八肋游仆虫γ微管蛋白cDNA序列及其在大肠杆菌中的表达

γ-tubulin cDNA of Euplotes Octocarinatus and its Heterologous Expression in E.coli

提取八肋游仆虫 (Euplotesoctocarinatus)的mRNA ,采用RT PCR技术对其γ微管蛋白基因的cDNA序列进行了分析。结果表明八肋游仆虫的微管蛋白编码基因至少使用 4个转录起始位点 ,分别位于起始密码子前的 32、 2 9、 2 4和 2 0bp处 ;该基因同时也有至少 4多聚腺苷酸加尾位点 ,分别位于终止密码子后的 2 1、 2 3、 2 9和 32bp处。由于多个转录起始位点和多聚腺苷酸加尾位点的存在 ,对应的mRNA长度在 142 7～1450核苷酸之间。此外 ,本研究对该基因中编码半胱氨酸的TGA密码子进行定点突变 ,然后在大肠杆菌中进行异体表达 ,SDS PAGE分析表明表达产物为

The γ tubulin encoding cDNA of Euplotes octocarinatus was isolated and analyzed by RACE PCR. Our result revealed that the γ tubulin encoding gene of E. octocarinatus uses at least 4 transcription initiation sites. They are located at positions 32, 29, 24 and 20 bp upstream of the translation start codon. On the other hand, 4 poly (A) addition sites were identified. They are present at positions 21, 23, 29 and 32 bp downstream of the translation stop codon. The corresponding γ tubulin mRNA is in length from 1427 to 1450 nucleotides. It codes for a protein of 461 amino acids with a calculated molecular mass of 52 kDa. In addition, after mutagenesis of the TGA codon in the open reading frame into TGC, the γ tubulin of E. octocarinatus was expressed in E. coli. As a result a γ tubulin fusion protein of about 92 kDa was obtained.