Study On The Regulation Mechanism Of LncRNA In Photosynthetic Hydrogen Production From Chlamydomonas Reinhardtii

Background:As important regulatory RNAs,long non-coding RNAs?lncRNAs?have been found and largely studied in animals and plants.However,reports on lncRNAs in model organism Chlamydomonas reinhardtii were still limited.Previously,we have performed RNA-seq with C.reinhardtii treated before and after the sulfur-deprivation to identify the lncRNAs and its predicted target genes.In this study,four lncRNAs[XLOC₀37917?lncRNA-A?,XLOC₀37246?lncRNA-B?,XLOC₀32037?lncRNA-C?and XLOC₀27503?lncRNA-D?]have been selected based on their predicted target gene?LHCBM2,LHCBM7,LHCA9 and FDX5,respectively?.In this study,we want to investigate the regulatory mechanism of lncRNAs potentially involved in photosynthetic hydrogen production in C.reinhardtii.To this end,over-expressed lncRNA vectors?pH-lncRNA?as well as knock-down lncRNA vectors?pH-amiR-lncRNA?have been constructed to regulate the corresponding lncRNA expression.After alga transformation,expression of the predicted target genes,photosynthetic efficiency and alga growth,as well as the photosynthetic hydrogen production were compared and analysed.Results:Over-expression and knock-down vectors were successfully expressed in C.reinhardtii.In over-expression transgenic alga strain,lncRNA was over-expressed,and target gene expression was up-regulated.While over-expressing lncRNA-A and lncRNA-B,LHCBM2 were up-regulated by 55.07%and 33.24%,respectively,and LHCBM7 was up-regulated by 221.44%and 155.33%,respectively.While lncRNA-C was over-expressed,LHCA9 was up-regulated by 137.00%.While over-expressing lncRNA-D was over-expressed,the expression of FDX5 increased to60.99 times.After knock-down lncRNA in C.reinhardtii,target gene expression was down-regulated.Silencing lncRNA-A and lncRNA-B,LHCBM2 were down-regulated by 47.03%and 40.40%,respectively.LHCBM7 was down-regulated by 39.20%and79.67%,respectively.Silenced lncRNA-C,LHCA9 was down-regulated by 23.90%.Silenced lncRNA-D.FDX5 was down-regulated by 69.85%.The photosynthetic efficiency of the over-expression lncRNA-A,lncRNA-B,and lncRNA-C transformants didn't change significantly,and the over-expression of lncRNA-D significantly decreased the photosynthetic efficiency.The photosynthetic efficiency of C.reinhardtii lncRNA-D transformed strains was significantly increased compared with CC849.Low light(50?mol·m^-2·s^-1)cultures significantly increased the hydrogen production of the lncRNA-D transformants and increased it by 5.67 times.The hydrogen production of the lncRNA-A and lncRNA-B transformants decreased by14.26%and 44.33%;There is no significant change in lncRNA-C over-expression transformants;Under high light(100?mol·m^-2·s^-1)conditions,hydrogen production increased by 51.02%and 74.90%in lncRNA-A,lncRNA-C over-expression transformants,respectively.Under low light(50?mol·m^-2·s^-1)conditions,gas production in lncRNA-D silenced transformants decreased by 32.56%.Conclusion:Over-expression and knock-down lncRNA vectors have been successfully constructed and transformed into C.reinhardtii.Up-regulation of predicted target genes has been observed after over-expression of lncRNAs whereas knock-down of lncRNAs can down-regulate the expression of these target genes.More interestingly,lncRNA-D showed a tight relationship with photosynthetic hydrogen production in C.reinhardtii.Over-expression of lncRNA-D resulted in an improved hydrogen production capacity.Mechanism of action of lncRNA-D involved in the pathway of photosynthetic hydrogen production should be studied in details for the next step.