Cloning And Characterization Of Genes Encoding Phytoene Desaturase And ζ-Carotene Desaturase From Chlorella Protothecoides

Carotenoids are an important group of natural isoprenoid-derivedpigments, which are widely distributed in living organisms including allphotosynthetic organisms, as well as in some nonphotosynthetic fungi andbacteria. They include non-oxygenic carotenes and xanthophylls withoxygenic functional group, and most of natural carotenoids demonstratehigh performance in anti-oxidation effect. Chlorella is notable for its highcellular carotenoid content, especially lutein, which makes it a potentialresource for lutein production. Comparing to other resources, Chlorella hasmany advantages such as the high specific growth rate and the ability ofgrowing heterotrophically. However, previous researches were focusedprimarily on the optimization of cultivation conditions and extractionmethods, rather than on the genetic modification of enzymes directlyinvolved in the carotenoid metabolic pathway.To preliminarily understand their regulatory expression mechanism atmolecular level by cloning of the full-length cDNA of PDS, ZDS genes from Chlorella protothecoides CS-41and their bioinformatics analysis. Thefollowing aspects were included in this study.1. A full-length cDNA of PDS (GenBank Accession No. FJ968162) andZDS (GenBank Accession No. GU269622) were cloned from Chlorellaprotothecoides CS-41by RACE (rapid-amplification of cDNA ends) andRT-PCR, which length are2054bp and2031bp, respectively. It was alsofound that there were two types of DNA sequences for PDS, named PDS1(3857bp, GenBank Accession No. GU269620) and PDS2(1802bp, GenBankAccession No. GU269621), one type of DNA sequence for ZDS (4713bp,GenBank Accession No. GU269623).2. Homology studies showed that the deduced amino acid sequences ofPDS and ZDS genes had a significant similarity with the correspondingsequences of other green algae and higher plants, respectively. It is predictedthat they share the three similar domains: one at the N-terminus for bindingdinucleotides; one in the middle of the sequence for carrying substrate; andthe third at the C-terminus for binding carotenoid. It was notable that theyalso have the transit peptide. The analysis of phylogenetic tree showed theclosest relationship between Chlorella protothecoides CS-41andChlamydomonas reinhardtii.3. The PDS gene expressed in Escherichia coli produced a protein with the expected size61kDa. Furthermore, the PDS protein expressed in the E.coli strain, which containing a plasmid pACCRT-EB, was able to transfer thestrain color from colorless to pale yellow. Taken as a whole, it wasdemonstrated from these results that the cloned cDNA sequence of thephytoene desaturase gene from C. protothecoides was actually a PDS-typegene.4. Sensitivities of Chlorella protothecoides to six common antibioticswere evaluated to find out an optimum concentration of effective antibiotic asa marker for its genetic modification. The results showed that10μg/ml G418completely inhibited the growth of algal cells in the solid and liquid cultures,and the inhibition ratio was about80%and up to about95%to the5μg/mlG418and10μg/ml G418after two weeks, respectively. Therefore, itsuggested that G418be the most suitable selective reagent, therefore NPT-IIgene is considered as best selective marker for genetic engineering ofChlorella protothecoides.5. Bivalent expression vector (pHB-PDS) was constructed whichcontained the PDS gene, and then successfully transformed into Arabidopsisthaliana by Agrobacterium-mediated method. The functional analysis of thePDS is being carried out in Arabidopsis thaliana.