| MicroRNAs(miRNAs)are a class of endogenous non-coding RNA with about 21-24 nucleotides.As a conservative post-transcriptional regulator,they play an important role in the regulation of gene expression in cells.Dunaliella salina is a kind of photosynthetic halophilic algae lacking rigid cell wall,which can adapt to various extreme environments,especially high light and high salt.When D.salina survives in this environment,a large amount of ?-carotene accumulates in cells with the form of lipid globules,which are essential for cellular protection.In this study,to find out whether miRNAs are involved in the regulation of intracellular ?-carotene accumulation in D.salina,the miRNA expression profiles of strain CCAP19/18 were analyzed before and after high-light and high-salt stress treatment using highthroughput sequencing technology.A series of differentially expressed miRNAs were identified and their target genes were predicted.Interestingly,miRNA novel-m0533-3p might promote carotenoids synthesis by inhibiting its target gene.The results are as follows:1.Observation of D.salina cells by transmission electron microscopy showed that the number of ?-carotene lipid globules in the treatment group was four times as much as that in the control group,indicating that the content of ?-carotene in D.salina increased significantly after high light and high salt treatment.Further analysis by high performance liquid chromatography(HPLC)showed that the intracellular ?-carotene content of D.salina increased from 44.90 mg/g to 305.63 mg/g dry weight after treatment.2.High throughput sequencing was used to analyze the expression profiles of small RNAs in D.salina before and after treatment.11,542,169 and 16,877,598 clean tags were obtained respectively.The results showed that the length of small RNA in D.salina was mainly 21 nt,and its first nucleotide preferred Uridine(U).3.By analyzing the expression profile of small RNA in D.salina,84 known miRNAs and 1054 unknown miRNAs were identified in this study.Among them,9 known microRNAs and 39 novel microRNAs were significantly altered after high light and high salt stress(32 differentially expressed miRNAs were up-regulated and 16 novel miRNAs were down-regulated).Prediction,annotation and enrichment of target genes of miRNAs indicate that there may be multiple predictive target genes for miRNAs of D.salina.Differential expression of target genes of miRNAs is related to biological and molecular functional processes.Among them,most of the target genes are involved in the binding process,followed by the catalytic activity process,cell process and metabolic process.4.Twenty-four randomly selected miRNAs were verified by qRT-PCR.The results showed that 17 miRNAs could be successfully identified in the control group and the treatment group.Five of them were significantly up-regulated in the treatment group.In particular,novel-m0857-5p and novel-m0533-3p achieved by 7.47 and 3.83 times respectively in the treatment group.Correspondingly,the four predictive target genes of novel-m0783-5p were down-regulated by 71.72%,57.01%,53.14% and 42.93%,respectively.Interestingly,the malic dehydrogenase,as predicted target gene of novel-m0533-3p,was down-regulated by 68.88%.5.Western blot was result support the protein expression level of malic dehydrogenase decreased.Combining with the results of qRT-PCR,this result indicates that miRNA novel-m0533-3p might play a role in splicing target gene.6.The expression levels of several key genes involved in carotenoid metabolic pathway in D.salina were also analyzed by qRT-PCR.The expression levels of PSY,PDS and LCYB were up-regulated in the treatment group.PSY,in particular,increased 17 times compared with the control group,indicating that the metabolic pathway of carotenoids was significantly strengthened.In conclusion,D.salina accumulates ?-carotene under high light and high salt stress,and our results confirmed that miRNAs involved in the metabolic pathway of ?-carotene synthesis.This study preliminarily elucidated the molecular regulation mechanism of miRNAs involved in the accumulation of ?-carotene in D.salina.Moreover,this study provided an alternative to improve the content of ?-carotene in D.salina by gene regulation. |
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