| Lutein is a natural pigment belonging to the xanthophyll family of carotenoids, which is often found in higher plants and algae. Chlorella, a small green alga, is a good source of lutein because it can be heterotrophically-cultivated which makes it a more efficient way of producing lutein. Chlorella protothecoides CS-41 is able to accumulate a considerable amount of this kind of xanthophyll. Current studies on C. protothecoides CS-41 are focused on optimizing the culture conditions and extraction techniques to improve lutein yields. Previous studies in our lab showed that C. protothecoides CS-41 could produce much higher level of natural lutein. The objective of this research was to understand more about the lutein biosynthesis pathway in C. protothecoides CS-41, especially to find out the mechanism on the the expression and regulation of these key genes involved in lutein biosynthesis pathway in C. protothecoides CS-41, and to establish a genetic transformation system for the lutein production by C. protothecoides CS-41. The following are the main results of this study:1. The gene encoding phytoene synthase( psy) was isolated from C. protothecoides CS-41. The full-length DNA was 2488 bp, and the corresponding c DNA was 1143 bp,which encoded 380 amino acids. Southern blot analysis indicated that the psy gene was single copied in this alga. Blast alignment results showed that this PSY belonging to Isoprenoid_Biosyn_C1 subfamily, in which there were three predicted Mg2+binding sites( DXXXD). Phylogenetic analysis of the amino acid sequence revealed that the phytoene synthase belongs to Chlorophyta, which is a sister group to higherplants. Phytoene synthase gene was expressed in Escherichia coli. The product of psy gene was a single protein of 43 k Da. The PSY activity of the expressed protein was confirmed by the production of phytoene, which was identified by high-performance liquid chromatography( HPLC) analysis. The promoter of phytoene synthase gene was isolated from C. protothecoides CS-41 using genomic walking kit, then the promoter core was analyzed by fusion expression of shortened psy gene promoter fragments with gus gene in tobacco suspension cells through Agrobacterium-mediated transient transformation. GUS activity was detected to inform the promoter activity.The results showed that-774 bp upstream of the gene had a strong promoter activity.Light-inducing experiment showed there were some light-induced components in this promoter. Quantitative PCR analysis of gene expression confirmed that psy gene expression was induced by methyl jasmonate.2. The psy gene from C. protothecoides CS-41 was transformed into lettuce using Agrobacterium-mediated transformation method. Regular PCR and semi-quantitative RT-PCR results indicated that seven positive transgenic plants were obtained. In most of the transgenic lettuces, the contents of lutein were higher than that in wildtype, the hightest one was 1.8 times higher than the wildtype lettuce, indicating that the psy gene from C. protothecoides CS-41 could be expressed in higher plants, and over-expression of this gene could increase the lutein content. These data suggested that development of useful genes from this alga was possible. This psy gene could also complement the psy mutant of Chlamydomonas reinhardtii, and made it possible to grow and show a green color under light.3. The phytoene desaturase gene( pds) has been isolated from C. protothecoides CS-41 recently in our lab. The PDS desaturation activity of the expressed protein in phytoene-accumulated Escherichia coli was confirmed by the resulting production ofζ-carotene, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis,a single amino acid mutation( N144D) at the 430 thnucleotide was identified and confirmed. This mutant encoded an inactive enzyme, impling that the 144 thamino acid was crucial to the activity of the PDS enzyme. The promoter of phytoene desaturase gene was also isolated from C. protothecoides CS-41, which contained some light induction and methyl jasmonate(Me JA)-response elements, but the expression of pds gene was down-regulated by Me JA.4. The environmental factors, sodium chloride( Na Cl), ABA and light were involved in this study. Their effect on the growth of C. protothecoides CS-41 and the expression of key genes involved in carotenoid biosynthesis pathway were studied. The results showed that C. protothecoides CS-41 could grow well in the media with Na Cl concentration less than 300 m M, but not when more than 600 m M, which will seriously affect the growth of this alga. Na Cl treatment caused the phenomenon,inducing cell wall thickening and lipid accumulation. Na Cl has little effect on the expression of psy, pds, zds, lcye genes of C. protothecoides CS-41 after treated with tested concentrations, while the expression of zep gene was upregulated and lutein in C. protothecoides CS-41 was accumulated when treated with Na Cl concentration less than 300 m M. The expression of all these genes was down-regulated when treated with exogenous ABA. Light upregulated the expression of all these genes involved in carotenoid biosynthesis with different levels of promotion, especially the expression of psy gene could be upregulated by light. In addition, the expression of these genes under two conditions of light or dark were compared when treated with salt stress, and the result showed that light could alleviate the effects of salt stress on C.protothecoides CS-41.5. C. vulgaris C-27 was chosen to set up the chloroplast transformation system for Chlorella, since its chloroplast genome has been fully sequenced. According to the chloroplast genome information, the gap between trn L and psb H genes was selected to insert the aad A gene expression cassette. The chloroplast transformation was doneby gene gun, and some resistant clonies were obtained, although they need to be proven as real transformants. The transformation system also requires optimization. |
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