Identification And Functional Analysis Of MicroRNAs And Their Targets Related To Eu-rubber Biosynthesis In Eucommia Ulmoides

Natural rubber is an isoprenoid polymer.According to statistics in 2019,the global demand for natural rubber has reached 30 million tons and is still under steadily growing.As the main source of natural rubber,Hevea brasiliensis is susceptible to diseases and pests and has a narrow geographic distribution range,which causes enormous challenges for the rubber industry.Eu-rubber is a kind of trans-polyisoprene with the dual characteristics of rubber and plastic.The new material developed by Eu-rubber has the advantages of high insulation and acid and alkali corrosion resistance.Moreover,Eucommia ulmoides Oliver has strong adaptability,wide geographic distribution range,large planting area and produces high quality trans-gum.It is an internationally recognized rubber source tree which an alternative to Hevea brasiliensis,and is an important guarantee for the sustainable development of rubber industry in China.Understanding the molecular regulation mechanism of Eu-rubber biosynthesis is beneficial to improve the content of Eu-rubber in E.ulmoides.However,the biosynthesis mechanism of Eu-rubber is very complicated,and the related research is still very limited.In this study,high throughput sequencing technique was used to sequencing the transcriptome,small RNA,and degradome using leaves of high yield Eu-rubber variety “Qinzhong 2” and low yield variety “Xiaoye”,and to analyze the regulatory network between miRNA and mRNA from genome-wide.The cleavage and interaction between n-eu-miR15 and its target GGPS6 were confirmed by RLM-5' RACE assay and agrobacterium-mediated transient co-transformation assay.The precursor gene of n-eu-miR15 was transformed into Arabidopsis thaliana and E.ulmoides for functional studies.The results of this study provide a theoretical basis for further elucidation of the molecular regulation mechanism of Eu-rubber biosynthesis,and provide new insights for the use of genetic engineering techniques to improve the content of Eu-rubber in E.ulmoides.The main results are as follows:1.The qRT-PCR reference gene of E.ulmoides with different variety / genotype,different develop period,different tissue,cold treatment,drought treatment and salinity treatment were screened.The stability of 11 commonly used candidate reference genes under the above experimental conditions was analyzed by geNorm,Normfinder,Bestkeeper,RefFinder software,and the suitable reference genes and the best reference gene combinations were selected for each experimental condition.The results are as follows:(1)For different varieties / genotypes,UBC and UBC E2 are the most stable reference genes,and UBA80 + UBC is the best reference gene combination;(2)for different developmental stages,HIS4 and UBC are the most stable reference genes,and UBC + UBC E2 is the best reference gene combination;(3)for different tissues,UBC and UBC E2 are the most stable reference genes,and UBA52 + UBA80 is the best reference gene combination;(4)under cold treatment,TUB and UBC are the most stable reference genes,and TUB + UBC is the best reference gene combination;(5)under drought treatment,UBA80 and HIS4 are the most stable reference genes,and HIS2 B + UBC E2 is the best reference gene combination;(6)under sailinity treatment,UBA80 and HIS4 are the most stable reference genes,and UBC E2 + TUB is the best reference gene combination.In addition,three functional genes were simultaneously used to verify the selected reference genes under various experimental conditions,and the results showed that the selected reference genes in this study were accurate and reliable.2.The key genes of Eu-rubber biosynthesis of E.ulmoides were identified by transcriptional sequencing.A total of 153,241 transcripts were obtained by transcriptome sequencing and further assembled into 82,065 unigenes.By comparing transcriptomics analysis,a total of 568 differentially expressed genes(DEGs)were identified(HR vs LR),of which 341 unigenes were up-regulated and 227 unigenes were down-regulated.Through GO enrichment analysis,these DEGs were mainly involved in metabolic process,single-organism process,celler process,catalytic activity and binding,etc.Through KEGG enrichment analysis,125 unigenes were enriched to 49 pathways,including celler process,environmental information process,genetic information process,metabolism and organism composition,etc.Among them,the metabolic process contains 119 DEGs,which was the most abundant.And there are three DEGs(ACCT2,DXS2,GGPS6)were enriched in terpenoid synthesis(ko00900).3.Identification of miRNA in E.ulmoides leaves.A total of 221 miRNAs were identified by small RNA sequencing,including 68 known miRNAs and 153 novel miRNAs.The psRNATarget software was employed to predict the targets.A total of 20,815 targets were predicted for all of the 221 miRNAs,of which 779 miRNA-targets were verified by degradome sequencing.Through GO enrichment analysis,it was found that targets are mainly involved in single-organism process,positive regulation of cellular processes,single-cell biological processes,positive regulation of cellular process,single-multicellular organism process,developmental process,multicellular organismal process,metal ion transport,response to stimulus,etc.Through KEGG annotation,a total of 1866 targets were assigned to 107 pathways,17 targets were assigned to the Eu-rubber biosynthesis pathway,and 43 miRNAs were involved in the regulation of these targets.4.The key genes and corresponding miRNAs of Eu-rubber biosynthesis were identified by transcriptomes and sRNAs sequencing data integration analysis.In combination with gene expression profiling and miRNA expression profiling,31 of 221 miRNAs are differentially expressed miRNAs(DEMs),including four known miRNAs and 27 new miRNAs;19 of these miRNAs were up-regulated in HR library,and 12 miRNAs down-regulated.Of all targets,214 of them were DEGs,among which 37 DEGs were targeted by 22 DEMs,forming 44 miRNA-target pairs,including 19 pairs of positive regulation,and 25 pairs of negative regulation.Among them,the n-eu-miR15 and its target(GGPS6,c133948.graph_c0)was involved in Eu-rubber biosynthesis.5.The biological function of n-eu-miR15 was revealed.The cleavage and interaction of n-eu-miR15 with its target(GGPS6)was verified by RLM-5' RACE and agrobacterium-mediated transient co-transformation assay,it proved that n-eu-miR15 could reduce the expression levels of GGPS6.We cloned the precursor gene of n-eu-miR15—MIR15,and transformed it into Arabidopsis thaliana and hypocotyls of E.ulmoides by agrobacterium-mediated.The results showed that the overexpression of miR15 in Arabidopsis thaliana would lead to the decrease of polyisoprene content;and overexpression of miR15 in E.ulmoides would lead to a significant reduction in the number and volume of gutta cells in E.ulmoides leaves.The results indicated that n-eu-miR15 could strongly inhibited the Eu-rubber biosynthesis in E.ulmoides.