Cloning Of Photoreceptor Genes From Haematococcus Pluvialis And Functional Study On Blue Light Photoreceptors HpChR1 And HpCRY4

Haematococcus pluvialis is a type of freshwater unicellular green algae,which is known to accumulate a large amount of astaxanthin under some conditions.Astaxanthin is a super antioxidant with high commercial value which is widely used in aquaculture,food industry,medical care,cosmetics and health products.It has been demonstrated that astaxanthin content can reach 3-5% of cell dry weight under some stresses.High temperature,high salinity,light treatment and nutrient deficiency are all able to affect astaxanthin accumulation in Haematococcus pluvialis,and light treatment is one of the most important factors for astaxanthin biosynthesis.It is a worldwide hot research topic for how Haematococcus pluvialis senses light,and subsequently light signal transmits and activates astaxanthin biosynthesis in algae cells.In higher plants and Chlamydomonas reinhardtii,photoreceptor-mediated signaling pathways regulate the transcrips of genes related to carotenoid metabolism.Therefore,photoreceptor mediated phototransduction is likely the key pathway for astaxanthin production in Haematococcus pluvialis cells under light treatment.Thus,the aim of this study is to identify the putative photoreceptors in Haematococcus pluvialis and to investigate their functions.After the study of the effect of different wavelengths on astaxanthin accumulation in Haematococcus pluvialis cells,the photoreceptor genes of Haematococcus pluvialis were cloned and analyzed by molecular biology and bioinformatics techniques.The biological functions of two photoreceptors HpChR1 and HpCRY4 were intensively investigated.The results are as follows:(1)The biological characteristics of the Haematococcus pluvialis cells under light treatment with different wavelengths was firstly examined.The results showed that red light promoted the proliferation of Haematococcus pluvialis cells,but had no obvious effect on the accumulation of astaxanthin.In contrast blue light had no significant effect on the proliferation algal cells,but remarkably increased the accumulation of astaxanthin,which was consistent with the reported results.(2)Based on the conserved domains of well-studied photoreceptor proteins in Chlamydomonas reinhardtii,11 EST sequences of photoreceptors in Haematococcus pluvialis were obtained by bioinformatical approaches.Then,the full-length rhodopsins(HpChR1 and HpHKR2)and cryptochrome HpCRY4 genes as well as the full ORF of ultraviolet.Furthermore,these photoreceptor protein sequences were analyzed by bioinformatics approaches,including three-dimensional structure,functional domains,phosphorylation sites and post-translational modification recognition motifs.(3)Then functional study was performed on blue light photoreceptor HpChR1.Similar to its homologue,CrChR2,in Chlamydomonas reinhardtii,HpChR1 possesses seven transmembrane domains and two putative Med15 domains.However,unlike CrChR2,HpChR1 is mainly localized at the endoplasmic reticulum(ER)and may act as a calcium channel.Blue light may activate either HpChR1 at ER to cause intracellular calcium signal transduction,or nuclear HpChR1,both of which lead to the expression changes of astaxanthin synthesis-related genes.In addition,we identified five interacting proteins of HpChR1 by yeast two-hybrid screening.Among them,Ran may facilitate HpChR1 to enter the nucleus and RACK1 may be involved in the regulation of gene expression together with HpChR1.(4)Functional study was also carried out on blue light photoreceptor HpCRY4.HpCRY4 exhibited four types of subcellular distributions in different cell cycle phases.In the progression of cell cycle,the abundance of HpCRY4 protein was decreased from G1 to M phase.During G1/S transition,HpCRY4 in the nucleus decreased significantly.The degradation of nuclear HpCRY4 might be regulated by ubiquitination pathway,since Cop1,an E3 ligase,was demonstrated to interact with HpCRY4 by yeast two-hybrid.In M phase,HpCRY4 was completely eliminated.Since HpCRY4 possesses a putative D-box at its N-terminus,APC/C may be responsible for its degradation.The elimination of HpCRY4 may be a necessity for cell cycle of mitosis.In summary,this study demonstrated the existence of multiple photoreceptors in Haematococcus pluvialis and the function of two blue light photoreceptors HpChR1 and HpCRY4 were intensively investigated.The results showed that photoreceptor-mediated phototransduction may play an important role in calcium signaling,gene expression and cell cycle progression,which are closely related to astaxanthin biosynthesis in Haematococcus pluvialis.Therefore,this study laid an important foundation for elucidating the mechanism of lightinduced astaxanthin biosynthesis.