蓖麻蚕和柞蚕烯醇化酶基因与草地螟线粒体基因组

发布时间：2018-03-20 17:24

本文选题：蓖麻蚕　切入点：柞蚕　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：烯醇化酶是一种多功能蛋白,在生理病理中有重要作用。目前,对烯醇化酶的了解主要来源于人和病原微生物方面的研究工作,而来自昆虫方面的研究工作极少。我们实验室最先开展了昆虫烯醇化酶的鉴定工作。从柞蚕中分离出2个烯醇化酶基因,分别命名为烯醇化酶Ⅰ和烯醇化酶Ⅱ,并发现烯醇化酶Ⅱ很可能是鳞翅目昆虫特异的,且仅在生殖腺中表达,而烯醇化酶Ⅰ则广泛存在于各种昆虫中。为了探讨烯醇化酶的起源进化,仍需要在更多物种中调查烯醇化酶Ⅱ的序列特征和组织表达特征,同时需要对烯醇化酶Ⅰ和Ⅱ的酶学特性进行鉴定和比较。本论文中,我们以蓖麻蚕为研究材料,比较了烯醇化酶Ⅰ和Ⅱ的序列特征、发育表达特征;以柞蚕为研究材料,利用qRT-PCR技术进一步调查了烯醇化酶Ⅰ和Ⅱ的组织表达特征,进行了原核表达和抗体制备。昆虫线粒体基因组测序可以为了解线粒体基因组结构、构建物种系统发育关系等提供基础数据。本论文中,我们对鳞翅目重要害虫之一的草地螟线粒体基因组做了生物信息学分析,并利用线粒体基因组全序列重建了 24种螟蛾总科(来源于2个家族10个亚家族)昆虫的系统进化关系。本论文取得的研究结果如下:1.蓖麻蚕烯醇化酶Ⅰ和Ⅱ分别编码433和431个氨基酸。蓖麻蚕烯醇化酶Ⅰ与Ⅱ的序列一致性仅为60%,而蓖麻蚕烯醇化酶Ⅰ与已经鉴定的鳞翅目昆虫烯醇化酶Ⅰ在蛋白水平上的序列一致性为91%～97%,蓖麻蚕烯醇化酶Ⅱ与已经鉴定的鳞翅目烯醇化酶Ⅱ的序列一致性为84%～92%。进化分析表明,昆虫烯醇化酶Ⅱ和Ⅰ是独立进化的。qRT-PCR检测结果表明,在胚胎期,烯醇化酶Ⅱ属于痕量表达,而烯醇化酶Ⅰ的表达水平很高;蛹期精巢中烯醇化酶Ⅰ的表达量较低,而烯醇化酶Ⅱ的表达量远高于Ⅰ。这一结果说明烯醇化酶Ⅱ与生殖发育有关。2.在柞蚕胚胎发育期和幼虫期,烯醇化酶Ⅰ的表达量均高于烯醇化酶Ⅱ;在蛹期,烯醇化酶Ⅰ的表达量也微高于烯醇化酶Ⅱ。同时,我们成功构建了重组表达质粒pET28b(+)-ApenoⅠ和Ⅱ,并获得了以可溶性或包涵体形式表达的重组蛋白rApenoⅠ和rAenoⅡ。Western blot检测结果表明,rApeno Ⅰ和rApenoⅡ能正确表达。利用重组蛋白经4次免疫家兔后,获得了高滴度的抗体。免疫学试验结果表明,ApenoⅠ和Apeno Ⅱ均在柞蚕的血液、生殖腺及脂肪体中表达,进一步试验发现所制备的两个抗体存在严重的交叉反应。柞蚕烯醇化酶Ⅰ和Ⅱ原核表达的成功为下一步开展酶学特性的研究奠定了基础。3.草地螟线粒体基因组中AT含量及偏好性与螟蛾总科的线粒体基因组基本相似。基因间隔区虽然分布于15个区域,但只有位于tRNAGln ND2之间和tRNASer(UCN)和ND1之间的2个区域的基因序列在螟蛾总科均存在。鉴定了草地螟A+T富集区的3个保守序列。基于线粒体基因组全序列的系统进化分析结果支持螟蛾总科分为螟蛾科和草螟科两个家族;进一步将草螟科分为两个进化枝,PS进化枝和非PS进化枝,PS进化枝包括野螟亚科Pyraustinae和斑野螟亚科Spilomelinae,非PS进化枝包括草螟亚科Crambinae、水螟亚科 Acentropinae、苔螟亚科 Scopariinae、禾螟亚科 Schoenobiinae。这是首次利用线粒体基因组全序列探讨螟蛾总科的系统发育关系。
[Abstract]:Enolase is a multifunctional protein that plays an important role in the physiology and pathology. At present, understanding of enolase mainly originates from the research work and the pathogenic microorganism, and the research work from the aspects of insects. We carried out the first few laboratory identification of insect enolization enzyme. The separation of 2 enolase gene from silkworm, named as enolase I and II enolase, and found that the enolase II is likely Lepidoptera specific, and only expressed in the gonad, and enolase 1 is widespread in a variety of insects. In order to investigate the origin and evolution of enolase the enzyme, the expression characteristics of sequence and organization still need to investigate enolase II in more species, and the need for the enol characterization of enzyme I and II were identified and compared. In this thesis, we study on castor silkworm material ratio The sequence characteristics of enolase I and II is developmental expression characteristics; tussah as research materials, further investigation of enolase I and II expression characteristics using qRT-PCR technology, the prokaryotic expression and antibody preparation. Insect mitochondrial genomes can be sequenced in order to understand the structure of the mitochondrial genome, to construct phylogenetic species the relationship can provide the basic data. In this thesis, we on the one of the important pests of Lepidoptera of the meadow moth mitochondrial genome were analyzed using bioinformatics, and mitochondrial genome sequence reconstruction 24 pyraloidea (from 2 families, 10 subfamilies) phylogenetic relationships of insects. The research results achieved are as follows: 1. castor silkworm enolase I and II respectively encoding 433 and 431 amino acids. Sequence eri-silkworm enolase I and II consistency is only 60%, while the enolization enzyme of castor silkworm And has been the identification of lepidopteran enolase 1 at the protein level of sequence identity is 91% ~ 97%, sequence consistency eri-silkworm enolase II and had identified the lepidopteran enolase II is 84% ~ 92%. phylogenetic analysis showed that insect enolase II and I is.QRT-PCR independent test results evolution showed that in the embryo, enolase II belongs to trace expression, while the expression levels of enolase 1 high expression in testis; enolase of amount of pupae was low, whereas the expression of enolase II is much higher than 1. This result indicated that enolase of reproduction and development the.2. in the larval period and silkworm embryonic development, the expression of enolase 1 were higher than that of enolase II; in the pupal stage, the expression of enolase 1 is slightly higher than the enolase II. At the same time, we successfully constructed the recombinant expression plasmid pET28b (+) -Apeno I and II, and The recombinant protein rApeno I and rAeno II.Western blot results to soluble or inclusion bodies expressed that rApeno I and rApeno II can be expressed correctly. Using the recombinant protein after 4 times of immunization of rabbits after obtained high titer antibody. Immunological test results show that the Apeno I and Apeno II in Tussah blood the expression, gonad and fat body, further test the cross reaction exists two antibody preparation. Antheraea pernyi enolase I and II successfully prokaryotic expression to study the enzymatic properties of the next step to lay the foundation of the mitochondrial genome.3. loxostegesticticalis mitochondrial genome AT content and preference and family families are basically similar. Although the intergenic region distributed in 15 regions, but only in the tRNAGln between ND2 and tRNASer (UCN) and 2 gene sequences of ND1 region between the pyraloidea are kept in In. 3 conserved sequences of meadow moth A+T rich regions were identified. The complete sequence of the mitochondrial genome phylogenetic analysis supports pyraloidea divided into Pyralidae and Crambidae two based on family Crambidae; will be further divided into two clades, PS clade and non PS clade, PS in branch including pyraustinae Pyraustinae and spilomelinae Spilomelinae, non PS clade including crambinae Crambinae, nymphulinae Acentropinae, moss moth subfamily Scopariinae, schoenobiinae Schoenobiinae. this is the first use of the mitochondrial genome system pyraloidea full phylogenetic relationship.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.4;Q966

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