美洲大蠊生殖发育相关基因的筛
本文选题:美洲大蠊 切入点:性别差异基因 出处:《南京师范大学》2015年博士论文 论文类型:学位论文
【摘要】:美洲大蠊Periplaneta americana属节肢动物门Arthropoda,昆虫纲Insecta,蜚蠊目Blattaria,蜚蠊科Blattidae,大蠊属Periplaneta,俗称蟑螂,是我国室内优势卫生害虫种群之一。美洲大蠊有着极强的繁殖与适应能力,随着比较基因组学的迅速发展以及高通量测序技术的发展,关于美洲大蠊生殖发育及翅发育的研究越来越引起人们的关注。因此,本论文采用抑制消减杂交技术和高通量测序技术构建美洲大蠊EST数据库,通过分子克隆、实时定量、RNA 干扰、比较基因组学等检测手段做了几个方面的研究内容:1.使用抑制消减杂交技术构建了美洲大蠊卵巢和精巢差异基因的两个cDNA文库,通过组装和聚类分析,在这2个正、反向文库中分别得到了433条和599条序列,一共是1,032条EST序列。通过对这些序列进行Gene Ontology分析,它们可以被分为3个类别:生物过程、分钟功能和细胞组成。这些差异表达的基因参与了美洲大蠊的性腺发育。我们随机挑选了 12个差异基因,使用实时定量(qRT-PCR)进行了验证。同时,通过进一步的生物信息学研究,从这些EST 序列中预测了2个microRNA 序列,pam-miR100-3p 和pam-miR7,并使用了qRT-PCR方法在美洲大蠊中对这两个microRNA进行了验证。本研究对探索美洲大蠊生殖差异基因的分子机制奠定了基础,同时是首次在美洲大蠊中对miRNA进行了预测。本文可以为进一步阐明蜚蠊生殖发育相关基因提供线索。2.为了进一步对美洲大蠊生殖发育相关基因进行研究,我们使用高通量测序技术对美洲大蠊的精巢进行了Illumina测序。排除低质量的序列之后,一共获得了64,954,709条序列,它们进一步被组装成为125,390条基因序列,平均长度为711 bp。通过同源性比对分析,有48,300条基因序列与NCBI数据库中的已知基因比对成功。我们对这些基因序列进行了GO、COG和KEGG分析,同时对2个明显的生殖差异基因(fru和Spag6)使用实时定量方法进行了特异性验证。另外,我们在这些序列中预测到了 14,195个潜在的微卫星(SSR),通过验证,最终在随机挑选的63个潜在的SSR中,成功扩增了41个SSR,其中有7个SSR具有明显的多态性。本文为美洲大蠊构建了转录组序列库,在无参考基因组的情况下成功地对美洲大蠊转录组序列进行了de novo组装,同时为美洲大蠊预测了许多分子标记,为进一步研究美洲大蠊种群遗传结构以及参与美洲大蠊生殖发育的基因奠定了基础。3.我们对美洲大蠊wingless基因进行了全长序列的克隆及序列分析,命名为PaWg,并使用RNA干扰技术和原位杂交技术对该基因的功能进行了进一步的研究,同时对昆虫中的wg基因进行了适应性进化分析。该基因全长1,456 bp,ORF包含1,194个碱基,编码了397个氨基酸。序列分析表明该基因与其它物种的氨基酸序列相似性在68-76%之间。对美洲大蠊进行wg基因的RNA干扰实验和原位杂交实验,结果表明:wg基因不仅对美洲大蠊的翅发育起到一定的调控作用,同时对美洲大蠊的触角、下唇须和腿部的发育,以及在生殖发育方面均具有重要的调控作用。本文获得的结果为今后深入探讨wg基因在昆虫中的作用及机制奠定了重要基础。
[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.
【学位授予单位】:南京师范大学
【学位级别】:博士
【学位授予年份】:2015
【分类号】:R384;Q78
【参考文献】
相关期刊论文 前10条
1 房学爽;徐刚标;;表达序列标签技术及其应用[J];经济林研究;2008年02期
2 肖小芹;汪世平;徐绍锐;刘雪琴;罗臣;吴仕筠;曾少华;;美洲大蠊提取物抗炎、镇痛作用的实验研究[J];中国病原生物学杂志;2007年02期
3 唐新燕;张才乔;;鸡胚原始生殖细胞体外的分化[J];农业生物技术学报;2007年01期
4 肖小芹;汪世平;罗臣;徐绍锐;吴仕筠;钟飞;;美洲大蠊抗胃溃疡作用的初步研究[J];热带医学杂志;2006年12期
5 丁鸿娟;赵凯;;蜚蠊及其危害和治理[J];医学动物防制;2006年12期
6 蒋永新;王熙才;金从国;陈晓群;李佳;伍治平;刘光明;李树楠;;美洲大蠊提取物对小鼠3LL肺癌的抑制作用及其机制探讨[J];中国肺癌杂志;2006年06期
7 王振兴;王璞;姚玲;曾智东;徐汉福;段军;王根洪;夏庆友;;家蚕细胞凋亡相关基因BmICAD的克隆、序列和功能分析及其在精巢中特异表达的初步研究[J];遗传;2006年07期
8 翟宗昭;杨星科;;Hox基因与昆虫翅的特化[J];昆虫学报;2006年06期
9 项朋志,戴云,张娅;羧甲基壳聚糖对微量铜离子吸附性能研究[J];云南中医中药杂志;2005年05期
10 吴绍强,邹丰才,翁亚彪,宋慧群,林瑞庆,朱兴全;利用抑制消减杂交技术筛选猪蛔虫性别差异表达基因[J];中国农业科学;2005年05期
相关重要报纸文章 前1条
1 张智;;第一个抗乙肝昆虫类药物上市[N];科技日报;2006年
相关博士学位论文 前1条
1 马长艳;中华绒螯蟹卵巢发育相关基因的研究[D];南京师范大学;2003年
相关硕士学位论文 前2条
1 项方;斑马鱼vasa基因和gcl基因在卵母细胞发生过程中的表达[D];武汉大学;2004年
2 周玉峰;美洲大蠊诱捕器及诱饵的研究[D];福建农林大学;2004年
,本文编号:1643534
本文链接:https://www.wllwen.com/yixuelunwen/jichuyixue/1643534.html