组蛋白脱乙酰化酶HDAC1 cDNA的克隆及其酵母表达质粒的构建

目的: 获得HDAC1 的cDNA及构建酵母双杂合系统中的靶基因。方法: 利用RT-PCR方法,从人Jurkat 细胞中扩增出一约1.45kb的DNA片段,作全自动测序,重组入pLexA载体,构建成pLexA-HDAC1, 并用醋酸锂法转化酵母菌EGY48, 在选择性培养基上观察pLexA-HDAC1 在EGY48中的表达情况。结果: 获得的1.45kb的DNA编码的氨基酸序列与文献报道的HDAC1一致,转化的酵母菌在选择性培养基SD/Gal/Raf/-Ura/-His/上培养3d后, 长出约1mm大小的白色菌落。结论：获得了HDAC1的cDNA,pLexA-HDAC1对EGY48没有毒性作用,也没有自身激活LacZ功能,可作为酵母双杂合系统中的靶基因。

Cloning of histone deacetylase 1 cDNA and construction of its yeast expression plasmid

Objective: To obtain the histone deacetylase 1(HDAC1) cDNA and construct the target gene of yeast two-hybrid system. Methods: About 1.45kb DNA fragment was amplified from the human Jurkat cell by RT-PCR. After being automaticly sequenced, the fragment was ligased with the vector pLexA to construct pLexA-HDAC1. The recombinant plasmid was transformed into yeast EGY48,and the expression of pLexA-HDAC1 in the yeast was observed. Results: The amino acid sequence encoded by RT-PCR product was the same as reported HDAC1,and about 1 mm white yeast clone grew in the selective medium after 3d. Conclusion:The HDAC1 cDNA has been obtained. pLexA-HDAC1 has nontoxic to yeast, which can serve as a target gene of yeast two-hybrid system.