柠条锦鸡儿响应干旱的miRNA表达谱分析及Cko-miR2119与靶基因克隆鉴定

发布时间：2018-11-28 18:21

【摘要】：干旱导致我国荒漠化和沙化土地总面积占国土面积的近45%。柠条作为防风固沙的优势物种,近年来从生态价值、生理特征及形态解剖等方面进行的抗旱机制研究较多,而在分子水平的机制阐释较少,从miRNA调控入手对干旱响应的解释更少。本研究应用高通量测序技术对自然降水梯度下的三个柠条分布区的柠条叶样进行miRNA测序,预测与柠条抗旱相关的miRNAs,结合miRNA调控的靶基因对柠条的干旱适应机制进行研究。同时为排除其他非生物胁迫的干扰,我们设计室内单因素控水实验,将柠条分别置于土壤相对含水量为75%,55%和35%三个不同的水分梯度下模拟野外干旱胁迫。通过检测野外样品和室内控水实验样品中与干旱响应有关的miRNA和靶基因的表达,为阐明柠条抗旱的分子机制扩充有关miRNAs调控靶基因响应干旱机理的内容。论文主要结果如下:(1)野外选取黄土高原降水依次减少的三个试验点(黄陵、榆林和达拉特旗)柠条叶片进行mi RNA转录组测序。三个样品分别获得了13,710,681,15,048,945和15,198,442条reads。共鉴定获得490个已报道的miRNAs,预测96个未报道的新miRNAs。(2)对已鉴定和预测的586个miRNA进行GO功能富集分析和KEGG通路显著性富集分析,获得39个可能与水分胁迫应答相关的miRNA。利用stem loop qPCR分析验证了miRNA高通量测序预测的9个差异表达miRNAs(miR390,miR394,miR398,miR529,miR530,miR2119,miR5232,miR5559,miR5770),结果表明9个miRNAs的差异表达趋势与测序预测数据一致。(3)对响应干旱较为关键的5个miRNA(miR390,miR398,miR530,miR2119,miR5559),室内设计三个水分梯度(75%,55%,35%)模拟野外干旱胁迫进行验证。利用qPCR同时对野外和室内柠条样品mi RNA表达进行定量分析,结果表明这5个miRNA对自然干旱和室内干旱梯度具有相同的响应趋势,即随干旱加剧柠条miRNA表达量均呈下调趋势。(4)进而对其中2个鲜有报道的miRNA(miR2119和miR5559)预测的靶基因进行定量分析,野外和室内结果表明随水分胁迫的加剧这2个miRNAs的表达量降低,而各自2个靶基因却呈现上调趋势。相关性分析表明:miR2119与靶基因BI-1和CAP的表达趋势相关性R2达到了0.942和0.852;miR5559与靶基因HIRA和SEO的表达趋势相关性R2分别达到了0.960和0.966。(5)进一步克隆miR2119和靶基因BI-1和CAP。构建pCMBIA-1304-amiR2119、pCMBIA-1304-BI-1和pCMBIA-1304-CAP载体,利用农杆菌介导的烟草叶片瞬时转染法证实miR2119与靶基因BI-1和CAP存在互作。利用农杆菌介导的烟草遗传转化法,获得转miR2119、BI-1和CAP基因的烟草转基因系。综合上述实验结果得出结论:我们证实高通量测序预测的可能对水分胁迫存在应答的39个柠条miRNA中的5个均对干旱有响应;进而确定其中一个鲜有报道的mi R2119是通过负调控其靶基因BI-1和CAP的上调表达响应干旱的加剧。从而提出柠条通过miRNA调控与干旱响应相关的靶基因实现对干旱环境的适应。
[Abstract]:Drought leads to desertification and desertification in China, the total land area accounts for nearly 45000. Caragana korshinskii, as a dominant species of windbreak and sand fixation, has been studied in recent years in the aspects of ecological value, physiological characteristics and morphology and anatomy, but there is little explanation of molecular mechanism. There are fewer explanations for drought response based on miRNA regulation. In this study, high-throughput sequencing technique was used to sequence the leaf samples of three Caragana korshinskii under natural precipitation gradient, and to predict the drought adaptation mechanism of Caragana korshinskii by miRNAs, associated with drought resistance and target genes regulated by miRNA. At the same time, in order to eliminate the interference of other abiotic stresses, we designed a single factor water control experiment in our design room. Caragana korshinskii was placed under three different water gradients of 75% and 35% relative water content in the soil, respectively, to simulate field drought stress. In order to elucidate the molecular mechanism of drought resistance of Caragana korshinskii by detecting the expression of miRNA and target genes related to drought response in field samples and indoor water control experiment samples the mechanism of miRNAs regulating target gene response to drought was expanded in order to elucidate the molecular mechanism of drought resistance of Caragana korshinskii. The main results are as follows: (1) in the field, the leaves of Caragana korshinskii were sequenced from the three experimental sites (Huangling, Yulin and Dalat Banner) in order to reduce precipitation in the Loess Plateau. Three samples have been obtained, and 15198442 reads. samples have been obtained, respectively. A total of 490 reported miRNAs, predictions were obtained for 96 unreported new miRNAs. (2) GO functional enrichment analysis and KEGG pathway significant enrichment analysis of 586 miRNA identified and predicted, and 39 miRNA. which may be related to water stress response. Nine differentially expressed miRNAs (miR390,miR394,miR398,miR529,miR530,miR2119,miR5232,miR5559,miR5770) predicted by miRNA high-throughput sequencing were verified by stem loop qPCR analysis. The results showed that the differential expression trend of 9 miRNAs was consistent with the predicted data of sequencing. (3) for 5 miRNA (miR390,miR398,miR530,miR2119,miR5559) that were key to drought response, three water gradients were designed for indoor design. 35%) simulated field drought stress. Quantitative analysis of mi RNA expression in field and indoor samples of Caragana korshinskii was carried out by using qPCR. The results showed that the five miRNA had the same response trend to natural drought and indoor drought gradient. That is, the miRNA expression of Caragana korshinskii was down-regulated with drought exacerbation. (4) quantitative analysis of two target genes predicted by miRNA (miR2119 and miR5559) was carried out. The results of field and laboratory showed that the expression of miRNAs decreased with the increase of water stress, but the two target genes were up-regulated. Correlation analysis showed that the correlation between miR2119 and target gene BI-1 and CAP was 0.942 and 0.852 respectively. The trend correlation between miR5559 and target gene HIRA and SEO was 0.960 and 0.966 respectively. (5) further cloning of miR2119 and target gene BI-1 and CAP. PCMBIA-1304-amiR2119,pCMBIA-1304-BI-1 and pCMBIA-1304-CAP vectors were constructed and transient transfection of tobacco leaves mediated by Agrobacterium tumefaciens was used to confirm the interaction between miR2119 and target gene BI-1 and CAP. Transgenic tobacco lines with miR2119,BI-1 and CAP genes were obtained by Agrobacterium tumefaciens mediated tobacco genetic transformation. Based on the above experimental results, we confirmed that 5 of the 39 miRNA of Caragana korshinskii that were predicted by high-throughput sequencing were responsive to drought. Thus, it is confirmed that one of the rarely reported mi R2119 is response to drought by negatively regulating the up-regulation of the expression of its target genes BI-1 and CAP. It is suggested that Caragana korshinskii adapts to drought environment through target genes related to drought response regulated by miRNA.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S793.3;Q943.2
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本文编号：2363850