To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii,a high efficient expression vector was constructed.Green fluorescent protein(GFP) was expressed in C.reinhardtii under the control of promoters:RBCS2 and HSP70A-RBCS2.Efficiency of transformation and expression were compared between two transgenic algae:RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ.Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2,and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ.In addition,a threefold increase of GFP in Tran-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.
The cDNA of the metallothionein-like (MT-like) gene from Festuca rubra cv. Merlin was optimized with bias codon of Chlamydomonous reinhardtii chloroplast genome. The optimized MT-like gene was de-livered into C. reinhardtii chloroplast and the transgenic strains expressing MT-like gene was obtained. PCR-Southern blot and RT-PCR-Southern blot analysis demonstrated that the MT-like gene was inte-grated into chloroplast genome of C. reinhardtii and expressed at the transcriptional level. The cad-mium binding capacity of the transgenic C. reinhardtii was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) and the binding properties were analyzed. Results showed that the transgenic C. reinhardtii expressing the MT-like gene exhibited remarkably higher Cd2+ binding capacity and grew to higher densities at toxic Cd2+ concentrations (40-100 μmol/L) than the wild type strain, and that the IC50 of Cd2+ (3-d treating ) to algal cell growth of transgenic strain was 55.43% higher than that of the wild type strain, indicating that the Cd2+ binding capacity and Cd2+ tolerance of C. reinhardtii was enhanced through the expression of the foreign MT-like gene in chloroplast.
HAN SiHai1,2, HU ZhangLi1 & LEI AnPing1 1 Institute of Eco-Environment, College of Life Science, Shenzhen University, Shenzhen 518060, China
