摘要
[目的]构建绒山羊FGF5基因打靶载体,以期获得基因敲除的绒山羊个体,为研究绒山羊FGF5基因的功能以及培养长绒毛型绒山羊新品种提供新思路。[方法]通过PCR法扩增并克隆了绒山羊FGF5基因启动子区和部分第一外显子的1.6 kb片段和部分第一内含子的4.5 kb片段,并将1.6 kb片段插入到打靶载体p Lox的XbaⅠ/ClaⅠ位点,获得了9.6 kb的中间打靶载体p Lox5;再将4.5 kb片段插入到p Lox5的NotⅠ位点,通过酶切和PCR法验证获得了插入方向正确的打靶载体p Lox5-33。[结果]经过酶切和PCR法鉴定,最终获得了大小为14.1 kb的p Lox5-33。[结论]成功构建了绒山羊FGF5基因包含启动子区第一外显子部分序列和第一内含子部分序列的大小为14.1 kb的基因打靶载体p Lox5-33,为进一步获得基因敲除绒山羊个体以及研究绒山羊FGF5基因的功能奠定了基础。
[Objective]In this study a goat FGF5 gene targeting vector was constructed in order to obtain the cashmere goats individuals of FGF5 gene targeting and provide new ideas and methods for identify the function of cashmere goats FGF5 gene as well as develop new breeds of cashmere goats. [Methods]The 1. 6 kb fragment of the goat FGF5 gene which include the promoter and 1stexon was amplified with the method of PCR and inserted into XbaⅠ/ClaⅠ sites of the p Lox successively and a intermediate targeting vector p Lox5 with the size of 9. 6 kb was obtained; The fragment with 4. 5 kb was obtained by PCR,which is a part of intron 1 of goat FGF5 gene. And it was inserted into the NotⅠ site of the p Lox5 successively. Finally,the selected the recombinant cloning vector with correct direction was named p Lox5-33 with 14. 1 kb according PCR and restriction enzymes digesting. The analysis of the sequence of the vector showed that the target vector was correct as design. [Results]p Lox5-33 was obtained by PCR and restriction enzyme digestion identification. [Conclusion]A cashmere goat FGF5 gene targeting vector named p Lox5-33 with 14. 1 kb was constructed successfully,which contained the partial exon1 and intron1 from FGF5 gene. It provided a vector for cloning Cashmere goat that target FGF5 gene from genomic chromosome and was base for developing the role of FGF5 gene.
出处
《生物技术》
CAS
CSCD
北大核心
2015年第1期1-7,共7页
Biotechnology
基金
国家转基因生物新品种培育重大专项("优质高产绒量转基因绒山羊新品种的培育"
2008ZX08008-002)
国家科技支撑计划项目("重点牧区‘生产生态生活’配套保障技术及适应性管理模式研究"
No.2012BAD13B07)
国家自然科学基金项目("BMP信号在绒山羊绒毛再生周期波中调节干细胞激活的研究"
No.30960244)
内蒙古自然科学基金重大项目(No.3013ZD06)资助~~
