本文關(guān)鍵詞： 版納微型豬近交系 Fosmid基因文庫(kù) 分級(jí)PCR　出處：《農(nóng)業(yè)生物技術(shù)學(xué)報(bào)》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(gè)中大型哺乳類實(shí)驗(yàn)動(dòng)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(yīng)用潛力。但因高度近交衰退導(dǎo)致后代存活率低,基因資源非常珍貴,對(duì)這些遺傳信息的保存具有重要科學(xué)和實(shí)際意義。本研究從染色體低熔點(diǎn)膠預(yù)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(guò)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(gè)亞系豬的基因組Fosmid文庫(kù)。隨機(jī)選取3管文庫(kù)菌液涂布平板,通過(guò)計(jì)算單位菌液?jiǎn)慰寺?shù)估算文庫(kù)克隆數(shù),清瘦亞系豬約30萬(wàn)個(gè)克隆,肥胖亞系豬約40萬(wàn)個(gè)克隆,對(duì)基因組的覆蓋率分別達(dá)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(duì)象,從構(gòu)建的文庫(kù)中篩選目的基因陽(yáng)性克隆。設(shè)計(jì)該基因片段上下游引物,采用"文庫(kù)菌液PCR——含陽(yáng)性管菌液稀釋——稀釋菌液PCR——單克隆菌液PCR"的分級(jí)菌液PCR方法進(jìn)行篩選。逐級(jí)將PCR陽(yáng)性菌液進(jìn)行稀釋,直到含目的基因的陽(yáng)性管菌液滴度約100~1 000 CFU/10μL時(shí),將陽(yáng)性管菌液涂布到含氯霉素LB平板37℃過(guò)夜培養(yǎng),之后將單菌落轉(zhuǎn)移到96孔板進(jìn)行單克隆培養(yǎng),再通過(guò)菌液PCR篩選目的基因單克隆,測(cè)序確定。最終從清瘦亞系豬Fosmid文庫(kù)中篩選到5個(gè)目的基因單克隆。利用同樣方法可從肥胖亞系豬文庫(kù)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(kù)的構(gòu)建,對(duì)基因組的覆蓋率高,理論上篩選到目的基因的概率達(dá)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(kāi)展分子遺傳育種、基因序列功能等研究工作奠定重要基礎(chǔ)。
[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(hào)：1483059