本文選題：美洲大蠊　切入點：性別差異基因　出處：《南京師范大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：美洲大蠊Periplaneta americana屬節(jié)肢動物門Arthropoda,昆蟲綱Insecta,蜚蠊目Blattaria,蜚蠊科Blattidae,大蠊屬Periplaneta,俗稱蟑螂,是我國室內(nèi)優(yōu)勢衛(wèi)生害蟲種群之一。美洲大蠊有著極強(qiáng)的繁殖與適應(yīng)能力,隨著比較基因組學(xué)的迅速發(fā)展以及高通量測序技術(shù)的發(fā)展,關(guān)于美洲大蠊生殖發(fā)育及翅發(fā)育的研究越來越引起人們的關(guān)注。因此,本論文采用抑制消減雜交技術(shù)和高通量測序技術(shù)構(gòu)建美洲大蠊EST數(shù)據(jù)庫,通過分子克隆、實時定量、RNA 干擾、比較基因組學(xué)等檢測手段做了幾個方面的研究內(nèi)容:1.使用抑制消減雜交技術(shù)構(gòu)建了美洲大蠊卵巢和精巢差異基因的兩個cDNA文庫,通過組裝和聚類分析,在這2個正、反向文庫中分別得到了433條和599條序列,一共是1,032條EST序列。通過對這些序列進(jìn)行Gene Ontology分析,它們可以被分為3個類別:生物過程、分鐘功能和細(xì)胞組成。這些差異表達(dá)的基因參與了美洲大蠊的性腺發(fā)育。我們隨機(jī)挑選了 12個差異基因,使用實時定量(qRT-PCR)進(jìn)行了驗證。同時,通過進(jìn)一步的生物信息學(xué)研究,從這些EST 序列中預(yù)測了2個microRNA 序列,pam-miR100-3p 和pam-miR7,并使用了qRT-PCR方法在美洲大蠊中對這兩個microRNA進(jìn)行了驗證。本研究對探索美洲大蠊生殖差異基因的分子機(jī)制奠定了基礎(chǔ),同時是首次在美洲大蠊中對miRNA進(jìn)行了預(yù)測。本文可以為進(jìn)一步闡明蜚蠊生殖發(fā)育相關(guān)基因提供線索。2.為了進(jìn)一步對美洲大蠊生殖發(fā)育相關(guān)基因進(jìn)行研究,我們使用高通量測序技術(shù)對美洲大蠊的精巢進(jìn)行了Illumina測序。排除低質(zhì)量的序列之后,一共獲得了64,954,709條序列,它們進(jìn)一步被組裝成為125,390條基因序列,平均長度為711 bp。通過同源性比對分析,有48,300條基因序列與NCBI數(shù)據(jù)庫中的已知基因比對成功。我們對這些基因序列進(jìn)行了GO、COG和KEGG分析,同時對2個明顯的生殖差異基因(fru和Spag6)使用實時定量方法進(jìn)行了特異性驗證。另外,我們在這些序列中預(yù)測到了 14,195個潛在的微衛(wèi)星(SSR),通過驗證,最終在隨機(jī)挑選的63個潛在的SSR中,成功擴(kuò)增了41個SSR,其中有7個SSR具有明顯的多態(tài)性。本文為美洲大蠊構(gòu)建了轉(zhuǎn)錄組序列庫,在無參考基因組的情況下成功地對美洲大蠊轉(zhuǎn)錄組序列進(jìn)行了de novo組裝,同時為美洲大蠊預(yù)測了許多分子標(biāo)記,為進(jìn)一步研究美洲大蠊種群遺傳結(jié)構(gòu)以及參與美洲大蠊生殖發(fā)育的基因奠定了基礎(chǔ)。3.我們對美洲大蠊wingless基因進(jìn)行了全長序列的克隆及序列分析,命名為PaWg,并使用RNA干擾技術(shù)和原位雜交技術(shù)對該基因的功能進(jìn)行了進(jìn)一步的研究,同時對昆蟲中的wg基因進(jìn)行了適應(yīng)性進(jìn)化分析。該基因全長1,456 bp,ORF包含1,194個堿基,編碼了397個氨基酸。序列分析表明該基因與其它物種的氨基酸序列相似性在68-76%之間。對美洲大蠊進(jìn)行wg基因的RNA干擾實驗和原位雜交實驗,結(jié)果表明:wg基因不僅對美洲大蠊的翅發(fā)育起到一定的調(diào)控作用,同時對美洲大蠊的觸角、下唇須和腿部的發(fā)育,以及在生殖發(fā)育方面均具有重要的調(diào)控作用。本文獲得的結(jié)果為今后深入探討wg基因在昆蟲中的作用及機(jī)制奠定了重要基礎(chǔ)。
[Abstract]:Periplaneta americana Periplaneta genus Americana arthropod animal Arthropoda, Insecta Blattodea Insecta, Blattaria, Blattidae blattidae, Periplaneta Periplaneta, commonly known as cockroaches, is one of the population in China. The indoor dominant pests of Periplaneta americana had strong reproduction and fitness, along with the rapid development of comparative genomics and high throughput sequencing technology, on Periplaneta americana on reproductive development and wing development more and more attentions. Therefore, suppression subtractive hybridization and high-throughput sequencing technology to construct the Periplaneta americana EST database used in this paper, through molecular cloning, real-time, RNA interference, comparative genomics and other detection methods to do the research several aspects: 1. using suppression constructs two cDNA Library of differentially expressed genes of Periplaneta americana ovary and testis by subtractive hybridization, assembly and clustering analysis, in this 2 positive, reverse library were obtained 433 and 599 series, a total of 1032 EST sequences. Through Gene Ontology analysis of these sequences, they can be divided into 3 categories: biological process, composition and function of minutes cells. These differentially expressed genes participated in gonad America cockroach development. We randomly selected 12 genes, using quantitative real-time (qRT-PCR) is verified. At the same time, through the study of biological information further, from the EST sequence predicted 2 sequences of microRNA, pam-miR100-3p and pam-miR7, and the use of qRT-PCR method in Periplaneta americana was verified on the two microRNA. This research laid the foundation for exploring the molecular mechanism of Periplaneta americana reproductive differential genes were predicted at the same time, is the first time for miRNA in Periplaneta americana. This paper can further elucidate the reproductive development related cockroaches In order to provide clues to the.2. gene of Periplaneta americana reproductive development related gene, we used high-throughput sequencing technology on the Periplaneta americana testis performed Illumina sequencing. After exclusion of low quality sequences, won a total of 64954709 sequences, they were further assembled into 125390 sequences, the average length of 711 by bp. homology analysis with known genes than 48300 gene sequences in the NCBI database. We were on the GO gene sequence analysis of COG and KEGG, while the 2 obvious differences in reproductive genes (FRU and Spag6) were used to validate specific real-time quantitative methods. In addition, we in these sequences predicted 14195 potential microsatellite (SSR), through the verification, the final in the 63 potential SSR randomly selected, amplified 41 SSR, including 7 with SSR Obvious polymorphism. This paper constructs the transcriptome Library of Periplaneta americana, with no reference genome successfully under the condition of Periplaneta americana transcriptome sequences were de and novo assembly, many molecular markers for prediction of Periplaneta americana, laid the foundation for our.3. on Periplaneta americana wingless gene were cloned and sequence analysis of full-length sequences of America for further Study on population genetic structure of cockroach Periplaneta americana and involved in reproductive development gene, named PaWg, and the functions of the gene were studied further using RNA interference technique and in situ hybridization, and WG gene in insects of adaptive evolution analysis. The gene is 1456 BP, ORF contains 1194 nucleotides, encoding 397 amino acids. Sequence analysis showed that the amino acid sequence of the gene and other species similarity between 68-76%. In Periplaneta americana WG gene RNA interference experiments and in situ hybridization. The results showed that the WG gene is not only a regulatory role of Periplaneta americana wing development, at the same time of Periplaneta americana tentacles, palpus and leg development, both play an important role in the reproductive development and the results obtained in this paper lay. An important basis for further research on the insects of the role and mechanism of WG gene.

【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R384;Q78

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本文編號：1643534