本文關(guān)鍵詞： 版納微型豬近交系 Fosmid基因文庫 分級PCR　出處：《農(nóng)業(yè)生物技術(shù)學報》2017年12期 　論文類型：期刊論文

【摘要】：版納微型豬(Sus scrofa)近交系是世界上第一個中大型哺乳類實驗動物近交系,其基因高度純合、遺傳背景清楚,在遺傳育種、功能基因研究、人類疾病模型和異種器官移植等方面有巨大應用潛力。但因高度近交衰退導致后代存活率低,基因資源非常珍貴,對這些遺傳信息的保存具有重要科學和實際意義。本研究從染色體低熔點膠預包埋片中提取版納微型豬近交系清瘦亞系豬和肥胖亞系豬DNA,通過物理方法剪切DNA到合適大小片段(約36~48 kb),使用Copy Control?Fosmid Library Production Kit試劑盒構(gòu)建了這兩個亞系豬的基因組Fosmid文庫。隨機選取3管文庫菌液涂布平板,通過計算單位菌液單克隆數(shù)估算文庫克隆數(shù),清瘦亞系豬約30萬個克隆,肥胖亞系豬約40萬個克隆,對基因組的覆蓋率分別達到4.4倍和5.9倍。以豬的心肌脂肪酸結(jié)合蛋白(heart-type fatty acid-binding protein,H-FABP)基因調(diào)控序列為研究對象,從構(gòu)建的文庫中篩選目的基因陽性克隆。設計該基因片段上下游引物,采用"文庫菌液PCR——含陽性管菌液稀釋——稀釋菌液PCR——單克隆菌液PCR"的分級菌液PCR方法進行篩選。逐級將PCR陽性菌液進行稀釋,直到含目的基因的陽性管菌液滴度約100~1 000 CFU/10μL時,將陽性管菌液涂布到含氯霉素LB平板37℃過夜培養(yǎng),之后將單菌落轉(zhuǎn)移到96孔板進行單克隆培養(yǎng),再通過菌液PCR篩選目的基因單克隆,測序確定。最終從清瘦亞系豬Fosmid文庫中篩選到5個目的基因單克隆。利用同樣方法可從肥胖亞系豬文庫中篩選目的基因克隆。版納微型豬近交系清瘦亞系豬和肥胖亞系豬全基因組Fosmid文庫的構(gòu)建,對基因組的覆蓋率高,理論上篩選到目的基因的概率達99%,為保存版納微型豬近交系這一特色種質(zhì)資源基因組以及深入開展分子遺傳育種、基因序列功能等研究工作奠定重要基礎。
[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，

本文編號：1483059