Analysis Of The Role Of DpAP2,a Transcription Factor Gene Related To Carotenoid Biosynthesis And Regulation In Dunaliella Parva

Carotenoids are important bioactive substances in Dunaliella parva,which can improve immunity,resist oxidation,eliminate free radicals,inhibit cardiovascular and cerebrovascular diseases,prevent cancer,and are widely used in medicine,health care,food,cosmetics and other industries.However,its low content has greatly restricted the development of the health value of Dunaliella parva.How to increase the carotenoid content in microalgae is an important problem at this stage.Three genes in carotenoid biosynthesis pathway and their promoters were cloned,and the responses of these genes to salt oscillation and nitrogen limitation were studied in Dunaliella parva.At the same time,it was found that some cis-acting elements widely existed in these promoters.But the regulatory mechanism of transcription factors binding to these elements for carotenoid biosynthesis was not yet clear in Dunaliella parva.The AP2/ERF superfamily is one of the largest transcription factors（TFs）families in plants,including AP2,CBF/DREB,ERF and RAV subfamily,which is related to plant growth and development,carotenoid metabolism and the response to various stresses.Investigating the mechanism of DpAP2 is of great scientific significance for elucidating the carotenoid metabolism in D.parva.In this study,the effects of DpAP2 gene on carotenoid synthesis in D.parva were studied through molecular biology,bioinformatics and biochemical techniques to explore the important key enzyme and regulatory genes of the carotenoid synthesis pathway.The core fragment of DpAP2 obtained in the differentially expressed genes was used to amplify by RACE to obtain the full-length DpAP2 sequence and perform bioinformatic analysis.The c DNA library and the yeast two-hybrid system bait vector were constructed.Yeast toxicity and self-activation were tested,and the transcription factor DpAP2 interaction was screened using the yeast two-hybrid technology.In addition,DpAP2 protein was purified and DpAP2antibodies were prepared.Ch IP-Seq was performed with DpAP2 antibody and algae samples to obtain gene fragments bound to DpAP2 protein,to understand downstream target genes regulated by DpAP2 protein.The DpAP2 promoter sequence was obtained and the active function was verified using Genome Walking.We constructed high-yielding carotenoid algae plants and treated D.parva cells with PEG,Ca Cl_2,GA3 and Me JA to increase the content of carotenoids.The response mechanism of microalgae carotenoid synthesis was further analyzed.The study results are as follows:The c DNA of the DpAP2 gene was 3129 bp,the open reading frame（ORF）was 2331 bp,encoding 776 amino acids,the 3′untranslated region（UTR）was 582 bp,and 5′UTR was 216bp.The genbank of the DpAP2 gene was the ON548537.By biological information analysis,the DpAP2 protein p I was 9.14,the relative molecular mass is 83915.48,and the total number of atoms is 11578.The DpAP2 protein is hydrophilic and unstable.DpAP2 contains two AP2domains（AP2-R1 and AP2-R2）,which is highly similar to the domain of Arabidopsis thaliana.It lays the foundation for the next identification of interacting proteins and target genes.The storage capacity of the primary library reaches 2.9x108 pfu/m L,which is much higher than that of the standard library.The gene recombination rate of clones was 99%.The interacting proteins of DpAP2 which may be related to carotenoid biosynthesis were screened by yeast two-hybrid technology,and three interacting proteins were screened.Bioinformatics analysis predicted that they had DNA binding transcription factor activity,protein kinase activity and alpha-D-phosphohexomutase activity.However,there is still a lack of detailed understanding of its functions.A total of 902 target genes of DpAP2 were identified by Ch IP-sequencing.Gene enrichment analysis showed that these target genes were related to RNA transport,carbon metabolism,biosynthesis of secondary metabolites,fatty acid metabolism and other functions.In particular,some important target genes regulated by DpAP2 may affect carotenoid content by participating in carbohydrate metabolism（including starch and sucrose metabolism,photosynthesis,glycolysis/gluconeogenesis,tricarboxylic acid cycle）and carotenoid biosynthesis（such as Z-ISO,CRTISO,AOG）.Overexpression of DpAP2 showed that the carotenoid accumulation in D.parva cells reached 3.17 mg/g DW on the 9th day,which was 46.76%higher than that of WT cells（2.16mg/g DW）.The content of carotenoids increased significantly after PEG（80 ppm）and Ca Cl₂（40 ppm）treatment,reaching 3.1mg/g DW and 2.78mg/g DW respectively.and the antioxidant activity（superoxide dismutase,reducing capacity）of carotenoids and protein content in the treatment group were significantly higher than those in the control group,and the relative expression levels of PDS and GGPS increased significantly by quantitative fluorescence analysis.The DpAP2 promoter was cloned and 1881 bp specific promoter fragment was obtained.Through Plant CARE analysis,the basic elements of the promoter（CAAT box and TATA box）were found.In addition,the promoter also contains stress induced elements,such as light response element（G-box）,gibberellin response element（P-box）,abscisic acid response element（ABRE）and drought response element（MBS）.The inducer gibberellin（GA3）and methyl jasmonate（Me JA）affect carotenoid metabolism in many plants.Therefore,the dosage effect of exogenous substance Me JA was found in algal cells treated with different concentrations of Me JA（10,20,50,100μM）and GA3（10,20,50,100μM）.In other words,a high concentration of Me JA（10-100μM）inhibited the accumulation of carotenoids,and the relative expressions of DpAP2,PSY,PDS and GGPS decreased significantly,inhibiting the accumulation of carotenoids.On the contrary,the relative expressions of DpAP2,PDS and GGPS increased significantly when GA3 was treated with GA3,which promoted the synthesis of carotenoid.