Cloning And Functional Analysis Of The Precursor Sequence Of Salvia MiR408

Plant miRNAs are a class of 20-24 nt small non-coding RNAs,which reglute target gene expression by guiding direct cleavage of mRNAs or hampering mRNAs translation.MiR408 is a highly conserved family that has been identified in many plants.The function of miR408 in plant is mainly involved in abiotic stress response and maintaining the balance of copper homeostasis.It was found that the anthocyanin content of miR408 in overexpressing Arabidopsis seedlings was 7-8 times higher than wild-type,suggesting that miR408 might be involved in the regulation of plant secondary metabolism.Salvia miltiorrhiza Bunge,a traditional perennial medicinal plant of Chinese,has a good effect on cardiovascular,cerebrovascular diseases,tissue damage,high blood lipids and so on,known as a new type of "mode medicinal plant".Recently,there is no report about the function of miRNA in Salvia miltiorrhiza.In this study,we successfully cloned miR408 precursor from S.miltiorrhiza,the transgenic over-expression tobacco and transgenic Salvia miltiorrhiza lines were obtained to study the biological function of secondary metabolites.The main contents and results are listed as follows:1.Based on the genomic survey database of S.miltiorrhiza established in our lab,a 366 bp miR408 precursor sequence was cloned and named Sm-MIR408.The 723 bp promoter upstream region of Sm-MIR408 was obtained by PCR.Bioinformatics analysis showed that Sm-MIR408 can form stable stem loop structure and mature miR408 was highly conserved.There are several light responsive elements and methyl jasmonate responsive elements in the promoter region by PlantCARE database analysis.The real-time quantitative PCR analysis showed that Sm-MIR408 was expressed in roots,stems,leaves and flowers of S.miltiorrhiza and influenced by copper,light and methyl jasmonate,etc.2.Promoter-GUS recombinant plasmid was constructed by the traditional double enzyme digestion method.Then we generated Sm-MIR408-Promoter:GUS transgenic lines with the method of Agrobacterium-mediated transformation of tobacco leaf discs.Based on the DNA and GUS histochemical staining identification,11 positive transgenic lines were chose to study the activity of the promoter region of Sm-MIR408.According to the analysis of cis-acting elements in promoter region,transgenic tobacco were treated with different concentrations of copper,methyl jasmonate,injury and light,and the results of GUS staining showed that promoter region of Sm-MIR408 was regulated by the above factors.Compared with the control(0.1 uM Cu2+),the expression of GUS in tobacco seedlings was increased under the condition of copper deficiency(0 uM Cu2+),but in the condition of multi copper(5 uM Cu2+),the expression of GUS in tobacco seedlings was decreased.3.The Sm-MIR408 overexpression vector was transferred into wild type tobacco plants using Agrobacterium-mediated transformation method.According to the results of DNA assay,we obtained 9 overexpression positive transgenic lines.The results of real-time quantitative PCR showed that the expression of Sm-MIR408 and Sm-miR408 in tobacco were higher than that of the control strain.These results indicate that Sm-MIR408 gene had been successfully integrated into tobacco genome.Detection of target of miR408 gene expression in tobacco,the results showed that after heterologous expression of Sm-MIR408,the expression of Copper-transporting ATPase PAA2 and Uclacyanin-2 were significantly down regulated.4.Using artificial microRNAs(amiRNAs)mediated gene silencing method,Sm-MIR408 interference vector was successfully constructed.The RNAi transgenic lines were obtained by Agrobacterium-mediated transformation of S.miltiorrhiza,and 5 positive lines were obtained by DNA and RNA level detection.The amiR-408-3,amiR-408-4 transgenic lines with low expression of Sm-MIR408 were selected for the following experiment.Using bioinformatics to predict the target of Sm-miR408,real-time quantitative PCR results showed that the expression of Sm-LAC13 in the RNAi transgenic lines were significantly higher than the control lines.Thus Sm-LAC13 may be a target for miR408.Analysis of total phenolic acids content and total flavonoids content in the interference lines and control lines,the results showed that total phenolic acids content and total flavonoids content of the interference lines were higher than that of the control lines.HPLC was used to determine the content of active ingredient in vitro culture of 60 d S.miltiorrhiza,the results showed that the contents of rosmarinic acid and salvianolic acid B in the roots of RNAi transgenic lines were significantly higher than the control lines.The content of rosmarinic acid was 1.4?1.8 times of the control lines,and the content of salvianolic acid B was 2.5?4.1 times of the control lines.While for the content of tanshinone ? A,cryptotanshinone,there was no significant difference between transgenic lines and control lines.Furthermore,the key enzyme genes on the secondary metabolism pathway in the RNAi transgenic lines and control lines were analyzed through real-time PCR method.The results showed that expression of phenolic acids biosynthesis genes including TAT?HPPR?PAL2?C4H?4CL2 were significantly up-regulated in the RNAi lines compared with control.In summary,Sm-miR408 may play a role in the regulation of secondary metabolism of Salvia miltiorrhiza,and lay a foundation for further exploring the function of miR408.