鱼类自主pogo转座子的挖掘、进化及活性验证

发布时间：2018-01-25 14:59

本文关键词： 转座子进化转座活性斑马鱼硬骨鱼　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：Tc1转座子超家族广泛分布于细菌、真菌及昆虫等各种生物中,有些Tc1转座子超家族成员已经被开发为重要的遗传育种操作工具,如SB转座子已经作为基因治疗和转基因的介导载体得到广泛应用。pogo转座子是Tc1转座子超家族的重要成员,但对其分布、进化和活性研究报道很少。本研究通过硬骨鱼转座组注释,并在脊椎动物中针对Tc1转座子超家族的重要成员pogo转座子进行分布、进化和活性研究,以挖掘活性pogo转座子,研发自主知识产权的遗传操作工具,为动物转基因和基因治疗等研究提供技术支撑。主要研究内容如下:(1)通过生物信息学手段对9种硬骨鱼转座组进行注释。结果表明9种硬骨鱼类转座组大小和构成差异显著,其转座组含量从高到低分别为斑马鱼、矛尾鱼、青溕鱼、花斑剑尾鱼、大西洋鳕鱼、三刺鱼、金娃娃、罗非鱼和红鳍东方渶,转座子含量和基因组大小呈正相关。DNA转座子在硬骨鱼类中具有多样性高和含量差异大的特点(0.50%-38.37%),是硬骨鱼类转座组差异的主要决定因素,其中hAT和Tc/Mariner超家族是硬骨鱼类主要的DNA转座子。RNA转座子在硬骨鱼类中也具有多样性高的特点,其中LINE转座子占硬骨鱼类基因组0.53%-5.66%,共检测到 12 个(I、CR1、L1、L2、R1、R2、Jockey、RTE 和Rex)超家族分布,其中L1、L2、RTE和Rex转座子扩增较为明显,LTR转座子除了在斑马鱼和三刺鱼中含量达到、5.58%和2.51%,在大多硬骨鱼类基因组中的含量低于2%,在硬骨鱼类中共检测到6个LTR转座子(Copia、DIRS、ERV、Gypsy、Ngaro和Pao)超家族分布,其中扩增最为明显的是Gypsy。而SINE转座子在硬骨鱼类中扩增最弱,仅在斑马鱼和矛尾鱼中分别达到3.28%和5.64%,在其它7个品种中低于1%。SINE中tRNA、5S和MIR三个超家族在部分硬骨鱼类中有一定程度扩增。本研究表明硬骨鱼类转座组具有多样性丰富、差异大的特点,转座组差异与硬骨鱼基因组大小有很强的相关,转座组是决定硬骨鱼基因组大小的重要因素。(2)通过TBLAST程序对NCBI数据库中脊椎动物基因组比对发现pogo转座子主要分布在鱼类基因组中,其中在28个鱼物种中发现了 pogo转座子的分布,在2种两栖动物(西方爪蛙和高山蛙)和4种爬行动物中发现了 pogo转座子的分布,在鸟类和哺乳动物未能发现者典型的pogo转座子结构。pogo转座子在进化中衍生出POGK和POGZ功能基因,POGZ在脊椎动物中分布广泛,而POGK主要分布在哺乳动物中。POGK和POGZ功能基因含有保守的pogo转座酶DBD和DDE结构域,且DDE氨基酸残基高度保守,提示POGK和POGZ可能具有DNA酶活性。(3)为验证人源POGK和POGZ在哺乳动物细胞上的活性,构建了 pogo转座子介导的新霉素报告基因表达质粒pPG-PGK-NEO,及转座酶表达质粒pT2-CMV-POGK和pT2-CMV-POGZ,同时构建斑马鱼pogo转座酶表达质粒pT2-CMV-Pgase。将pPG-PGK-NEO与分别上述三个转座酶表达质粒共转染Hela细胞,通过G418筛选获得稳定表达Neo-基因的阳性细胞,阳性细胞克隆数比较表明,pT2-CMV-POGK和pT2-CMV-POGZ组显著高于对照组,而pT2-CMV-Pgase组显著高于POGK、POGZ和对照组,提示pogo转座酶、POGK、POGZ基因均具有转座活性。(4)为了进一步明确斑马鱼pogo转座酶PGase转座子时空表达特性,以PGase正、反义RNA为探针,对不同发育阶段的斑马鱼胚胎进行原位杂交,结果表明,PGase在胚胎早期发育各阶段具有表达活性,正向表达水平明显高于反向,且表达无明显组织特异性,本研究表明,pogo转座子主要分布在鱼类、两栖类和爬行类动物中,具有较高的转座活性,且其衍生基因POGK和POGZ也具有转座活性。
[Abstract]:The Tc1 transposon superfamily are widely distributed in bacteria, fungi and insects and other organisms, some of the Tc1 transposon superfamily has been developed as an important tool for genetic breeding operation, such as the SB transposon has been used as gene therapy and transgenic vector mediated widely used transposon.Pogo is an important member of Tc1 transposon superfamily but, the distribution, evolution and activity are rarely reported. This study through the teleost transposable group notes, pogo and important members in vertebrates for Tc1 transposon superfamily transposons distribution, evolution and research activity, in order to tap the active pogo transposon, genetic manipulation tool research and independent intellectual property rights. Provide technical support for the research of transgenic animal and gene therapy. The main contents are as follows: (1) notes on 9 species of teleost fish transposable group by means of bioinformatics. The results show that 9 kinds of hard Fish bone transposition group size and constitute a significant difference, the transposition group content from high to low was the fish, green Mengyu, Latimeria chalumnae, Xiphophorus maculatus, the Atlantic cod, three fish, gold doll, tilapia and t.rubripes Ying, transposon content and genome size of Cheng Zhengxiang.DNA transposon has the characteristics of diversity and high the content in teleost fishes (0.50%-38.37%), is a major determinant of differences in teleost transposable group factors, including hAT and Tc/Mariner superfamily is DNA major teleost transposon.RNA also has the characteristics of high diversity in teleost fishes, which accounted for LINE transposon in teleost genomic 0.53%-5.66% were detected 12 (I, CR1, L1, L2, R1, R2, Jockey, RTE and Rex) super family distribution, which L1, L2, RTE and Rex transposon was more obvious, LTR transposon except in zebrafish and three spined stickleback in content reached 5.5. 8% and 2.51%, in most teleost fish genome content of less than 2%, in the teleost were detected 6 transposon LTR (Copia, DIRS, ERV, Gypsy, Ngaro and Pao) super family distribution, which was the most obvious is the Gypsy. and SINE transposon in teleosts was the most weak, 3.28% and 5.64% respectively in zebrafish and Latimeria chalumnae, in the other 7 varieties in less than 1%.SINE in tRNA, 5S and MIR three superfamily in teleosts in part with a certain degree of amplification. This study shows that teleost transposable group has rich diversity, the characteristics of big differences, there is a strong correlation of transposition group differences and teleost genomes, transposable group is an important factor in determining the teleost genome size. (2) through the TBLAST program of NCBI database in the vertebrate genome comparison showed that pogo transposon is mainly distributed in the fish genome, one in 28 fish species Found in the distribution of pogo transposon, 2 species of amphibians in animal (Western clawed frog and alpine frog) and 4 reptile species were found in the distribution of pogo transposon in birds and mammals, who failed to find the typical pogo transposon structure of.Pogo transposon in evolution derived from POGK and POGZ genes, POGZ in vertebrates, POGK mainly.POGK in mammals and POGZ genes containing pogo transposase DBD and DDE conserved domains, and highly conserved amino acid residue DDE, suggesting that POGK and POGZ may have DNA activity. (3) in order to verify the witness source POGK and POGZ in mammalian cells activity. The construction of gene expression vector pPG-PGK-NEO neomycin report pogo transposon mediated, and transposase expression plasmid pT2-CMV-POGK and pT2-CMV-POGZ, and construct the zebrafish pogo transposase expression plasmid pT2-CMV-Pgase. pPG-PGK-NEO And respectively above three transposase expression plasmids were transfected into Hela cells by G418 screening for stable expression of Neo- gene positive cells, positive cells number comparison showed that pT2-CMV-POGK and pT2-CMV-POGZ were significantly higher than the control group, and pT2-CMV-Pgase group was significantly higher than that of POGK, POGZ and the control group, suggesting that pogo transposase, POGK, POGZ gene transposition activity. (4) in order to further clarify the zebrafish pogo transposase PGase transposon expression characteristics, PGase, antisense RNA probe of zebrafish embryos at different developmental stages by in situ hybridization. The results show that PGase has expression in the early stages of embryonic development, the positive expression level was significantly higher than that of the reverse. And the expression had no obvious tissue specificity, this study showed that pogo transposon mainly distributed in fish, amphibians and reptiles in animal, transposition activity is high, and its derivative matrix POGK and POGZ also have transposable activity.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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