Tol2转座子介导的斑马鱼诱变及nup107突变体研究

发布时间：2018-03-27 06:45

本文选题：斑马鱼　切入点：Tol2基因捕获　出处：《清华大学》2012年博士论文

【摘要】：出生缺陷严重危害人类健康。全世界人类出生缺陷率在3~4%之间，，且居高不下。大部分出生缺陷是遗传和环境因素的综合结果，如染色体异常、单基因突变以及环境致畸因素的影响。为了研究调控胚胎早期发育的重要基因，我们利用斑马鱼作为模式生物开展了相关工作。在本课题中，我们进行了Tol2转座子介导的基因捕获及突变体筛选，并利用突变体对被捕获基因的功能及分子机制进行了深入研究。本课题共筛选出195个转基因家系，大部分荧光在母源和合子期都表达。其中，约40个家系荧光表达有时空特异性，其余为全身性表达。筛选到4个突变体，且都是合子纯合隐性突变体。本课题主要对nup107tsu068Gt突变体家系进行了深入研究。该家系中，EGFP在母源和合子期都表达，早期表现为全身性表达，24hpf后逐渐集中在头部和肠道区域。Tol2转座子插入至斑马鱼nucleoporin107（nup107）基因第一个内含子中，破坏了野生型转录本的表达。纯合体从3dpf开始表现出组织特异性发育缺陷，包括咽弓软骨缺失、肠道和眼变小、鱼鳔消失等表型，纯合体在5~6dpf之间死亡。野生型胚胎中用反义吗啉环寡核苷酸敲低nup107的表达，胚胎显示出与突变体类似的表型，表明nup107缺失直接导致了突变表型。在发育缺陷的组织中，细胞过量凋亡，并且这些细胞凋亡部分依赖p53信号通路。在受影响的细胞中，FG-repeat核孔蛋白定位紊乱，核孔数量减少，mRNA出核受到抑制，但蛋白入核不受影响。在有丝分裂细胞中，nup107缺失并未影响纺锤体的装配和染色单体的分离。同时，细胞周期和增殖也是正常的。另一方面，nup107tsu068Gt突变体中γ-H2A.X水平显著上调，表明DNA双链缺口的大量积累。突变体对紫外辐射和hydroxyurea（一种DNA复制抑制剂）高度敏感，显示nup107在修正DNA复制错误以及维持基因组稳定和完整性中发挥作用。同时， DNA损伤应答ATM-Chk2-p53通路被上调。我们认为nup107缺失破坏了核孔复合体装配和功能，同时使DNA损伤得不到有效修复，二者共同造成了特异组织的细胞凋亡。本研究第一次报道了nup107在脊椎动物中的功能和分子机制。
[Abstract]:Birth defects are a serious hazard to human health. The rate of human birth defects is between 3 and 4 per cent worldwide and remains high. Most birth defects are the result of a combination of genetic and environmental factors, such as chromosomal abnormalities, Single gene mutation and environmental teratogenic factors. In order to study the important genes regulating early embryonic development, we used zebrafish as a model organism to carry out related work. Tol2 transposon mediated gene capture and mutants screening were carried out, and the function and molecular mechanism of the captured genes were studied by using the mutants. A total of 195 transgenic families were screened in this study, most of which were expressed in the mother and daughter stages. Among them, about 40 families had space-time specific fluorescence expression, and the rest were systemic. Four mutants were screened. All of them were homozygous recessive mutants. In this study, we studied the nup107tsu068Gt mutants in the pedigree. In the early stage, after 24 HPF, the transposon of Tol2 was inserted into the first intron of the nucleoporin 107 nup107) gene in zebrafish. Homozygote showed tissue specific developmental defects from 3dpf, including loss of pharyngeal arch cartilage, small intestine and eyes, disappearance of swim bladder, and so on. Homozygote died between 5~6dpf. In wild type embryos, antisense morpholine ring oligonucleotides were used to knock down the expression of nup107. The embryos showed a phenotype similar to that of mutants, indicating that nup107 deletion directly resulted in mutant phenotypes. The apoptosis of these cells partly depends on p53 signaling pathway. In the affected cells, the localization of nuclear pore protein of FG-repeat is disordered, and the decrease of nuclear pore number is inhibited. The deletion of nup107 in mitotic cells did not affect the assembly of spindle and the separation of chromatid. At the same time, cell cycle and proliferation were normal. On the other hand, the level of 纬 -H2A.X in nup107tsu068Gt mutant was significantly up-regulated. The results showed that the DNA double strand gap accumulated a lot. The mutants were highly sensitive to ultraviolet radiation and hydroxyurea (a DNA replication inhibitor). The results show that nup107 plays a role in correcting DNA replication errors and maintaining genomic stability and integrity. At the same time, the ATM-Chk2-p53 pathway of DNA damage response is up-regulated. We believe that nup107 deletion destroys the assembly and function of nuclear pore complexes. At the same time, the damage of DNA could not be repaired effectively, and both of them caused apoptosis of specific tissues. In this study, the function and molecular mechanism of nup107 in vertebrates were reported for the first time.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R3411

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