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  

Construction of the Fosmid Genomic Library for the Lean Subline and the Fat Subline of Banna Miniature Inbred Pig(Sus scrofa)

作　　者：苏俊 律娜[2] 朱宝利[2] 刘维全[1] 

机构地区：[1]中国农业大学生物学院/国家级实验教学示范中心,北京100193 [2]中国科学院微生物研究所,北京100101

出　　处：《农业生物技术学报》2017年第12期2052-2057,共6页Journal of Agricultural Biotechnology

基　　金：国家转基因重大专项(No.2009ZX08009-140B)

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。Banna Miniature Inbred Pig（Sus scrofa） is the first large inbred mammal experimental animal. Its gene is highly homozygous, and genetic background is clear. And its gene has great potential in genetic breeding, functional gene research, human disease model and xenotransplantation. However, due to the high degree of inbreeding depression, the survival rate of offspring is low and genetic resources are very precious,and the preservation of the genetic information has scientific and practical significance. In this paper, the leansubline pig and the fat subline pig were taken as the research objects. The two subline pigs＇ DNA was extracted from agarose pre-embedding lumps, and sheared to the appropriate size（about 36-48 kb） by physical method.And then, the DNA fragments were used to construct the two sublines＇ Fosmid libraries by Copy Control^TM Fosmid Library Production kit. There were about 300 000 and 400 000 clones of the lean subline pig＇s and the fat subline pig＇s Fosmid library respectively, which covers the genome up to 4.4 and 5.9 fold. Basing on the upstream sequence of lean subline pig＇ heart-type fatty acid-binding protein gene, we designed the specific primers for grading PCR to screen the target clone in the library. Firstly, the first round PCR reaction using the original library bacteria solutions as template were conducted, which could screen the tube containing the target clones. When the tube which contained the positive target clones was confirmed, and then the positive tube bacterium were cultured. Then, the colonies of every plate were collected to one EP tube, and used as template to do PCR, to continue to screen the target tube. Like this, several rounds grading PCR were done until the target tube which contained 100-1 000 CFU per 10 μL LB medium was screened. Then this positive tube bacterium were cultured on LB medium plates. After 12-16 hours, the single colonies on LB medium plate were transferred to 96-well plate continued to culture as the same conditions as before. Th

关 键 词：版纳微型猪近交系 Fosmid基因文库 分级PCR 

分 类 号：S828[农业科学—畜牧学] Q812[农业科学—畜牧兽医]