAC conserved motif. The similarity between PxMet-1 and PxMet-2 and Bombyx mori, Helicoverpa armigera and Inchworm in Lepidoptera was more than 50. The result of phylogenetic tree analysis showed that PxMet-1 of Plutella xylostella was similar. PxMet-2 was clustered into two different branches, indicating that PxMet-1 PxMet- may have different biological functions and evolutionary models, but both of them were clustered with Lepidoptera insects. And with silkworm PxMet-1 and PxMet-2. It is suggested that PxMet- and Mets of Lepidoptera and PxMet-2 may play the same role. PxMet-1 and PxMet-2 may play the same role in pupa stage (1-3 d) and female adult stage (0-72 h). All expressed. There was no significant difference in the expression of PxMet-1 in pupa stage, but it was significantly higher than that in female adult stage from 0 to 48 hours. However, the expression of PxMet-2 increased first and then decreased at 0-48 h after female adult stage, and reached a peak at 12 h after emergence. And the expression level of the adult stage was significantly higher than that of the pupa stage. Both PxMet-1 and PxMet-2 were significantly increased at 72 hours after emergence. It is speculated that the expression of .PxMet-1 and PxMet-2 in fat body is significantly higher than that in other tissues (head, intestine, ovary), which may be related to the energy tradeoff between reproduction and longevity of Plutella xylostella. Both PxMet-1 and PxMet-2 were interfered with by RNAi technique. The results showed that after Met-l Met-2 dsRNA24 h injection, both PxMet-1 and PxMet-2 were interfered with. The expression of PxMet-1 and PxMet-2 were significantly inhibited, but at 48 and 72 hours, the interference effect was significantly weakened. After interfering with PxMet-1 and PxMet-2, the ovary development of Plutella xylostella was significantly inhibited, the egg yolk deposition slowed down, and the number of mature eggs decreased. The molecular structure, expression pattern and gene function of Plutella xylostella Met were determined. It can lay a foundation for elucidating the molecular signal pathway of JH regulating the reproductive process of Plutella xylostella and is helpful to screen the potential targets for controlling Plutella xylostella in practice. To effectively control and sustainable control of diamondback moth population outbreak and harm to provide innovative ideas and ways.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.4

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