【摘要】：干旱導(dǎo)致我國(guó)荒漠化和沙化土地總面積占國(guó)土面積的近45%。檸條作為防風(fēng)固沙的優(yōu)勢(shì)物種,近年來從生態(tài)價(jià)值、生理特征及形態(tài)解剖等方面進(jìn)行的抗旱機(jī)制研究較多,而在分子水平的機(jī)制闡釋較少,從miRNA調(diào)控入手對(duì)干旱響應(yīng)的解釋更少。本研究應(yīng)用高通量測(cè)序技術(shù)對(duì)自然降水梯度下的三個(gè)檸條分布區(qū)的檸條葉樣進(jìn)行miRNA測(cè)序,預(yù)測(cè)與檸條抗旱相關(guān)的miRNAs,結(jié)合miRNA調(diào)控的靶基因?qū)帡l的干旱適應(yīng)機(jī)制進(jìn)行研究。同時(shí)為排除其他非生物脅迫的干擾,我們?cè)O(shè)計(jì)室內(nèi)單因素控水實(shí)驗(yàn),將檸條分別置于土壤相對(duì)含水量為75%,55%和35%三個(gè)不同的水分梯度下模擬野外干旱脅迫。通過檢測(cè)野外樣品和室內(nèi)控水實(shí)驗(yàn)樣品中與干旱響應(yīng)有關(guān)的miRNA和靶基因的表達(dá),為闡明檸條抗旱的分子機(jī)制擴(kuò)充有關(guān)miRNAs調(diào)控靶基因響應(yīng)干旱機(jī)理的內(nèi)容。論文主要結(jié)果如下:(1)野外選取黃土高原降水依次減少的三個(gè)試驗(yàn)點(diǎn)(黃陵、榆林和達(dá)拉特旗)檸條葉片進(jìn)行mi RNA轉(zhuǎn)錄組測(cè)序。三個(gè)樣品分別獲得了13,710,681,15,048,945和15,198,442條reads。共鑒定獲得490個(gè)已報(bào)道的miRNAs,預(yù)測(cè)96個(gè)未報(bào)道的新miRNAs。(2)對(duì)已鑒定和預(yù)測(cè)的586個(gè)miRNA進(jìn)行GO功能富集分析和KEGG通路顯著性富集分析,獲得39個(gè)可能與水分脅迫應(yīng)答相關(guān)的miRNA。利用stem loop qPCR分析驗(yàn)證了miRNA高通量測(cè)序預(yù)測(cè)的9個(gè)差異表達(dá)miRNAs(miR390,miR394,miR398,miR529,miR530,miR2119,miR5232,miR5559,miR5770),結(jié)果表明9個(gè)miRNAs的差異表達(dá)趨勢(shì)與測(cè)序預(yù)測(cè)數(shù)據(jù)一致。(3)對(duì)響應(yīng)干旱較為關(guān)鍵的5個(gè)miRNA(miR390,miR398,miR530,miR2119,miR5559),室內(nèi)設(shè)計(jì)三個(gè)水分梯度(75%,55%,35%)模擬野外干旱脅迫進(jìn)行驗(yàn)證。利用qPCR同時(shí)對(duì)野外和室內(nèi)檸條樣品mi RNA表達(dá)進(jìn)行定量分析,結(jié)果表明這5個(gè)miRNA對(duì)自然干旱和室內(nèi)干旱梯度具有相同的響應(yīng)趨勢(shì),即隨干旱加劇檸條miRNA表達(dá)量均呈下調(diào)趨勢(shì)。(4)進(jìn)而對(duì)其中2個(gè)鮮有報(bào)道的miRNA(miR2119和miR5559)預(yù)測(cè)的靶基因進(jìn)行定量分析,野外和室內(nèi)結(jié)果表明隨水分脅迫的加劇這2個(gè)miRNAs的表達(dá)量降低,而各自2個(gè)靶基因卻呈現(xiàn)上調(diào)趨勢(shì)。相關(guān)性分析表明:miR2119與靶基因BI-1和CAP的表達(dá)趨勢(shì)相關(guān)性R2達(dá)到了0.942和0.852;miR5559與靶基因HIRA和SEO的表達(dá)趨勢(shì)相關(guān)性R2分別達(dá)到了0.960和0.966。(5)進(jìn)一步克隆miR2119和靶基因BI-1和CAP。構(gòu)建pCMBIA-1304-amiR2119、pCMBIA-1304-BI-1和pCMBIA-1304-CAP載體,利用農(nóng)桿菌介導(dǎo)的煙草葉片瞬時(shí)轉(zhuǎn)染法證實(shí)miR2119與靶基因BI-1和CAP存在互作。利用農(nóng)桿菌介導(dǎo)的煙草遺傳轉(zhuǎn)化法,獲得轉(zhuǎn)miR2119、BI-1和CAP基因的煙草轉(zhuǎn)基因系。綜合上述實(shí)驗(yàn)結(jié)果得出結(jié)論:我們證實(shí)高通量測(cè)序預(yù)測(cè)的可能對(duì)水分脅迫存在應(yīng)答的39個(gè)檸條miRNA中的5個(gè)均對(duì)干旱有響應(yīng);進(jìn)而確定其中一個(gè)鮮有報(bào)道的mi R2119是通過負(fù)調(diào)控其靶基因BI-1和CAP的上調(diào)表達(dá)響應(yīng)干旱的加劇。從而提出檸條通過miRNA調(diào)控與干旱響應(yīng)相關(guān)的靶基因?qū)崿F(xiàn)對(duì)干旱環(huán)境的適應(yīng)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S793.3;Q943.2
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本文編號(hào)：2363850