Gene Cloning And Expression Analysis Of Carotenoid Hydroxylase From Two Green Algae

Carotenoids are a group of lipophilic terpenoids with antioxidant properties that are widely distributed in photosynthetic organisms, including higher plants and algae. They are composition and functional components of the photosynthetic reaction center and play an important role in light harvesting and transmission, in protecting chlorophyll from photo damage. In addition, carotenoids exert important physiological functions as well, such as enhancing immunity and preventing cardiovascular diseases and cancer. Meanwhile they have been widely used in many industrial fields of food, cosmetic and animal feeds. Recently, the carotenoids biosynthetic pathways and related enzymes and genes have been extensively studied. Carotenoid hydroxylase is the key enzyme in xanthophylls biosynthetic pathways because they can introduce hydroxyls in β-ring and ε-ring of carotein to form many kinds of intermediate products which are necessary for xanthophyll synthesis.Based on homologous cloning and RACE methods, two full-length cDNA of carotenoid hydroxylase Ckecyp97a and Haecyp97c were obtained from green alga Chlorella kessleri CGMCC 4917 and Haematococcus pluvialis NIES-1844, respectively. Furthermore gDNA sequences of Ckecyp97a and Haecyp97c were cloned and analyzed. Then a series of bioinformatics methods were applied to analyse nucleotide and protein sequences, to predict the physicochemical properties, transmembrane regions and subcellular locations. The results indicated that Ckecyp97a and Haecyp97c were probably located in the chloroplast.Multiple sequence alignment of Ckecyp97a and Haecyp97c with other predicted CYP97Cs from several green algae and higher-plants, revealed that these two genes contained all the conserved domains associated with active sites of the P450 family. It is likely that the results supply an important clue that they belong to P450 family. The phylogeneic analysis shows that genes coding CYP97A is evolutionarily more closely to CYP97C and CYP97B is an ancient gene.The 5’-flanking regions of Haecyp97c were obtained by genomic walking methods which showed a great variety of cis-acting elements including various hormone response elements, numerous promoter/enhancer regions and high light responsive elements. The prediction of these cis-acting elements laid the foundation for research on regulatory mechanism of carotenoids synthesis.To further explore the effects of high light on mRNA expression levels of Ckecyp97a /Haecyp97c and composition of carotenoids, the photoheterophic green alga C. kessleri and photoautotrophic green algae H. pluvialis in exponential growth status were treated with different high light (white and blue) stresses. In H. pluvialis the transcriptional levels of Haecyp97c increased markedly throughout the course of high light stress conditions, in addition zeaxanthin and astaxanthin concentration under both high light were much higher than that of the control and Haematococcus cells reacted quickly and more sensitive to blue HL induction compared to white HL stress. But in C. kessleri Ckecyp97a transcript increased strongly under white HL treatment, while decreased under blue HL treatment. Furthermore white HL stimulation enhanced the biosynthesis of zeaxanthin under photoheterotrophic culture conditions, while blue HL may be without impact on the biosynthesis of zenxanthin. Our results demonstrate that Ckecyp97a and Haecyp97c are the enzymes responsible for carotenoid hydroxylation involved in HL acclimation for C. kessleri and H. pluvialis respectively and they respond differently not only to distinct stresses but also to culture condition at transcriptional level.Three prokaryotic expression vectors of Haecyp97a/b/c were constructed and recombinant proteins were detected by SDS-PAGE after IPTG induction. Then prokaryotic expression vectors were transformed into E.coli which can produce (5-carotene and analysis of carotenoids composition showed that β-carotene concentration declined but objective products were not detected. Therefore, the functions of these genes need to be studied further.