| The green alga Chlamydomonas reinhardtii is an ideal model organism, that is widely used in the research of cellular and molecular biology as well as in the elucidating of photosynthetic pathway. It has many virtues: short life cycle, the only one big chloroplast, great convenience for experimental manipulation, and various kinds of mutants already obtained. Nuclear transformation provides powerful approaches for assessing gene function, generating large numbers of mutants, and isolating the genes that are affected in these mutants. In order to improve the application of Chlamydomonas reinhardtii in biological technology and in genetics of photosynthetic, I had estimated the optimal transformation conditions for Chlamydomonas reinhardtii, and I had transformed the cbn1gene and the EGF gene successfully into Chlamydomonas reinhardtii. I have proved the EGF gene expression in Chlamydomonas reinhardtii transforming. First, I optimized the transformation conditions for glass bead assay during my research. I carried out transformation experiments with the p389ARG7 vector many times. From my experiments I had drawn a conclusion that the optimum concentrations of agar powder in medium is 1.8%. I had also found out that the time of autolysin treatment should be about 40min. The transformation rate of linear plasmids is not better than that of cyclic plasmids. After estimating the most favorable transformation conditions, I used glass bead assay to transform a double mutant strain, which is an arginine deficient as well as cell wall-defective with the p389ARG7, I gained in my experiments more than 3000 transformants. When using differences fluorescence emission to detect mutants I selected two low-fluorescing chlorophyll b-deficient strains, ART3and ART4. The lack of chlorophyll b in these strains has been proved by HPLC analysis. In order to clone cbn1 gene, I used glass bead method and transformed nitrate reductase defective and cell wall-deficient mutant strain CC-2677 (cw15, arg7, mt-) with the p387NIT1 vector. I isolated 1800 transformants of carrying many different insertion mutations. I have selected one chlorophyll b-deficient mutant NIT17-1 by detection of diminished fluorescence and subsequent HPLC analysis. Successful transformation by two vectors not only validated the effectiveness of the glass bead approach, but also paved the road for further experiments aimed to clone the chlorophyll b biosynthetic gene. In my research, I had also selected the best experimental conditions to perform the electrotransformation of Chlamydomonas reinhardtii cells. According to my knowledge, no one had yet used the BIO-RAD165-2110 type of eletrotransformation apparatus to transform Chlamydomonas reinhardtii. My experimental results showed that the optimum voltage is 900V. Using algae to produce protein drugs is an important challenge of gene engineering. In order to express the EGF gene in Chlamydomonas reinhardtii, I had constructed the EGF gene vector successfully, with a 3'RBCS2 terminator and a Zeocin resistance selective marker on it. Using this vector I had achieved my goal by transforming the EGF gene into Chlamydomonas reinhardtii nuclear genome using the method of electrotransformation, and I gained one transformant. The subsequentPCR analysis confirmed the existence of EGF gene in transformed genome. This result paves the road for the expression of EGF protein in Chlamydomonas reinhardtii. It is an efficient transformation technique vital part in the biotechnological production of any other proteins.
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