The Study Of Transcription Factor HpHB1 And The Regulational Mechanism Of Astaxanthin Synthesis In Haematococcus Pluvialis

As a unicellular eukaryote,Haematococcus pluvialis is a kind of green algae.When it is exposed to environmental stress(high light,high temperature,high salt,nitrogen starvation,etc.),it will synthesize and accumulate a large amount of astaxanthin,which can occupy the2-5%dry weight of algae cell.So H.pluvialis is a good source of natural astaxanthin.Astaxanthin is a compound with high-strength antioxidant capacity and is widely used in various industries,including aquaculture,cosmetics and health care products.The synthetic pathway of astaxanthin in H.pluvialis has been elucidated basically,among which BKTs and Crt R-B are the key rate-limiting enzymes.However,this synthetic pathway involves a large number of genes,and its complex regulatory networks,as well as the molecular mechanisms(such as the regulation of key rate-limiting enzymes)still have many unclear details.Homeobox(HB)transcription factors are involved in plant growth and development,differentiation,morphogenesis,hormone response,and participate in abiotic stress processes of plants(including strong light,hormones,drought,cold,high temperature,high wet,and salt stress,etc.).At present,the research of HB transcription factors in algae is scarce,which mainly focuses on the origin and evolution of algae and plants basing on the phylogenetic analysis.The research on the structures,functions and the regulations of HB transcription factor in algae are still not clear.The regulatory role and functional position of this kind of transcription factors in algae,especially single-celled green algae are blank.To clarify the regulatory role and role of HpHB1 transcription factor in the astaxanthin synthesis network which responses to early stress of H.pluvialis,this study analyzed the regulation mode and transcription activation activity between HpHB1 and the key enzymes of astaxanthin synthesis,to analyze the function of HpHB1 transcription factors involved in the regulation of astaxanthin metabolism,and lay the foundation for the further research of the molecular regulation mechanism of astaxanthin synthesis.The research contents and results are showed:(1)Analysis of the transcription pattern of Hphb1 gene after high-light and high salt stress by fluorescent quantitative PCR.Under 50?mol/m~2/s intense light stress,the gene transcription levels were significantly up-regulated,reaching a peak at 3 h(up-regulation by11.5-fold).Under 45 m M/L Na AC stress,the gene transcription level was gradually increased,reaching a peak at 2 h(up-regulation by 3-fold).The results show that the expression of Hphb1 gene can be induced by high-light and high salt stress,and the response to high-light is very fast and strong.Therefore,Hphb1 gene may be involved in the regulation of the astaxanthin synthesis pathway.(2)The full length of Hphb1 gene(1429 bp)was obtained and its open reading frame(954 bp)was cloned.Bioinformatics analysis showed that HpHB1 protein has two homeobox domains:KNOX1 and HOX,and belongs to the knotted-1 homeobox transcription factor.(3)A plant subcellular localization expression vector was constructed,and the onion inner epidermal cells were transformed by a gene gun.The subcellular localization analysis of the HpHB1 protein showed that it was localized in the nucleus and conformed to the characteristic of transcription factors.(4)Screening for the interaction protein of HpHB1 by yeast two-hybrid,and verification of the interaction protein through bimolecular fluorescent complementary(Bi Fc).The results show that the heat shock protein(Hp HSP90)and the kinase(Hp PKTc)can interact with the HpHB1 transcription factor.Hp HSP90 may be involved in the folding of HpHB1transcription factor during synthesis,and Hp PTKc may participate in the activation of HpHB1,so that HpHB1 can promote the transcription and expression of target genes and exert its biological function.(5)Screening for DNA cis-acting elements that may bind to HpHB1 transcription factor by DNA-protein interaction.The results showed that the HpHB1 transcription factor may bind to the stress response element(STRE).Promoter analysis showed that the promoter regions of bkts and crt RB have STRE elements,so it is speculated that the HpHB1 transcription factor may use the STRE cis-acting element(A/T)GGGG(A/T)as the target sequence and bind the promoter regions of key rate-limiting enzyme genes(bkts and crt RB)to regulate astaxanthin synthesis.