| The area of desertification caused by drought accounts for nearly 45% of the total land area of China.In recent years,Caragana korshinskii,as the dominant species of windbreak and sand fixation,has been studied more about the drought resistance mechanism,such as ecological value,physiological characteristics and morphological anatomy,also a few explanations for drought resistance mechanism at the molecular level,but less explanations of the drought resistance mechanism with miRNA regulation.In this study,high-throughput sequencing technology was used to perform miRNA sequencing of Caragana korshinskii from three experimental sites across the natural precipitation gradient.We analysed and predicted the drought-related of miRNA,and combined with miRNAs target genes to explore the drought adaptation mechanism of Caragana korshinskii.In order to eliminate the interference of other abiotic stress,we designed the indoor water control experiment,Caragana korshinskii seedlings were planted in the soil with relative water content of 75%,55% and 35% as the simulation of natural drought and water scarcity environment in Caragana korshinskii.The molecular mechanism of Caragana korshinskii miRNAs in drought resistance of was then elucidated by detecting the expression mi RNAs and target genes in natural samples and indoor samples.The main experimental results are as follows:(1)We selected and plotted the three experimental sites(HuL,Yu L and DaB),which were located in the Loess Plateau where distributed widely Caragana korshinskii.Then sampled leaves from 3 sites and sequenced by miRNA transcriptome.After sequenced,we obtained 13,710,681,15,048,945 and 15,198,442 reads respectively.A total of 490 known miRNAs genes were screened and 96 novel miRNAs were predicted.(2)There were 586 identified and predicted mi RNAs for GO functional enrichment analysis and KEGG significant difference pathways analysis.And 39 miRNAs related to water stress response were obtained.The expression of miRNAs(miR390,miR398,miR398,miR530,miR2119,miR5232,miR5559,miR5770)was verified by stem loop qPCR.The qPCR results were consistent with the sequencing data.(3)Three water conditions of plant growth were designed(soil with relative water content of 75%,55%,35%),which was a simulation for natural drought and water scarcity environment of Caragana korshinskii.We used stem-loop qRT-PCR to validate the expression of 5 miRNAs(e.g.miR390,miR398,miR530,miR2119,and miR5559)of 39 miRNAs that obviously responded to water stress during plants grown both under natural and experimental water stress conditions.The result indicated the expression of 5 miRNAs were down-regulated with theincreasing drought.(4)The expression of two key and few reported(miR2119 and miR5559)miRNAs was carried out by qPCR.The results both in natural and indoor miRNAs showed that the expression of these two miRNAs decreased with the increase of water stress,while the two target genes showed an up-regulation.Correlation analysis of miRNA expression and their target genes showed that the correlation coefficient R2 between miR2119 and target gene BI-1 and CAP was 0.942 and 0.852;The correlation coefficient R2 between miR5559 and target gene HIRA and SEO reached 0.960 and 0.966,respectively.(5)We cloned miR2119 and its two target genes(BI-1,CAP)and constructed the expression vector of pCMBIA-1304-amiR2119,pCMBIA-1304-BI-1 and pCMBIA-1304-CAP.The interaction of miR2119 with target gene BI-1 and CAP was confirmed by tobacco leaf transient transfection mediated by Agrobacterium tumefaciens.Meanwhile,tobacco transgenic lines of miR2119,BI-1 and CAP genes were obtained by Agrobacterium tumefaciens-mediated tobacco genetic transformation.In conclusion,5 mi RNAs of the 39 miRNAs responded to increasing drought;furthermore,the few reported mi R2119 was negatively regulated its target genes expression of BI-1 and CAP to respond to drought stress.It is suggested that Caragana korshinskii was able to adapt to the arid environments by curb miRNA expression to up-regulate their target genes to respond drought. |
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