花絨寄甲線粒體基因組與合成調(diào)控基因及其COXI基因的種群遺傳結(jié)構(gòu)研究
發(fā)布時間：2018-08-01 09:02
【摘要】：花絨寄甲(Dastarcus helophoroides)屬鞘翅目寄甲科(Coleoptera:Bothrideridae),是各類天牛的重要天敵。線粒體是細胞及生命體活動所需能量的主要來源,其擁有分子量相對較小、突變率高、呈母系遺傳及在細胞中拷貝數(shù)量多等眾多特點,常被用于譜系地理學、種群遺傳學、種系發(fā)生、分子進化和比較進化基因組學等方面的研究。線粒體編碼基因不能獨立完成氧化磷酸化產(chǎn)生ATP的過程,細胞核通過各種轉(zhuǎn)錄因子和輔酶因子對線粒體基因進行調(diào)控。本文通過長片段PCR-步移測序法研究了花絨寄甲線粒體基因組全序列,得到了其序列全長并對其所包含的分子生物學信息進行了詳細分析;在線粒體基因組全序列的基礎(chǔ)上,研究基于COXI基因的不同種群花絨寄甲的種群遺傳結(jié)構(gòu)和遺傳差異;利用花絨寄甲轉(zhuǎn)錄組數(shù)據(jù),對調(diào)控線粒體生物合成的AMPK基因和PGC-1α信號通路基因進行了篩選、鑒定和實時定量表達分析。分析結(jié)果如下:1)花絨寄甲線粒體基因組全長序列為15878bp(GenBank:KF811054.1),由13個蛋白質(zhì)編碼基因(PCGs)、22個tRNA基因、2個rRNA基因和非編碼區(qū)(D-loop環(huán))組成的。除了COI基因擁有非規(guī)范的起始密碼子,其他PCGs均以ATN密碼子起始并以TA(A)或TAG密碼子終止。rrnL和rrnS的二級結(jié)構(gòu)分別由48個螺旋(包含四個新提出的螺旋)和35個螺旋(包含兩個新提出的螺旋)構(gòu)成。除trnS1(AGN)因缺少二氫尿嘧啶臂(DHU臂)不能形成穩(wěn)定的莖-環(huán)結(jié)構(gòu)外,其余21個tRNAs均能折疊成典型的三葉草型二級結(jié)構(gòu)。運用蛋白質(zhì)編碼基因的核苷酸和氨基酸數(shù)據(jù)集分別分析物種間的系統(tǒng)發(fā)育關(guān)系結(jié)果表明,花絨寄甲屬于鞘翅目扁甲總科的寄甲科,與形態(tài)學分類結(jié)果一致。為探索種群遺傳結(jié)構(gòu)、種系鑒定以及花絨寄甲的進化和種系發(fā)生提供有效的分子生物學基礎(chǔ)信息。2)通過COXI基因研究不同寄主花絨寄甲種群的遺傳學關(guān)系,共得到具有51個核苷酸多態(tài)性位點的26個單倍型,種群間遺傳多樣性指數(shù)較低,遺傳變異主要發(fā)生在種群內(nèi)部。種群間存在較低的遺傳分化系數(shù)和較高的基因流動,表明種群間的基因交換明顯。共有單倍型DH01是祖先的單倍型,其他單倍型通過不同的突變從其進化而來。在五個種群之間,特別是在群體AG、MA和MR之間基因交換程度較高,表明在野外這些種群之間可能存在交叉混合。為花絨寄甲遺傳差異提供了理論基礎(chǔ),同時也為有效利用天敵進行生物學防治提供了分子生物學佐證。3)通過花絨寄甲轉(zhuǎn)錄組文庫篩選和RACE擴增,得到3條花絨寄甲AMPK基因的cDNA序列全長。系統(tǒng)發(fā)育分析表明,花絨寄甲AMPK亞基與其他昆蟲相應(yīng)序列的同源性較高,尤其與鞘翅目的赤擬谷盜的同源性達到85%以上。通過實時定量qPCR,發(fā)現(xiàn)在不同處理條件下均檢測到AMPK基因的表達,且表達水平存在顯著的差異性。3條AMPK基因均表現(xiàn)為在雄性生殖系統(tǒng)和脂肪體中表達相對較高;在一齡幼蟲和蛹中的表達量較高;隨著花絨寄甲存活時間的延長,各基因的表達有明顯的上升趨勢,可能與其應(yīng)對環(huán)境變化和緩解衰老有關(guān);在不同脅迫條件下,各基因在雌成蟲中的表達量均高于雄成蟲,隨著處理時間的增加表現(xiàn)出先上升后降低的趨勢,顯示AMPK基因在昆蟲變態(tài)發(fā)育、壽命延伸和應(yīng)激過程中發(fā)揮重要作用。4)從花絨寄甲轉(zhuǎn)錄組文庫和線粒體基因組中篩選出PGC1-α、NRF1、mtTFA、COXI和ATP6基因,5條基因的氨基酸序列均表現(xiàn)出與其他物種相應(yīng)氨基酸序列的同源性較高。實時熒光定量qPCR結(jié)果表明,5個線粒體合成基因的表達存在顯著的組織特異性和蟲態(tài)特異性:在花絨寄甲雄性生殖系統(tǒng)和脂肪體中的表達量明顯高于其他組織;在1齡幼蟲中的表達量高于其他幼蟲期,在蛹中的表達量最小;花絨寄甲成蟲羽化20個月后,各基因的表達量隨著蟲齡的增加呈上升趨勢,在30個月達到最大值。核基因編碼的PGC1-α、NRF1和mtTFA基因表達量高的組織、蟲態(tài)和蟲齡中,線粒體編碼基因COXI和ATP6的表達量也相對較高,為進一步研究花絨寄甲在較長壽命中的能量合成奠定了理論基礎(chǔ)。
[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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S763.306.4
，
本文編號：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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S763.306.4
，
本文編號：2157070
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