Cloning And Functional Study Of The Precursor Sequence Of Salvia MiR156

Salvia miltiorrhiza Bunge is an important Chinese herbal plant and its dried root has been widely used in modern and traditional Chinese medicine(TCM)for the treatment of cardiovascular and cerebrovascular diseases and various inflammation symptoms.Modern pharmacology study has confirmed that the main medicinal ingredients of S.miltiorrhiza can be divided into water-soluble phenolic acid compounds and fat-soluble tanshinone.MiR156,the only age factor affecting plants growth and development,is a kind of small noncoding RNA whose evolution is very conservative.Existing researches have showed that miR156 not only regulated the growth and development of plants(including transformation,morphogenesis,female gametogenesis reproductive,trichome development,lateral root growth,flowering time,biomass),but also played an important regulatory role in the accumulation of secondary metabolites.To study the function of miR156 in S.miltiorrhiza,we firstly cloned miR156 precursor sequence Sm-MIR156a.Then Sm-MIR156a was overexpressed in tobacco and suppressed in S.miltiorrhiza to investigate the effects of miR156 on plants growth and the contents of secondary metabolitesThe main results and conclusions are as follows:1.Based on a conservative mature Sm-miR156 sequence,S.miltiorrhiza miR156 precursor-Sm-MIR156a,a 465 bp sequence,was cloned from S.miltiorrhiza genome survey database search.Bioinformatics analysis found that the secondary structure of Sm-MIR156a can form stable stem ring structure.Then we used some MiR156 precursor sequences from different plants to build evolutionary tree,finding that Sm-MIR156a evolution is very conservative and it has a high similarity with other sequences.Real-time quantitative PCR results showed that Sm-MIR156a expressed in the root,stem,leaf and flower.The expression was highest in stem and lowest in root.With the growth of S.miltiorrhiza,the expression of MIR156a gradually declined.2.Sm-MIR156a interference vector was constructed using artificial small RNA interference technology.Then we got transgenic S.miltiorrhiza through the method of agrobacterium mediated transformation.Subsequently,we used real-time quantitative PCR to detect Sm-MIR156a expression in different interference strains.As a result,Sm-MIR156a expression were 40%,27%,35%and 35%of control in interference lines A,B,E and K.The expression of 15 predicted target genes SmSPLs in transgenic strains B and E were significantly increased,which showed that the 15 SmSPLs may be the targets of miR156.Fresh weight and chlorophyll content of Sm-MIR156a transgenic interference lines were significantly reduced compared with the control.3.The content of total phenol and total flavonoids in Sm-MIR156a transgenic interference lines(B and E)in root were significantly improved compared with the control.The total phenolic acid content of ground part is significantly higher than control,but the total flavonoids content was significantly lower than control.HPLC results showed that interference of Sm-MIR156a not only had distinct effect on the phenolic acids material content,but had a marked change in tanshinone content.Rosmarinic acid,salvianolic acid B,tanshinone I and cryptotanshinone contents had obvious improvement in interference lines B,E.4.We used real-time quantitative PCR to detect transcript levels for key structural genes in the flavonoids and phenolic acids biosynthesis pathways in interference strain and control.RT-qPCR results showed that the expression of TAT1,HPPR1,PAL2,C4H1 and 4CL2 in phenolic acids biosynthesis pathway in interference strain E were 20.5,5.9,1.8,13.5 and 8.4 times than control,respectively.The expression of ANS,CYP98A14,F3'H,F3' 5 'H,FLS and DFR in flavonoids biosynthesis pathway in interference strain E were 3.8,2.7,6.6,28.9,47.6 and 8.3 times than control,respectively.The expression of CCR and COMT of lignin synthesis pathway in interference strain E were 25.7 and 4.2 times than control,respectively.Comprehensive above analysis,miR156 had a function on regulating the expression of key enzyme genes in phenolic acids,flavonoids and lignin metabolism pathways.5.Sm-MIR156a overexpression vector was constructed and we got a series of transgenic overexpression tobaccos through agrobacterium mediated transformation.Phenotype of transgenic tobaccos showed that the branches and the number of leaves in Sm-MIR156a overexpression transgenic tobaccos significantly increased,but leaves were smaller and younger than control.Fresh weight of transgenic tobaccos increased significantly.We detected the expression of four miR156 target genes,the result showed that the expression of NbSPL4,NbSPL5a,NbSPL5b and NbSPL9 in transgenic overexpression tobaccos were 20%,40%,37%,16%of control.RT-qPCR results showed that the expression of CCD7,CCD8 and BCR1 in strigolactones pathway in OE-1 were 25%,49%and 67%of control.HPLC was used to detect secondary metabolites changes in transgenic tobacco OE-1 and control.As a result,the content of nicotine,caffeic acid and Scopoletin in OE-1 were 47%,30%,28%of control.In summary,Sm-miR156 may involved in plant growth and play a role in the regulation of secondary metabolism of S.miltiorrhiza.Our results lay a foundation for S.miltiorrhiza breeding by plant genetic engineering.