一种莱茵衣藻启动子功能检测系统的构建

Construction of Chlamydomonas reinhardtii system for analyzing the function of algal promoter

选用莱茵衣藻(Chlamydomonas reinhardtii)作为检测微藻启动子功能的生物系统,以pSP124质粒为基础,雨生红球藻β-胡萝卜素酮化酶基因bkt1的启动子片段(450 base pair,记作450 bp)为间隔序列,引入两个Eam 1105Ⅰ限制性内切酶位点,构建莱茵衣藻启动子功能检测T载体.将聚合酶链式反应(polymerase chain reaction,PCR)获得的1 986 bp的bkt1启动子片段直接克隆到T载体上,通过"珠磨法"转化到莱茵衣藻CC-849中,经Zeomycin筛选获得了TranB-0.45和TranBle转基因藻,而1 986 bp的bkt1启动子无转化子,原因可能是其含有负调控元件.PCR结果显示,ble可稳定存在于转基因藻中,bkt1启动子的450bp片段具有启动子活性,能正确表达BLE蛋白.该研究表明,莱茵衣藻和pB-0.45T载体所组成的检测系统可用于藻类启动子研究,为启动子功能的研究提供了一条新途径.

Chlamydomonas reinhardtii was employed as a bio-system for analyzing the function of algal promoter in this study. The C. reinhardtii T-vector based on pSP124 plasmid was constructed using 450 bp sequences of β carotene ketolase gene （ bktl ） promoter as insert fragment. Two Eam1105 I restriction sites were introduced into this T-vector. 1 986 bp sequences of bktl promoter was obtained by PCR and cloned into the C. reinhardtii T-vec- tor directly. By ＂glass-bead method＂, transformants of TranB-0.45 and TranBle were generated from TAP containing 10 μg/mL Zeomycin. However, none of TranB2 were observed due to some negative regulatory elements in 1 986 bp-bktl promoter. Results of PCR confirmed that ble was integrated into the genome DNA of C. reinhardtii. The 450 bp sequences of bktl promoter were able to express BLE in transgenic algae, which verified that it owned pro- moter ability in C. reinhardtii. These results indicate that the C. reinhardtii and pB-0. 45T system for analyzing promoter function is workable. It is a new way for studying algal promoters.