Transcriptome Sequencing Of Haematoccus Pluvialis And Cloning And Analysis Of The Acetyltransferase Gene

Haematococcus pluvialis,a unicellular eukaryotic green algae,was concerned because it can accumulate a large number of astaxanthin under stress conditions such as strong light,high temperature,high salinity and nitrogen starvation.However,the molecular regulatory mechanisms are still unclear due to involving a large number of genes in astaxanthin biosynthesis and the complex regulatory network.At the same time,a new generation of high-sequencing technology has greatly facilitated the study of transcriptomics,without limit of species and the reference genome.By this way,we can quickly research the complex regulatory networks.In order to understand the molecular mechanism of astaxanthin biosynthesis,the total RNA of Haematococcus pluvialis 192.80 in the early stresses was RNA-seq.Through data analysis,key genes involved in transcription regulation were found,leading to understanding the regulatory network of astaxanthin biosynthesis.Results are as follows:(1)Both HpCrtR-B and Hpbkt1 were detected by semi quantitative RT-PCR under different stresses indicating that these key genes could express under the condition of 45 m M sodium acetate(Na AC)and 550 ?mol /m2/s high light.The transcription of HpCrtR-B and Hpbkt1 genes could be detected after 1.5 hours of stresses and the accumulation of astaxanthin was observed in algal cells after 6 hours;(2)To obtain total RNA with high quality from Haematococcus pluvialis 192.80,factors such as the different culture medium,the cell wall breaking methods and the extraction methods were compared.The optimum method for extracting total RNA was as that Haematococcus pluvialis cultured in MIX medium with continuous light25 ?mol/ m2/ s until to the logarithmic phase and total RNA was isolated with the improved Trizol method after collecting cells by centrifugation.(3)Total RNA of Haematococcus pluvialis 192.80 under early stress was extracted,sequenced,spliced and annotated,resulting in obtainment of 59057482 clean reads and 83869 unigenes with average length of 747 bp.Based on the functional classification of GO,KOG and KEEG in transcriptome,the data showed that large amounts of unigenes were associated with protein transport,catalytic activity and translation implying that the intracellular metabolism was very strong in order to address stresses.Through homologous searching(Blast X)against the Plant TFDB database,478 transcription factors(TFs)were found,including member of the transcription families AP2/EREBP,b HLH,b ZIP,C2H2,MYB and WRKY showing that a large number of transcription factors involved in the gene regulation and transcriptional activation were expressed in the early stage of stresses.(4)Moreover,a total of 4367 differentially transcribed genes(DTGs)were detected by comparing the gene expression levels after induction.Of them,2050 and2317 DTGs were un-regulated and down-regulated expression,respectively.According to results of fluorescent quantitative RT-PCR,75% of DTGs were identical with transcriptome data.Analysis of GO and KEEG showed that up-regulated genes were mainly related with catalysis and metal ion binding,while down-regulated genes were focused on small molecular metabolism.In the meantime,71 differentially expressed transcription factors were found.Of them,28 TFs related with catalytic activity were activated and 43 TFs involved in stress-tolerance were depressed.According to the analysis of transcription level,it was found that TFs showed difference when treated with high light and sodium acetate.Among them,the transcription level of HpGNAT-1 increased 300 times on the condition of sodium acetate for 6h,while 30 times after high light.(5)Finally,HpGNAT-1 gene was cloned and bioinformatics analysis was carried out.Results of homology analysis showed that the HpGNAT-1 with length of 493 bp might be the N-acetyltransferase(NAT).NAT which translated into 163 amino acids and held isoelectric point of 5.92 might be localized in the cytoplasm and have no signal peptide and transmembrace region.NAT contained 4 alpha helices and 7 beta folds might own HAT domain and Rim I domain.The prediction of HAT structure implied that it could bind with Co A and perform its function by acting on the ribosomal protein S18 subunit.In a word,Haematococcus pluvialis positively responded to stresses through signal transduction,transcriptional activation and protein catalysis in the early stage of high light and sodium acetate stress.A numerous of genes related with signal transduction,transcriptional activation and protein catalysis were un-regulated,while genes involved in small molecular metabolism were down-regulated.TFs played a role in transcriptional regulation by differentially responding to the stresses of high light and sodium acetate.