Molecular Mechanisms Of Drought Stress Affecting Saponins Synthesis Pathway Of Panax Notoginseng

Plant small RNA(sRNA)is a type of non-coding RNA with 18-24 nt long,which is involved in the regulation of plant growth and development,reproduction and genomic recombination.At the same time,small RNA also plays a very important role in plant response to abiotic stress and tolerance.Drought stress is the most common environmental stress encountered by plants,and the drought response of plants is a complex process.Panax notoginseng is one of the medicinal plants with a long history of planting in China and capable of large-scale planting with high economic value.With the increasing global warming effect and the frequent occurrence of extreme climate,studying how drought stress affects the growth and content of active constituents in Panax notoginseng and its related mechanisms can not only provide scientific basis for high quality cultivation management for Panax notoginseng production,but also expand the research of medicinal plant physiology and secondary metabolites.In this paper,we hope to explore the regulation pathway of saponin terpenoids skeleton pathway mediated by small RNA in Panax notoginseng by analyzing high-throughput sequencing data.Especially we will find some miRNA and their target genes which are in upstream of tetracyclic triterpenoids synthesis,which are in terpenoid backbone biosynthesis.In order to expand the understanding of miRNAs in the regulation of secondary metabolic pathways,and to explore relevant miRNA markers,to lay the foundation for high-quality Panax notoginseng breeding.This article mainly studies from the following aspects.1.After transcriptome sequencing of drought-treated Panax notoginseng leaves and roots,STAR-Cufflinks was used to analyze the transcriptome,and 3539 and 3179 genes with higher expression levels were obtained from leaves and roots,respectively.Gene cluster analysis was performed to obtain 5 and 2 gene classes with different expression trends respectively.The genes of these seven different expression patterns were analyzed by KEGG and GO cluster analysis,and the genes responsible for drought stress and their functions were obtained.In the secondary metabolic pathway,the terpenoids skeleton and the phenylpropanoid metabolic pathway were analyzed,and 15 and 13 drought-responsive genes were obtained.There are 149 and 67 stress-related transcription factors in Panax notoginseng leaves and root.Among them,24 families members are important response proteins in plant endogenous hormone response pathway,with the largest number of ABA and ethylene response protein members.The expression level increases with the increase of drought time.It can be seen that ABA and ethylene play an important role in the drought stress response of Panax notoginseng.2.Through high-throughput sequencing and data analysis of small RNAs,a total of 1584 conserved miRNAs were identified,and 256 conserved miRNA precursor structures were predicted,and 198 non-conserved miRNAs and their precursors were obtained.After predicting these miRNAs’ target genes by CleaveLand,584 target genes of conserved miRNAs and 254 target genes of non-conserved miRNAs were identified.After SOM cluster analysis of these target genes,the expression level under drought stress occurred.The miRNA target genes were analyzed by KEGG pathway and GO function analysis,and 43 miRNAs which can responded to drought stress and regulated transcription factors and have stress response function were obtained.After correlation analysis of the expression levels of these miRNAs and their target genes,8 pairs of miRNA-target genes with significant negative correlation were obtained.After analysis by the KEGG pathway,four pairs of miRNA-target genes related to steroid synthesis were found,which were MIR156aaai-GGPS,MIR482g-ispF,t15589037-ispH and t49562107-HMGCS.These miRNAs can be used as markers for screening droughttolerant or high-quality Panax notoginseng.By analyzing the Panax notoginseng genome,a TAS3 gene,TAS3b(-),was identified and it generated tasiRNA which targeted two ARF genes.The correlation analysis showed that TAS3b(-),TAS3b_D5(-)and TAS3b_D6(-)were negatively correlated with ARF expression,and TAS3b(-)and TAS3b_D5(-)in leaves increased with drought time.The amount of expression increased while the amount of ARF expression decreased.It can be seen that under drought stress,Panax notoginseng leaves activate TAS3 gene to inhibit the expression of ARF gene.3.In the saponin terpenoids skeleton pathway,the steroid skeleton synthesis pathway is an important process for the synthesis of precursors.First,qRT-PCR experiments were performed on all of these genes after short-term(0-24 h)and long-term drought treatment(0-30 d).The results of short-term treatment of Panax notoginseng leaves and root qRT-PCR were similar to those predicted by this study,indicating that the gene expression predicted by this study was more accurate.On the other hand,the long-term drought treatment,the expression of the genes related to the skeleton synthesis of steroids was studied by qRT-PCR,and the expression of other genes was increased in addition to PMK,MVK and CAS.4.In this study,the 4 predicted miRNA-regulated genes were subjected to RLM-5’RACE experiments to obtain a site of targeted miRNA and its target gene,t49562107 and HMGCS.After the full-length ORF sequences of t49562107 and HMGCS were obtained,the t49562107 precursor-RFP and HMGCS-GFP reporter vector plasmids were constructed and transferred into onion epidermal cells for transient expression.It was found that the GFP signal of onion was weakened after transfering into two plasmids.It is suggested that T49562107 has the effect of inhibiting HMGCS.In summary,by analyzing the transcriptome,degradation group and small RNA sequencing data of the leaves and roots of Panax notoginseng under drought stress,this study identified 40 miRNAs that respond to drought stress and participate in the regulation of related transcription factors and stress-related proteins,and 4 miRNAs that responded to drought stress and regulated saponin skeleton anabolism-related genes,and we identified miRNA cleavage sites of HMGCS gene.The study provides a basis for that miRNAs act on key genes for saponin skeleton synthesis.This study will also provide an experimental basis for the scientific control of water to improve the content of notoginsenosides in Panax notoginseng,and lay a foundation for the screening of drought-resistant or high-quality Panax notoginseng breeding.