花绒寄甲线粒体基因组与合成调控基因及其COXI基因的种群遗传结构研究
发布时间：2018-08-01 09:02
【摘要】：花绒寄甲(Dastarcus helophoroides)属鞘翅目寄甲科(Coleoptera:Bothrideridae),是各类天牛的重要天敌。线粒体是细胞及生命体活动所需能量的主要来源,其拥有分子量相对较小、突变率高、呈母系遗传及在细胞中拷贝数量多等众多特点,常被用于谱系地理学、种群遗传学、种系发生、分子进化和比较进化基因组学等方面的研究。线粒体编码基因不能独立完成氧化磷酸化产生ATP的过程,细胞核通过各种转录因子和辅酶因子对线粒体基因进行调控。本文通过长片段PCR-步移测序法研究了花绒寄甲线粒体基因组全序列,得到了其序列全长并对其所包含的分子生物学信息进行了详细分析;在线粒体基因组全序列的基础上,研究基于COXI基因的不同种群花绒寄甲的种群遗传结构和遗传差异;利用花绒寄甲转录组数据,对调控线粒体生物合成的AMPK基因和PGC-1α信号通路基因进行了筛选、鉴定和实时定量表达分析。分析结果如下:1)花绒寄甲线粒体基因组全长序列为15878bp(GenBank:KF811054.1),由13个蛋白质编码基因(PCGs)、22个tRNA基因、2个rRNA基因和非编码区(D-loop环)组成的。除了COI基因拥有非规范的起始密码子,其他PCGs均以ATN密码子起始并以TA(A)或TAG密码子终止。rrnL和rrnS的二级结构分别由48个螺旋(包含四个新提出的螺旋)和35个螺旋(包含两个新提出的螺旋)构成。除trnS1(AGN)因缺少二氢尿嘧啶臂(DHU臂)不能形成稳定的茎-环结构外,其余21个tRNAs均能折叠成典型的三叶草型二级结构。运用蛋白质编码基因的核苷酸和氨基酸数据集分别分析物种间的系统发育关系结果表明,花绒寄甲属于鞘翅目扁甲总科的寄甲科,与形态学分类结果一致。为探索种群遗传结构、种系鉴定以及花绒寄甲的进化和种系发生提供有效的分子生物学基础信息。2)通过COXI基因研究不同寄主花绒寄甲种群的遗传学关系,共得到具有51个核苷酸多态性位点的26个单倍型,种群间遗传多样性指数较低,遗传变异主要发生在种群内部。种群间存在较低的遗传分化系数和较高的基因流动,表明种群间的基因交换明显。共有单倍型DH01是祖先的单倍型,其他单倍型通过不同的突变从其进化而来。在五个种群之间,特别是在群体AG、MA和MR之间基因交换程度较高,表明在野外这些种群之间可能存在交叉混合。为花绒寄甲遗传差异提供了理论基础,同时也为有效利用天敌进行生物学防治提供了分子生物学佐证。3)通过花绒寄甲转录组文库筛选和RACE扩增,得到3条花绒寄甲AMPK基因的cDNA序列全长。系统发育分析表明,花绒寄甲AMPK亚基与其他昆虫相应序列的同源性较高,尤其与鞘翅目的赤拟谷盗的同源性达到85%以上。通过实时定量qPCR,发现在不同处理条件下均检测到AMPK基因的表达,且表达水平存在显著的差异性。3条AMPK基因均表现为在雄性生殖系统和脂肪体中表达相对较高;在一龄幼虫和蛹中的表达量较高;随着花绒寄甲存活时间的延长,各基因的表达有明显的上升趋势,可能与其应对环境变化和缓解衰老有关;在不同胁迫条件下,各基因在雌成虫中的表达量均高于雄成虫,随着处理时间的增加表现出先上升后降低的趋势,显示AMPK基因在昆虫变态发育、寿命延伸和应激过程中发挥重要作用。4)从花绒寄甲转录组文库和线粒体基因组中筛选出PGC1-α、NRF1、mtTFA、COXI和ATP6基因,5条基因的氨基酸序列均表现出与其他物种相应氨基酸序列的同源性较高。实时荧光定量qPCR结果表明,5个线粒体合成基因的表达存在显著的组织特异性和虫态特异性:在花绒寄甲雄性生殖系统和脂肪体中的表达量明显高于其他组织;在1龄幼虫中的表达量高于其他幼虫期,在蛹中的表达量最小;花绒寄甲成虫羽化20个月后,各基因的表达量随着虫龄的增加呈上升趋势,在30个月达到最大值。核基因编码的PGC1-α、NRF1和mtTFA基因表达量高的组织、虫态和虫龄中,线粒体编码基因COXI和ATP6的表达量也相对较高,为进一步研究花绒寄甲在较长寿命中的能量合成奠定了理论基础。
[Abstract]:Dastarcus helophoroides is an important natural enemy of various species of Coleoptera (Coleoptera:Bothrideridae). Mitochondria are the main source of energy for cell and life activities. It has a relatively small molecular weight, a high mutation rate, a large number of copies of the mother family and a large number of copies in cells. Genealogeography, population genetics, phylogeny, molecular evolution and comparative evolutionary genomics. Mitochondrial coding genes can not independently complete the process of oxidative phosphorylation to produce ATP, and the nucleus through a variety of transcription factors and coenzyme factors to regulate the mitochondrial gene. In this paper, a long fragment of PCR- step sequencing method was used. The complete sequence of mitochondrial genome of cashmere armour was studied, and its sequence length was obtained and the molecular biological information contained in it was analyzed in detail. On the basis of the complete mitochondrial genome sequence, the population genetic structure and genetic difference of different species of cashmere mailing based on COXI gene were studied. The AMPK and PGC-1 alpha signaling genes regulating mitochondrial biosynthesis were screened, identified and quantified in real time. The results were as follows: 1) the full-length sequence of mitochondrial genome of cashmere mailing was 15878bp (GenBank:KF811054.1), and 13 proteins encoded the gene (PCGs), 22 tRNA genes, 2 rRNA genes and non coding regions (D-). Loop rings. In addition to the COI gene having an irregular starting codon, the other PCGs initiates with the ATN codon and terminates.RrnL and rrnS with TA (A) or TAG codon with 48 helices (including four newly proposed helices) and 35 helices (including two newly proposed helix). Except trnS1 (AGN), there is a lack of two hydrogen urine. The DHU arm (DHU arm) can not form a stable stem and ring structure, and the rest of the other tRNAs can be folded into a typical trifoliate type two structure. The phylogenetic relationship between the nucleotide and amino acid data sets of the protein encoding genes shows that the genus cashmere is in the mailing family of the family Coleoptera and the morphological credit. In order to explore the genetic structure of the population, the identification of the species and the evolution and phylogeny of the cashmere mailing, the genetic information of the molecular biology.2) was provided by the COXI gene to study the genetic relationship of the different host cashmere populations, and a total of 26 haplotypes with 51 nucleotide polymorphisms were obtained, and the genetic diversity among the populations was found. The sex index is low, and the genetic variation mainly occurs within the population. There is a low genetic differentiation coefficient and high gene flow among the population, which indicates that the gene exchange among the population is obvious. The common haplotype DH01 is the haplotype of the ancestor, the other haplotypes evolve from the different mutations. Among the five populations, especially in the group AG, The high degree of gene exchange between MA and MR indicates that there may be a cross mixing between these populations in the field. It provides a theoretical basis for the genetic difference of cashmere mailing, and also provides a molecular biological proof of biological control for the effective use of natural enemies for biological control. 3 floral fluxes are obtained through the screening of the cashmere transcriptional library and the RACE amplification. The cDNA sequence of the AMPK gene was full. Phylogenetic analysis showed that the homology of the cashmere AMPK subunit was higher than that of other insects, especially more than 85% of the homology of the Coleoptera. The expression of the AMPK gene was detected under different processing conditions, and the expression level of the AMPK gene was detected under different conditions. The differential expression of.3 AMPK gene expressed relatively high expression in the male reproductive system and the fat body; the expression in the first instar larvae and pupae is higher; with the prolongation of the survival time of the cashmere mailing, the expression of each gene has an obvious upward trend, which may be related to its response to environmental changes and the mitigation of senility; under different stress conditions, the expression of the gene is higher. The expression of each gene in the female adult is higher than that of the male adult. With the increase of the processing time, it shows a trend of rising and decreasing. It shows that the AMPK gene plays an important role in insect metamorphosis, life extension and stress..4, PGC1- alpha, NRF1, mtTFA, COXI and A are screened from the cashmere transcriptome library and the grain genome. The TP6 gene, the amino acid sequence of the 5 genes showed high homology with the corresponding amino acid sequences of other species. Real time fluorescence quantitative qPCR results showed that the expression of the 5 mitochondrial synthetic genes had significant tissue specificity and insect specificity: the expression in the cashmere male colonization system and the fat body was significantly higher than that of them. The expression amount in the 1 instar larvae is higher than that in the other larval stages, and the expression in the pupa is the smallest. After 20 months, the expression of each gene increases with the increase of the age of the insect, and reaches the maximum in 30 months. The PGC1- a, NRF1 and mtTFA genes, which are encoded by the nuclear gene, are high in the tissues, the insect state and the age of the worm. The expression levels of COXI and ATP6 genes were also relatively high, which laid a theoretical foundation for the further study of energy synthesis in the longer life span.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.306.4
，
本文编号：2157070
[Abstract]:Dastarcus helophoroides is an important natural enemy of various species of Coleoptera (Coleoptera:Bothrideridae). Mitochondria are the main source of energy for cell and life activities. It has a relatively small molecular weight, a high mutation rate, a large number of copies of the mother family and a large number of copies in cells. Genealogeography, population genetics, phylogeny, molecular evolution and comparative evolutionary genomics. Mitochondrial coding genes can not independently complete the process of oxidative phosphorylation to produce ATP, and the nucleus through a variety of transcription factors and coenzyme factors to regulate the mitochondrial gene. In this paper, a long fragment of PCR- step sequencing method was used. The complete sequence of mitochondrial genome of cashmere armour was studied, and its sequence length was obtained and the molecular biological information contained in it was analyzed in detail. On the basis of the complete mitochondrial genome sequence, the population genetic structure and genetic difference of different species of cashmere mailing based on COXI gene were studied. The AMPK and PGC-1 alpha signaling genes regulating mitochondrial biosynthesis were screened, identified and quantified in real time. The results were as follows: 1) the full-length sequence of mitochondrial genome of cashmere mailing was 15878bp (GenBank:KF811054.1), and 13 proteins encoded the gene (PCGs), 22 tRNA genes, 2 rRNA genes and non coding regions (D-). Loop rings. In addition to the COI gene having an irregular starting codon, the other PCGs initiates with the ATN codon and terminates.RrnL and rrnS with TA (A) or TAG codon with 48 helices (including four newly proposed helices) and 35 helices (including two newly proposed helix). Except trnS1 (AGN), there is a lack of two hydrogen urine. The DHU arm (DHU arm) can not form a stable stem and ring structure, and the rest of the other tRNAs can be folded into a typical trifoliate type two structure. The phylogenetic relationship between the nucleotide and amino acid data sets of the protein encoding genes shows that the genus cashmere is in the mailing family of the family Coleoptera and the morphological credit. In order to explore the genetic structure of the population, the identification of the species and the evolution and phylogeny of the cashmere mailing, the genetic information of the molecular biology.2) was provided by the COXI gene to study the genetic relationship of the different host cashmere populations, and a total of 26 haplotypes with 51 nucleotide polymorphisms were obtained, and the genetic diversity among the populations was found. The sex index is low, and the genetic variation mainly occurs within the population. There is a low genetic differentiation coefficient and high gene flow among the population, which indicates that the gene exchange among the population is obvious. The common haplotype DH01 is the haplotype of the ancestor, the other haplotypes evolve from the different mutations. Among the five populations, especially in the group AG, The high degree of gene exchange between MA and MR indicates that there may be a cross mixing between these populations in the field. It provides a theoretical basis for the genetic difference of cashmere mailing, and also provides a molecular biological proof of biological control for the effective use of natural enemies for biological control. 3 floral fluxes are obtained through the screening of the cashmere transcriptional library and the RACE amplification. The cDNA sequence of the AMPK gene was full. Phylogenetic analysis showed that the homology of the cashmere AMPK subunit was higher than that of other insects, especially more than 85% of the homology of the Coleoptera. The expression of the AMPK gene was detected under different processing conditions, and the expression level of the AMPK gene was detected under different conditions. The differential expression of.3 AMPK gene expressed relatively high expression in the male reproductive system and the fat body; the expression in the first instar larvae and pupae is higher; with the prolongation of the survival time of the cashmere mailing, the expression of each gene has an obvious upward trend, which may be related to its response to environmental changes and the mitigation of senility; under different stress conditions, the expression of the gene is higher. The expression of each gene in the female adult is higher than that of the male adult. With the increase of the processing time, it shows a trend of rising and decreasing. It shows that the AMPK gene plays an important role in insect metamorphosis, life extension and stress..4, PGC1- alpha, NRF1, mtTFA, COXI and A are screened from the cashmere transcriptome library and the grain genome. The TP6 gene, the amino acid sequence of the 5 genes showed high homology with the corresponding amino acid sequences of other species. Real time fluorescence quantitative qPCR results showed that the expression of the 5 mitochondrial synthetic genes had significant tissue specificity and insect specificity: the expression in the cashmere male colonization system and the fat body was significantly higher than that of them. The expression amount in the 1 instar larvae is higher than that in the other larval stages, and the expression in the pupa is the smallest. After 20 months, the expression of each gene increases with the increase of the age of the insect, and reaches the maximum in 30 months. The PGC1- a, NRF1 and mtTFA genes, which are encoded by the nuclear gene, are high in the tissues, the insect state and the age of the worm. The expression levels of COXI and ATP6 genes were also relatively high, which laid a theoretical foundation for the further study of energy synthesis in the longer life span.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.306.4
，
本文编号：2157070
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