Analysis Of MiRNA Transcriptome From Haematococcus Pluvialis And Study On MiRNA-TF Co-regulated Networks

Although the biosynthesis pathway of astaxanthin in Haematococcus pluvialis is uknown,we still unknow the regulatory mechanism of astaxanthin synthesis.With the development of high-throughput sequencing technologies,small-RNA,transcriptome,and the other omics technology have become more popular.Multi-omics association analysis has become an essential tool for the study of complex gene regulatory networks.In this study,the sequence of known mi RNAs and novel mi RNAs were obtained after sequencing small RNAs from Haematococcus pluvialis 192.80 at the early phase of stresses.Through target gene prediction and enrichment analysis,we found mi RNAs that might involve in the regulation of astaxanthin synthesis.The negatively correlated mi RNA-target gene pairs were found through small RNA and transcriptome association analysis.Moreover,transcription factor?TF?genes were obtained by blast in database and the regulatory network were in further research associated with the relationship between transcription factors and m RNA.Finally,mi RNA-TF co-regulatory network on astaxanthin synthesis was constructed according to the above results.The specific research content and results are as follows:?1?A total of 9,363,208 clean tags were obtained after high-throughput small RNA sequencing of Haematococcus pluvialis at the early phase of high light and sodium acetate stress.Small RNAs showed peaks at 21 bp in length.A total of 276 known mi RNAs belonging to 259 families respectively were identified.In addition,300 novel mi RNAs were also predicted;?2?Nineteen differentially expressed mi RNAs were obtained,of which 14 were up-regulated and 5 were down-regulated.Down-regulated mi RNA expression patterns were the same,while up-regulated mi RNAs had different expression patterns.Through the prediction and enrichment of target genes,it was found 2122 target genes and some mi RNAs such as hp-mi R167-5p were involved in the synthesis of carotenoids and lipids;?3?Six negatively correlated mi RNA-target gene pairs were detected by m RNA-mi RNA association analysis.It was shown that mi RNA might involve in the response to stress and function in Haematococcus pluvialis;?4?Results of extracting small RNA demonstrated that both RNAiso for Small RNA?TAKARA?and Micro RNA Fast Extraction Kit?Bio Teke?could be used to extract small RNA of Haematococcus pluvialis.Relatively speaking,RNAiso for Small RNA?TAKARA?had better extraction efficiency.In addition,we obtained a stable internal reference M-3 for mi RNA q RT-PCR from Haematococcus pluvialis after screening,verification and comparison.Then,q RT-PCR was performed to verify the expression of negatively correlated mi RNA-target genes,showing the negative correlation between mi RNA and target genes;?5?Based on the transcriptome data of Haematococcus pluvialis under combined stress of strong sunlight and sodium acetate,13 key enzyme genes involved in the synthesis of astaxanthin including PDS,ZDS,LCY,BKT and crt R-B were found and their transcription were in high level when treated with 1.5h and 3h.Among them,the expression of crt R-B?Ch_GLEAN₁0006000?increased to 24 times at 1.5 h.Moreover,the transcription level of 11 key enzyme genes for the synthesis of astaxanthin including IPI?GGPS?PSY?BKT?CRTZ were on its peak when stress for24 h and 48 h.Among them,the expression of BKT?XLOC₀38901?increased to 19.6times at 24 h,and the expression of CRTZ?Ch_GLEAN₁0005232?increased to16.07 times.In addition,genes associated with TAG synthesis were also observed during different stress phases.In particular,the expression of 2 G3 PDH enzyme genes,4 GPAT genes,and 9 DGAT genes was in high levelwhen treated with 24 h and 48 h,showing that the synthesis of TAG in Haematococcus pluvialiswas active.?6?By blast in database,332 transcription factors belonging to 27 transcription factor families were found.According to the result of constructing transcription factor regulatory networks,there are 6 transcription factors involved in the regulation ofastaxanthin synthesis,mainly regulating carotenoid oxygenase.In addition,we also found that 5 transcription factors involved in the regulation of lipid synthesis,targeting diacylglycerol acyltransferase gene and so on.?7?In the studies of mi RNA-TF co-regulation network by small RNA analysis associated with transcriptome data,we found 4 mi RNAs and 9 transcription factors were involved in the regulation of astaxanthin and lipid synthesis in Haematococcus pluvialis.The enzyme genes associated with astaxanthin synthesis and lipid synthesis in Haematococcus pluvialis both positively regulated by transcription factors and negatively regulated by mi RNAs.Both pathways had their own unique transcription factors and mi RNAs,as well as common mi RNAs and transcription factors.These results showed that there was a close relationship between astaxanthin synthesis and lopid synthesis in Haematococcus pluvialis.In summary,mi RNAs were found in Haematococcus pluvialis cells and they showed function in gene regulation.Through the mi RNA-TF co-regulation network,we found that both mi RNAs and transcription factors were involved in the regulation of astaxanthin and lipid synthesis in Haematococcus pluvialis.Moreover,mi RNAs negatively regulated enzyme genes,while transcription factors positively regulated enzyme genes to achieve the aim of regulation at the transcriptional level.Some common mi RNAs and transcription factors might contribute to the synchronization in transcriptional levels,ultimately regulating astaxanthin synthesis at the transcriptional level.Hence,our studies established foundation to reveal the regulatory mechanism of astaxanthin synthesis in Haematococcus pluvialis.