| Carotenoids is one kind of pigment, which is numerous in photosynthetic organisms. Research shows that carotenoids are antioxidant and effective in disease prevention, which makes requirement promoting around the world. To increase the carotenoids production is a key to the industry. Microalgae contains plenty of specific carotenoids, gradually becoming the preferred source of carotenoids biosynthesis. In carotenoids biosynthesis, phytoene synthase(PSY) is a rate-limiting enzyme which catalyzes the key step and affects the efficency of synthesis. Further research on the encoding gene psy, and the structure and function of PSY protein will be helpful to improve carotenoids productivity by synthesis regulation or genetic engineering. In recent years, researches on psy gene of microalgae are mostly related to the cloning and identification of gene sequence and its use in genetic engineering, but studies on biological information and the structure and function of the corresponding protein are infrequent.Thus, purpose of the present study is to isolate the encoding gene psy of phytoene synthase in carotenoids-rich mciroalgae Chlorella vulgaris, and to obtain the gene information, protein information, and the expression regulation under various illumination.Results obtained can be used for genetic engineering such as constructing vector for overexpression. Furthermore, results provide sequence information for psy gene identification, and offer theoretical prediction of the protein structure and activity. In addition, the analysis results of growth characteristics and gene expression of algal cell can provide useful information for optimizing of the microalgae cultivation and carotenoid accumulation on gene expression level.1. cDNA cloning and deduced protein analysis of the phytoene synthase encoding gene psy in Chlorella vulgarisPSY can affect the efficiency of carotenoids synthesis as it is a rate-limiting enzyme that catalyzes the early step in the synthesis pathway. Therefore, a better understanding of PSY will help to figure out the mechanism and regulation of carotenoids synthesis pathway.In this chapter, the cDNA fragment of partial psy gene in C. vulgaris was cloned using degenerated primers at first. Then the full-length cDNA sequence was generated by RACE. The 1605 bp full-lenth sequence contained a 1245 bp open reading frame(ORF), which encoded a protein composed of 415 amino acids residues. Multiple sequence alignment and phylogenetic analysis of different PSY protein sequences from higher plants, Chlorophyta, Cyonophyta and bacteria was conducted. Results showed that the deduced sequence of the C. vulgaris PSY was homologous with other PSY protein, and clustered into the green algae with high similarity and identity. Analyses of conserved blocks and motifs showed that this sequence contained conserved blocks and characteristic motifs of PSY protein, and several potential modification sites. All the above results indicated that the corresponding full-length cDNA sequence encoded PSY protein in C. vulgaris.Physical and chemical properties of the deduced protein was predicted. Prediction result of subcellular location showed that the 1st to 45 th amino acid residues might constitute a chloroplast transit peptide, suggesting that PSY must be translated outside before entering the chloroplast and being active. By using homology-based and ab initio modelling, a tertiary structure of C. vulgaris PSY protein was constructed. There were two substrate-Mg2+ binding sites and two active site lid residues which might mediate binding of substrate and shield highly reactive carbocationic intermediates from solvent. A specific substrate-Mg2+ binding site was found in the C. vulgaris PSY protein, which formed a circle-like structure with the other two conserved binding sites. Activity and function of the third site was still unconfirmed.2. Study on the growth, form and chlorophyll fluorescence of C. vulgaris under different photoperiod using flow cytometryThe cell growth and accumulation of cellular nutrients can be affected by illumination. Adequate research on the growth characteristics of algal cell can provide reference for optimizing the microalgae culture and improving production of useful product such as carotenoids.In this chapter, characteristics of C. vulgaris cultured under 12 h light(12h dark) or 24 h continuous illumination was studied using flow cytometry. Results showed that C. vulgaris cells grown synchronicly under the 12 h light. During the illumination phase, algal cells converted light energy into cellar product through photosynthesis. During the dark phase, algal cells performed cell division within 6h. The volume, granularity and chlorophyll content of the daughter cells were low until starting growing under light in the next cell cycle. In addition, during the process of cell division, chlorophyll was distributed in two cell population. Chlorophyll content was high in mother cells while it was low in daughter cells. There was no other cell population besides them, which indicated presumably, that chlorophyll, or even with carotenoid, was sent in daughter cells with binding protein when chloroplast divided. Futher experiments will be necessary to verify whether pigment was degraded during cell division.When the algal cells were cultured under continuous illumination, cells didn’t grow synchronicly. Each algal cell grew and reproduced continuously and didn’t synchronize to each other. In this process, some cellular production synthesized, but the content was inferior significantly to that in algal cells under synchronized culture. Thus even though the algal cells could reproduced continually under continuous illumination, it seemd that it was bad for production accumulating such as pigment, in the cell.3 Ttranscriptional expression of relevant genes under various illumination.In this chapter, expression of psy, psbC, psaB and rbcL were measured using realtime quantitative PCR during the light phase under synchronized culture. The influence on gene expression of intense light irradiation exposure was also measured.Results showed that the expression of these 4 genes were up-regulated significantly during the process of photosynthesis. After being exposed to intense light irradiation for 30 min, transcript level of psy was up-regulated significantly to 26 times as high as control, while rbcL expression level was significantly suppressed to 0.01 times. On the other side, expression level of psbC and psaB did not change significantly. Results indicated that the photosynthesis process in algae cell may be affected under intense light irradiation, and high expression level of psy gene may increase the carotenoids products in response to the oxidative damage caused by strong light.To sum up, in this study the phytoene synthase gene psy from C. vulgaris has been isolated. The information of the gene and protein has been analyzed and predicted. Cell morphology of the C. vulgaris and cellular content of pigment under synchronized or nonsynchronized culture have been observed. Gene expression of psy, psbC, psaB and rbc L under different illumination during synchronized growth has been studied as well. |
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