版纳微型猪近交系清瘦亚系和肥胖亚系猪Fosmid基因组文库的构建

发布时间：2018-02-01 22:51

  本文关键词： 版纳微型猪近交系 Fosmid基因文库 分级PCR　出处：《农业生物技术学报》2017年12期 　论文类型：期刊论文

【摘要】：版纳微型猪(Sus scrofa)近交系是世界上第一个中大型哺乳类实验动物近交系,其基因高度纯合、遗传背景清楚,在遗传育种、功能基因研究、人类疾病模型和异种器官移植等方面有巨大应用潜力。但因高度近交衰退导致后代存活率低,基因资源非常珍贵,对这些遗传信息的保存具有重要科学和实际意义。本研究从染色体低熔点胶预包埋片中提取版纳微型猪近交系清瘦亚系猪和肥胖亚系猪DNA,通过物理方法剪切DNA到合适大小片段(约36~48 kb),使用Copy Control?Fosmid Library Production Kit试剂盒构建了这两个亚系猪的基因组Fosmid文库。随机选取3管文库菌液涂布平板,通过计算单位菌液单克隆数估算文库克隆数,清瘦亚系猪约30万个克隆,肥胖亚系猪约40万个克隆,对基因组的覆盖率分别达到4.4倍和5.9倍。以猪的心肌脂肪酸结合蛋白(heart-type fatty acid-binding protein,H-FABP)基因调控序列为研究对象,从构建的文库中筛选目的基因阳性克隆。设计该基因片段上下游引物,采用"文库菌液PCR——含阳性管菌液稀释——稀释菌液PCR——单克隆菌液PCR"的分级菌液PCR方法进行筛选。逐级将PCR阳性菌液进行稀释,直到含目的基因的阳性管菌液滴度约100~1 000 CFU/10μL时,将阳性管菌液涂布到含氯霉素LB平板37℃过夜培养,之后将单菌落转移到96孔板进行单克隆培养,再通过菌液PCR筛选目的基因单克隆,测序确定。最终从清瘦亚系猪Fosmid文库中筛选到5个目的基因单克隆。利用同样方法可从肥胖亚系猪文库中筛选目的基因克隆。版纳微型猪近交系清瘦亚系猪和肥胖亚系猪全基因组Fosmid文库的构建,对基因组的覆盖率高,理论上筛选到目的基因的概率达99%,为保存版纳微型猪近交系这一特色种质资源基因组以及深入开展分子遗传育种、基因序列功能等研究工作奠定重要基础。
[Abstract]:Banna Sus scrofa inbred line is the first medium and large mammal experimental animal inbred line in the world. Its gene is highly homozygous, its genetic background is clear, in genetics and breeding, functional gene research. Human disease model and xenogeneic organ transplantation have great application potential, but because of high inbreeding decline resulting in low survival rate of offspring, genetic resources are very valuable. This study is of great scientific and practical significance for the preservation of these genetic information. In this study, DNA was extracted from chromosome low melting point gel preembedded pieces of Banna miniature pig inbred line lean line and obese subline pig. Physically cut the DNA to the appropriate size fragment (about 36 ~ 48 kb / h, using Copy Control? Fosmid Library Production Kit kit was used to construct the genomic Fosmid library of the two sublines. The clone number of library was estimated by calculating the monoclonal number of unit bacterial fluid, about 300 000 clones of lean subline pigs and about 400,000 clones of obese subline pigs. The genomic coverage was 4.4 times and 5.9 times respectively. Heart-type fatty acid-binding protein. H-FABP- (H-FABP) gene regulatory sequence was selected from the constructed library to screen the target gene positive clones, and the upstream and downstream primers of the gene fragment were designed. The PCR method of "library bacteria PCR-containing positive tube bacterial dilution-dilution bacteria PCR-Monoclonal PCR" was used to screen. The PCR positive bacteria solution was diluted step by step. When the liquid titer of the positive tube bacteria containing the target gene was about 1 000 CFU/10 渭 L, the positive tube bacteria solution was coated with chloramphenicol LB plate for overnight culture at 37 鈩，

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