嗜热四膜虫δ微管蛋白基因的克隆、鉴定及细胞定位

Characterization and Cellular Localization of δ-tubulin from Tetrahymena thermophila

微管蛋白(tubulin)在细胞的结构和功能中发挥着重要作用,α微管蛋白和β微管蛋白是组成微管的主要因子,γ微管蛋白促使α和β微管蛋白二聚体组装为微管结构.然而,4种新的微管蛋白δ-,ε-,ζ-,和η-tubulin在细胞中的功能并不完全清楚.本研究从嗜热四膜虫大核基因组数据库中鉴定了一种新的编码δ微管蛋白基因(Tetrahymena delta tubulin 1,TDT1,TTHERM_00335970,http://www.ciliate.org),TDT1基因转录产生1 326 bp和1 363 bp两种不同的转录本,1 326 bp的转录本编码441个氨基酸的多肽;而1 363 bp的转录本含有37 bp未剪切的内含子序列,从而导致开发读框发生移码突变现象.实时荧光定量PCR结果表明,TDT1基因在四膜虫细胞营养生长和有性生殖过程中都有表达,且在有性生殖过程中的表达显著上调.免疫荧光定位表明,TDT1蛋白不仅定位于四膜虫基体和有性生殖期conjugation junction结构,而且在四膜虫的大核和小核中也有定位.TDT1基因敲除发现,该基因不能通过表型分配完全被巴龙霉素抗性基因替代,结果表明,TDT1蛋白在四膜虫细胞中可能具有多种不同的功能,它的正常表达对四膜虫细胞的生存是必需的.

Tubulin is a highly conserved heterodimeric protein comprised of monomeric globular polypeptides of or- and 13-tubulins. Besides ~/-tubulin, which is required to initiate the assembly of a-15 tubulin heterodimers to form microtubule polymers, 4 new members of tubulin superfamily and Xl-tubulin） were indentified. In this study, the 8-tubulin gene from Tetrahymena thermophia （Tetrahymena delta tubulin 1, TDT1, TTHERM_00335970, http：//www, ciliate, org） was isolated. Two different transcripts, 1 326 bp and 1 363 bp, were obtained by RT-PCR. The 1 326 bp transcript encodes 441 amino acids; the 1363 bp transcript is a frame-shift mutant containing a 37 bp intron. Quantitative reverse transcriptase PCR （qRT-PCR） analysis showed that TDT1 was expressed in both the growing and conjugation stage with higher levels in the latter. HA-tagged TDT1 localizeds at the basal bodies, macronuclei, micronuelei and conjugation junction. The TDT1 knockout strains showed that neo4 cassette could not fully replace the macronuclear TDTI with increasing paromomysin concentration. The results indicated that TDT1 was a multifunctional protein for normal functions of Tetrahymena cells.