The Studies On MiRNA Responsing To Salvia Miltiorrhiza Continuous Cropping Problem And Its Role In Secondary Metabolic Regulation

Salvia miltiorrhiza continuous cropping problem is a major factor limiting the artificial cultivation of the traditional Chinese medicinal herb Salvia miltiorrhiza.Continuous cultivation of Salvia miltiorrhiza not only impacts yield,but also reduces the production of salvianolic acid B and tanshinone.MiRNA is a kind of small molecule non-coding RNA,which negatively regulates the expression of target genes after transcriping.In order to explore the role of miRNA in salvia miltiorrhiza continuous cropping problem and secondary metabolism biosynthesis synthesis,this study identified the miRNA and compared differentially expression in continuous cropping and non continuous cropping through the miRNA high-throughput sequencing technology and molecular biology experiment,and study the tolerance of miR164 a to allelopathic autotoxicity and its role in secondary metabolism synthesis.The main results as follows:(1)Two small RNA libraries were obtained from first-year(FPR)and second-year plant(SPR)roots.Unique sequences of 2,050,638 and 1,512,866 were obtained from two libraries,respectively.Bioinformatics analysis revealed that 110 known and 7 novel miRNAs were annotated in the roots of salvia miltiorrhiza.Moreover,39 known and 2 novel miRNAs were identified and validated for differential expression in FPR compared with SPR.Eventually identified 110 from two library known miRNAs and seven new miRNAs.The target genes of differentially expressed miRNA were analyzed by bioinformatics software,degradation group data and qRT–PCR.At the same time,the content of salvianolic acid and tanshinone in the first year planting and continuous Danshen was determined.It was finally determined that miR160 a and miR164 a were most likely involved in the response to salvia miltiorrhiza continuous cropping and the regulation of secondary metabolism.(2)In order to verify the biological function and mechanism of miR164 a in salvia miltiorrhiza,we constructed the overexpression vector of miR164 a and obtained the overexpression transgenic hairy root of miR164 a.Then psRNAtarget was used to predict its target genes,and SmNAC2 and SmNAC3 were preliminarily determined.Quantitative PCR analysis showed that the expressions of SmNAC2 and SmNAC3 were significantly down-regulated in transgenic hairy roots.Compared with wild-type hairy roots,transgenic hairy roots overexpressing miR164 a were significantly tolerant after treating with coumaric acid(Simulating allelopathic substances in continuous cropping)with a final concentration of 5-100?g /mL.In addition,we also found that the biomass of hairy roots overexpressing miR164 a was only 32.43-47.84% of the wild-type.Tanshinone IIA(T-IIA)levels were also reduced to 53% to 66% of the wild-type.However,the content of salvianolic acid B in overexpressed hairy roots was significantly higher than that in wild type.The expression levels of PAL1,C4 H,CYP98A14,TAT and HPPR,key enzyme genes of salvianolic acid synthesis pathway,were up-regulation.The expression levels of HMGR1,DXS2,DXR,GGPPS1,CPS1,KSL2 and CYP76AH1 were significantly inhibited in the tanshinone synthesis pathway.These results suggest that miR164 a may increase resistance to continuous cropping problem,while simultaneously promote synthesis of salvianolic acid B and reduce the content of T-IIA.(3)To further investigate the role of these target genes in the regulation of secondary metabolism of salvia miltiorrhiza,we obtained the overexpression hairy roots of SmNAC2 and SmNAC3(OE-SmNAC2 and OE-SmNAC3)and silencing expression hairy roots(RNAi-SmNAC2 and RNAi-SmNAC3)by genetic transformation.The tanshinone ?(T-?)and T-?A content of transgenic hairy roots overexpressing OE-SmNAC2 decreased significantly compared with wild type,the T-I in the strains of OE-SmNAC2-1,2 decreasing to 59.31% and 52.68% of wild type,and T-IIA content to 65.4% and 59.28%,respectively.There was no significant change in the content of salvianolic acid B.However,the content of four tanshinone and salvianolic acid B in SmNAC2-RNA interference(RNAi-SmNAC2)transgenic hairy roots increased significantly,with the T-I of RNAi-NAC2-1 and RNAi-NAC2-2 increasing to 4.22 and 3.14 times of wild type,T-IIA increasing to 3.10 and 5.09 times,respectively,and the content of salvianolic acid B increasing by 2.43 and 3.21.times.The OE-SmNAC3 S.miltiorrhiza hairy roots improved the content of four tanshinone significantly,but the content of salvianolic acid B decreased(T-I increasing to 5.68 and 5.11 times,T-IIA increasing to 10.03 and 7.54 times,respectively,while content of salvianolic acid B decreasing to 54.5% and 75.26%).RNAi-SmNAC3 significantly decreased the T-?(24.56% and 23.34% of wild type)and T-? accumulation(46.15% and 30.5% of wild type)without obvious impact on DT-? and CT,but increased the content of salvianolic acid B(increasing 1.47 and 1.25times).Due to the negative regulation of miRNA on target genes,Because miR164 a positively regulated salvianolic acid B and negatively regulated the T-IIA,its target should negatively regulated salvianolic acid B and positively regulated the T-IIA according to the principle of miRNA negative regulation target genes.so it is possible that SmNAC3 in the two target genes of miR164 a plays a major role.(4)In order to verify the regulatory role of miR160 a in the synthesis pathway of tanshinone and salvianolic acid,overexpression vectors of miR160 a were constructed and transgenic hairy roots with overexpression miR160 a were obtained.Compared with wild-type hairy roots,the overexpression of miR160 a significantly increased the biomass of transgenic hairy roots,which was 1.5-1.9 times as much as that of wild-type hairy roots.The content of tanshinone was significantly decreased,and the expression of key enzyme genes in the synthesis pathway of tanshinone was also significantly inhibited.In addition,the tanshinones in the other two strains could hardly be detected.The content of salvianolic acid B increased by 1.3-1.9 times.Detection of plant hormones IAA,JA and SA showed that the content of IAA in overexpressed transgenic hairy roots increased,while the content of JA and SA decreased,suggesting that miR160 a may negatively regulate the biosynthesis of tanshinone by affecting the auxin signal.