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1、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 苏俊 律娜 朱宝利 刘维全 中国农业大学生物学院/国家级实验教学示范中心 中国科学院微生物研究所 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
2、(约 3648 kb) , 使用 Copy ControlFosmid 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
3、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 1001 000 CFU/10L 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
4、%, 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。关键词： 版纳微型猪近交系; Fosmid 基因文库; 分级 PCR; 作者简介：刘维全, 基金：国家转基因重大专项 (No.2009ZX08009-140B) Construction of the Fosmid Genomic Library for the Lean Subline and the Fat Subline of Banna Miniature Inbred Pig (Sus scrofa) SU Jun LV Na ZHU Bao-Li LIU Wei-Quan
5、 College of Biological Sciences, China Agricultural University/National Biology Experimental Teaching Demonstration Center; Institute of Microbiology, Chinese Academy of Sciences; Abstract： Banna Miniature Inbred Pig (Sus scrofa) is the first large inbred mammal experimental animal. Its gene is high
6、ly 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
7、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
8、size (about 3648 kb) by physical method.And then, the DNA fragments were used to construct the two sublines Fosmid libraries by Copy Control Fosmid Library Production kit. There were about 300 000 and 400 000 clones of the lean subline pigs and the fat subline pigs Fosmid library respectively, which
9、 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 bact
10、eria 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 a
11、s 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 1001 000 CFU per 10 L LB medium was screened. Then this positive tube bacterium were cultured on LB medium plates. After 12-16 hours, the single colonies
12、 on LB medium plate were transferred to 96-well plate continued to culture as the same conditions as before. Then, the single colony bacteria solutions were used to do PCR to screen positive colony, and sequenced to confirm the single colony of bacteria containing target gene. Five single colonies t
13、hat were identified by sequence analysis were abtained.Using the above method, we can get the fat subline pigs target single gene clone. We have successfully constructed the two subline pigs Fosmid libraries, with a high rate of genome coverage, and the probability of screening to the target gene is
14、 99% in theory. These Fosmid libraries can be used to preserve Banna Miniature Inbred Pigs genetic resources and carry out more deeply research on function and regulation of gene.Keyword： Banna Miniature Inbred pig; Fosmid genomic library; Grading PCR; 研究表明, 与小鼠 (Mus musculus) 相比, 猪 (Sus scrofa dome
15、stica) 在基因保守元件、mi RNAs、基因模式、解剖结构、生理指标、营养代谢、疾病发生机理等方面与人类 (Homo sapiens) 更相似 (Wernersson et al., 2005;张霞等, 2017;叶雷等, 2012) , 因此在功能基因研究、人类疾病模型和异种器官移植等方面具有巨大的应用潜力。版纳微型猪 (Sus scrofa) 近交系 (Banna Miniature Inbred pig) 是曾养志教授经过 20 多年连续近交而育成的世界上首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。2008 年, 版纳微型猪近交系已进入 25 世代, 其近交系数
16、达到 99.48%, 并分化为 18 个亚系, 每个亚系分别具有不同的表型和基因型 (霍金龙等, 2004;曾嵘等, 2005;叶雷等, 2012;霍金龙, 2012;邓景致, 2012) 。其中, 肥胖亚系是典型的遗传性肥胖, 极易沉积脂肪, 是研究人类肥胖和动物脂肪沉积功能基因的理想模型。清瘦亚系是典型的遗传性清瘦, 不易沉积脂肪, 不仅是研究肥胖和动物脂肪沉积功能基因的理想对照模型, 而且是研究瘦肉型猪肉品质性状相关主要基因及候选基因的极好材料。肥胖亚系的版纳微型猪胴体瘦肉率为 31%, 脂肪率为 51%, 平均背膘厚 4.87 cm, 肌内脂肪含量为 10.66%;而清瘦亚系的版纳微型猪胴体瘦肉率为 63%, 脂肪率为 19%, 平均背膘厚 2.14 cm, 肌内脂肪含量为 6.56% (未发表) 。版纳微型猪近交系因高度近交衰退等原因, 其后代存活率低, 且在选育过程中出现有脊柱弯曲、肿瘤、肥胖、O 型腿、耳聋等疾病特征个体, 但出现此类特征的个体数量较少, 几乎都为 1 头 (
