Target Gene Prediction And Analysis Of Chlamydomonas Reinhardtii MiRNA1166.1

Gene expression was regulated by diverse ways.The importance of non-encoding single stranded micro RNA(mi RNA)modulating gene expression has been widely recognized in animals and plants.Mi RNA was encoded by endogenous genes.In previous study in our laboratory,a high H2 production transformant-mi RNA1166.1(T-mi RNA1166.1),which expression backbone of mi RNA1166.1 was a high abundance cre-MIR1162 MI0006223,has been obtained through genetic engineering.On the basis of previous study,the target gene of endogenous mi RNA1166.1 were predicted,and the function of these predicted target genes were investigated in this study.Moreover,a luciferase reporter vector were established for the mi RNA1166.1 target genes verification.The results were as following:1.Use the WMD3 website to predict the target gene of mi RNA1166.1,the results showed 12 the most reliable target genes;2.Use the technology of quantitative real-time PCR(q RT-PCR)to verify the gene expression of mi RNA1166.1 in T-mi RNA1166.1-1 and T-mi RNA1166.1-2,the relative expression level of mi RNA1166.1 was up-regulated average of more than 5 times compared to control wild type algae,respectively;3.Use the technology of q RT-PCR to detect the target genes in T-mi RNA1166.1-1 and T-mi RNA1166.1-2,the gene expression of Cre11.g472700,Cre22.g763650,Cre09.g407501 showed a significant down-regulated trend.In T-mi RNA1166.1-1,the relative expression level of Cre11.g472700,Cre22.g763650,Cre09.g407501 was 0.310 ? 0.373 ? 0.476,respectively.In T-mi RNA1166.1-2,the relative expression level of Cre11.g472700,Cre22.g763650,Cre09.g407501 was 0.262?0.500?0.684,respectively.Comparing to control wild type algae,the relative expression level of Cre17.g696700 and Cre03.g189000 was up-regulated,Cre03.g169150 and Cre01.g064550 was no significant change,Cre03.g151000 and Cre13.g571400 showed different trend.4.A luciferase reporter vector was constructed the constitutive high expression Psa D 5' promoter,Luc,8 multiple Cre05.g238140 target gene sequence(8×target)and Psa D 3' terminator.This recombinant plasmid was next transformed into Chlamydomonas reinhardtii cells by glass-bead method;5.Use the technology of q RT-PCR to detect the gene expression of Luc in 7 Transformant-p DL-8×target(T-p DL-8×target),the results showed that a significant silence of Luc compared to control wild type algae;6.Detection of the luminescence in 7 T-p DL-8×target,the results showed that a significant decrease in luminescence,and the results were consistent with the expression of Luc.In conclusion,we have investigated the screening and validation of an endogenous mi RNA target gene of Chlamydomonas reinhardtii,and verified the expression of mi RNA1166.1 by using q RT-PCR technology and luciferase reporter vector.The function of non-coding RNA for single-cell eukaryotic green algae has opened up a new direction for biological prediction of target genes.