發(fā)布時間：2018-04-04 07:12

  本文選題：大麥　切入點：miRNA　出處：《西北農(nóng)林科技大學》2017年博士論文

【摘要】：microRNA(miRNA)是一類非編碼小RNA分子,其以堿基互補配對方式與基因mRNA結(jié)合,介導靶基因降解或抑制翻譯,進而參與基因表達調(diào)控,在植物生長、發(fā)育、逆境響應等過程中起著重要作用。大麥(Hordeumvulgare)是世界上第四大禾本科作物,同時也是最耐鹽的作物之一,廣泛種植于干旱、半干旱地區(qū),應用于人類食物、動物飼料和啤酒釀造。隨著新一代高通量測序技術(shù)的發(fā)展和相關(guān)生物信息學分析手段的提高,整合多組學數(shù)據(jù)(如小RNA測序、轉(zhuǎn)錄組、降解組等)為解析miRNA在大麥生長發(fā)育及逆境響應過程中的作用帶來了新的機遇。本研究采用多組學數(shù)據(jù)相結(jié)合的策略,一方面,比較分析大麥鹽脅迫響應及籽粒發(fā)育過程中的關(guān)鍵miRNAs及其調(diào)控靶基因,另一方面,系統(tǒng)分析野生大麥miRNAs,從miRNA角度比較分析野生大麥和栽培大麥間異同。本研究結(jié)果可豐富大麥miRNA數(shù)據(jù),并揭示miRNA在大麥逆境響應、生長發(fā)育及進化過程中的作用。主要研究結(jié)果如下:(1)大麥鹽脅迫相關(guān)miRNA的鑒定與功能分析我們以大麥栽培品種Morex為研究材料,取鹽脅迫(100mM)處理后3h、8h和27h及相應對照組樣品,分別構(gòu)建6個小RNA文庫和1個降解組測序文庫,然后,利用IlluminaHiseq2000測序平臺進行高通量測序。數(shù)據(jù)分析共鑒定到152個miRNAs,包括28個大麥knownmiRNAs、114個同源miRNAs和10個novelmiRNAs,隸屬于126個miRNAs家族。其中,44個miRNAs在鹽脅迫條件下顯著差異表達,隸屬于39個miRNA家族,并且分別有14、4和11個miRNA家族為植物保守性、禾本科保守性和大麥鹽脅迫特異性表達。與對照組相比,絕大部分鹽脅迫響應相關(guān)miRNAs在鹽脅迫處理8h后表現(xiàn)為顯著上調(diào)表達,而在3h和27h時表現(xiàn)為下調(diào)表達。隨后,結(jié)合降解組測序和生物信息學,我們對所有鹽脅迫相關(guān)miRNAs靶基因進行系統(tǒng)分析,結(jié)果發(fā)現(xiàn)分別有86和37個參與新陳代謝和逆境響應相關(guān)基因受到大麥鹽脅迫響應相關(guān)miRNAs的調(diào)控,這些基因在鹽脅迫條件下主要表現(xiàn)為抑制植物生長和降低代謝活動。最后,我們利用qRT-PCR對部分miRNAs及其靶基因表達模式進行比較分析。綜上所述,至少有39個miRNA家族及其調(diào)控的123個靶基因參與大麥苗期早期鹽脅迫響應,這些結(jié)果可為進一步解析大麥耐鹽機制提供參考。(2)利用高通量測序技術(shù)鑒定野生大麥miRNAs及其靶基因野生大麥是栽培大麥的祖先種,其也是大麥遺傳改良的重要基因庫。在本研究中,我們選用野生大麥MtGilboabarley3-25為材料,分別提取其不同發(fā)育階段根、莖、葉和穗等組織的總RNA,然后等量混合RNA并構(gòu)建小RNA文庫。高通量測序共獲得9885815條cleanreads,經(jīng)過數(shù)據(jù)分析,最終鑒定到55個knownmiRNAs、14個novelvariants和18個novelmiRNAs。序列分析發(fā)現(xiàn),本研究鑒定到的所有野生大麥miRNAs在4個大麥栽培品種中均具有高度保守性前體序列,尤其是在成熟miRNA區(qū)間,表明miRNAs功能上的重要性決定其在大麥不同品系間高度保守。隨后,我們比較分析了14個novelvariants和18個novelmiRNAs在栽培大麥中的表達情況,結(jié)果發(fā)現(xiàn)21個miRNAs在栽培大麥morex中表達,包括7個novelvariants和14個novelmiRNAs,并且絕大部分表達miRNAs在栽培大麥和野生大麥間顯著差異表達(95.24%,20/21),表明栽培大麥和野生大麥間表型變異可能大部分來源于miRNAs的差異表達,而非基因組miRNAs組成。靶基因分析發(fā)現(xiàn),有12個miRNA家族介導調(diào)控轉(zhuǎn)錄因子,包括8個植物保守性和4個wheat-barley保守性miRNA家族。此外,其它靶基因主要參與各種生理、代謝及逆境響應等過程。本研究首次在野生大麥中分析miRNAs,可為解析miRNAs在野生大麥中的調(diào)控作用提供參考。(3)整合mRNA和小RNA表達分析解析大麥籽粒發(fā)育大麥是研究禾本科作物籽粒發(fā)育的重要模式材料,在本研究中,我們結(jié)合轉(zhuǎn)錄組和小RNA高通量測序,比較分析大麥籽粒發(fā)育過程中基因和小RNAs的動態(tài)表達變化,包括籽粒發(fā)育4個重要階段:早期物質(zhì)存儲前期準備階段(stage01:0-5DPA,開花后0-5天),晚期物質(zhì)存儲前期準備階段或過渡階段(stage02:6-10DPA)、早期物質(zhì)存儲階段(stage03:11-15DPA)和levelsoff階段(stage04:16-20DPA)。轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn),在大麥籽粒發(fā)育過程中,初級和次級代謝相關(guān)基因表達活性均發(fā)生了顯著變化,并且轉(zhuǎn)錄活性變化與生理變化相一致,即隨著大麥籽粒的成熟,基因表達活性逐漸降低,變化最劇烈的時間出現(xiàn)在晚期物質(zhì)存儲前期準備階段(stage02)向早期物質(zhì)存儲階段(stage03)過渡時,并且ABA和糖信號轉(zhuǎn)導在階段轉(zhuǎn)換中起著重要作用。另外,籽粒自身光合作用相關(guān)基因表達最活躍時期出現(xiàn)在stage01和stage02,且主要參與脂肪代謝和核苷酸代謝,而非碳水化合物合成。整合小RNAs數(shù)據(jù)分析發(fā)現(xiàn),絕大部分miRNAs表達最活躍時期出現(xiàn)在基因表達最不活躍(stage04)和最活躍時期(stage01),而siRNAs主要在基因最不活躍時期(stage03和stage04)表達活性最高,表明在大麥籽粒發(fā)育過程中,miRNAs可能主要扮演著“regulator”的角色,其通過介導基因表達調(diào)控參與各種新陳代謝過程,而siRNAs主要扮演著“silencer”的角色,主要負責抑制基因活性,直至種子休眠。此外,我們鑒定到8908基因與大麥籽粒發(fā)育相關(guān),包括298個時期特異性表達和8610個顯著差異表達基因,其中,分別有131和1695個基因受miRNAs和siRNAs調(diào)控。本研究可為解析小RNAs介導的基因表達調(diào)控在大麥籽粒發(fā)育過程中的作用提供重要信息,也為大麥及其它禾本科作物籽粒相關(guān)性狀遺傳改良提供參考。
[Abstract]:MicroRNA (miRNA) is a kind of non encoding small RNA molecules, with its complementary base pairing mode combined with mRNA gene mediated target gene degradation or inhibition of translation, and then participate in the regulation of gene expression in plant growth, development, and plays an important role in the process of stress responses. Barley (Hordeumvulgare) is the fourth largest in the world gramineous crop, is also one of the most salt tolerant crops, widely planted in arid and semi-arid regions,, used in human food, animal feed and beer brewing. With the analysis method of the development of a new generation of high-throughput sequencing and bioinformatics of integration of multi omics data (such as small RNA sequencing. Transcriptome degradation group) has brought new opportunities for growth and development and stress analysis of miRNA in barley in response. This study used data sets combining strategy, on the one hand, a comparative analysis of barley under salt stress response The key of miRNAs and grain in the process of development and regulation of target genes, on the other hand, the system analysis of wild barley miRNAs, comparative analysis of the similarities and differences between wild barley and cultivated barley from the perspective of miRNA. The results of this study can enrich the barley miRNA data, and reveal miRNA in barley stress response, growth and evolution process. The main research the results are as follows: (1) identification and functional analysis of barley under salt stress related miRNA with barley cultivar Morex as research materials, the salt stress (100mM) after the treatment of 3H, 8h and 27h and the corresponding control samples and 6 small RNA libraries and 1 Degradome sequencing library were constructed, then high flux sequencing using IlluminaHiseq2000 sequencing platform. Data analysis identified 152 miRNAs, including 28 barley knownmiRNAs, 114 miRNAs and 10 novelmiRNAs respectively, which belong to 126 family of miRNAs. Among them, 44 miRNAs The significant difference expression under salt stress, which belong to 39 family and miRNA, respectively 14,4 and 11 miRNA family for plant conservation, Gramineae and conservation of barley under salt stress specific expression. Compared with the control group, most of the salt stress response is related to the expression of miRNAs 8h in the salt stress treatment showed significant increase. While 3H and 27h were decreased. Then, combined with the Degradome sequencing and bioinformatics, we all salt stress related target genes of miRNAs were analyzed, results showed that there were 86 and 37 in response to stress related genes by The new supersedes the old. and barley under salt stress response related to the regulation of miRNAs, these genes in salt under stress conditions mainly inhibited plant growth and decreased metabolic activity. Finally, we use the expression pattern of qRT-PCR were compared and analyzed on the part of miRNAs and its target gene. To sum up, There are at least 123 target genes of 39 miRNA family and its regulation in barley seedling early salt stress response, these results can provide a reference for the further analysis of the mechanism of salt tolerance in barley. (2) the use of high-throughput sequencing technology to identify wild barley miRNAs and its target genes in wild barley is the ancestor of cultivated barley, which is also an important gene pool of barley genetic improvement. In this study, we choose MtGilboabarley3-25 as the material of wild barley, which were extracted from different developmental stages of root, stem, leaf and total RNA and other organizations, and then mix RNA and construct small RNA library. High throughput sequencing received a total of 9885815 cleanreads, through data analysis, the final identification 55 knownmiRNAs, 14 novelvariants and 18 novelmiRNAs. sequence analysis showed that all wild barley miRNAs identified in this study that is highly conserved in 4 barley cultivars before The sequence, especially in the mature miRNA interval, indicating the importance of miRNAs on the function of the decision in the barley between different strains is highly conserved. Then, we compare the expression of 14 novelvariants and 18 novelmiRNAs in cultivated barley, results showed that the expression of 21 miRNAs in the cultivated barley morex, including 7 novelvariants and 14 novelmiRNAs, and the most significant difference in miRNAs expression between cultivated and wild barley (95.24%, 20/21), showed that the phenotypic variation of cultivated and wild barley may differentially expressed mostly from miRNAs, rather than miRNAs. The target gene of genome analysis found that there are 12 families of miRNA mediated regulation transcription factors, including 8 conserved plants and 4 wheat-barley conserved miRNA family. In addition, other genes are mainly involved in various physiological, metabolic and stress responses of the research process. For the first time in the analysis of miRNAs in wild barley, which can provide a reference for the regulatory role of miRNAs in the analysis of wild barley. (3) analysis of barley grain barley development is an important research materials for gramineous crop seed development expression of the integration of mRNA and RNA, in this study, transcriptome and small RNA high-throughput sequencing I we combine dynamic comparative analysis and small gene RNAs in the process of barley grain development expression, 4 important stages of grain development include: early material stored preparatory phase (stage01:0-5DPA, 0-5 days after flowering), advanced material storage preparation stage or the transitional stage (stage02:6-10DPA), early stage (stage03:11-15DPA) and material storage levelsoff stage (stage04:16-20DPA). The transcriptome data analysis found that during grain filling, primary and secondary metabolism related gene expression were varied significantly, and And the transcription activity and physiological changes consistent with barley grain, mature, gene expression activity decreased gradually, the most dramatic changes appeared in the late stage of preparation material storage (stage02) to the early stage of the storage material (stage03) transition, and the ABA and sugar signal transduction in the stage of transformation plays an important role in. In addition, the expression of its grain photosynthesis related genes in stage01 and stage02 is the most active period, and mainly involved in lipid metabolism and nucleotide metabolism, rather than carbohydrate synthesis. The integration of small RNAs data analysis found that most of the miRNAs expression of the most active period is not the most active in gene expression (stage04) and the most active period (stage01). While siRNAs is mainly in the least active period genes (stage03 and stage04) expression showed the highest activity, during grain filling, miRNAs may be the main play" The role of regulator, which mediate the regulation of gene expression in a variety of process and siRNAs The new supersedes the old., plays the role of the "silencer", is mainly responsible for the inhibition of gene activity, and seed dormancy. In addition, we identified 8908 genes with barley grain development, gene expression, 298 stage specific expression and 8610 significant the difference included, respectively 131 and 1695 genes by miRNAs and siRNAs control. This research can be of small RNAs mediated gene expression regulation during grain filling and provide important information, but also provide a reference for barley and other cereal crops, grain related traits genetic improvement.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S512.3
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本文編號：1708909