整合多组学数据解析miRNA在大麦盐胁迫响应和籽粒发育过程中的作用

发布时间：2018-04-04 07:12

  本文选题：大麦　切入点：miRNA　出处：《西北农林科技大学》2017年博士论文

【摘要】：microRNA(miRNA)是一类非编码小RNA分子,其以碱基互补配对方式与基因mRNA结合,介导靶基因降解或抑制翻译,进而参与基因表达调控,在植物生长、发育、逆境响应等过程中起着重要作用。大麦(Hordeumvulgare)是世界上第四大禾本科作物,同时也是最耐盐的作物之一,广泛种植于干旱、半干旱地区,应用于人类食物、动物饲料和啤酒酿造。随着新一代高通量测序技术的发展和相关生物信息学分析手段的提高,整合多组学数据(如小RNA测序、转录组、降解组等)为解析miRNA在大麦生长发育及逆境响应过程中的作用带来了新的机遇。本研究采用多组学数据相结合的策略,一方面,比较分析大麦盐胁迫响应及籽粒发育过程中的关键miRNAs及其调控靶基因,另一方面,系统分析野生大麦miRNAs,从miRNA角度比较分析野生大麦和栽培大麦间异同。本研究结果可丰富大麦miRNA数据,并揭示miRNA在大麦逆境响应、生长发育及进化过程中的作用。主要研究结果如下:(1)大麦盐胁迫相关miRNA的鉴定与功能分析我们以大麦栽培品种Morex为研究材料,取盐胁迫(100mM)处理后3h、8h和27h及相应对照组样品,分别构建6个小RNA文库和1个降解组测序文库,然后,利用IlluminaHiseq2000测序平台进行高通量测序。数据分析共鉴定到152个miRNAs,包括28个大麦knownmiRNAs、114个同源miRNAs和10个novelmiRNAs,隶属于126个miRNAs家族。其中,44个miRNAs在盐胁迫条件下显著差异表达,隶属于39个miRNA家族,并且分别有14、4和11个miRNA家族为植物保守性、禾本科保守性和大麦盐胁迫特异性表达。与对照组相比,绝大部分盐胁迫响应相关miRNAs在盐胁迫处理8h后表现为显著上调表达,而在3h和27h时表现为下调表达。随后,结合降解组测序和生物信息学,我们对所有盐胁迫相关miRNAs靶基因进行系统分析,结果发现分别有86和37个参与新陈代谢和逆境响应相关基因受到大麦盐胁迫响应相关miRNAs的调控,这些基因在盐胁迫条件下主要表现为抑制植物生长和降低代谢活动。最后,我们利用qRT-PCR对部分miRNAs及其靶基因表达模式进行比较分析。综上所述,至少有39个miRNA家族及其调控的123个靶基因参与大麦苗期早期盐胁迫响应,这些结果可为进一步解析大麦耐盐机制提供参考。(2)利用高通量测序技术鉴定野生大麦miRNAs及其靶基因野生大麦是栽培大麦的祖先种,其也是大麦遗传改良的重要基因库。在本研究中,我们选用野生大麦MtGilboabarley3-25为材料,分别提取其不同发育阶段根、茎、叶和穗等组织的总RNA,然后等量混合RNA并构建小RNA文库。高通量测序共获得9885815条cleanreads,经过数据分析,最终鉴定到55个knownmiRNAs、14个novelvariants和18个novelmiRNAs。序列分析发现,本研究鉴定到的所有野生大麦miRNAs在4个大麦栽培品种中均具有高度保守性前体序列,尤其是在成熟miRNA区间,表明miRNAs功能上的重要性决定其在大麦不同品系间高度保守。随后,我们比较分析了14个novelvariants和18个novelmiRNAs在栽培大麦中的表达情况,结果发现21个miRNAs在栽培大麦morex中表达,包括7个novelvariants和14个novelmiRNAs,并且绝大部分表达miRNAs在栽培大麦和野生大麦间显著差异表达(95.24%,20/21),表明栽培大麦和野生大麦间表型变异可能大部分来源于miRNAs的差异表达,而非基因组miRNAs组成。靶基因分析发现,有12个miRNA家族介导调控转录因子,包括8个植物保守性和4个wheat-barley保守性miRNA家族。此外,其它靶基因主要参与各种生理、代谢及逆境响应等过程。本研究首次在野生大麦中分析miRNAs,可为解析miRNAs在野生大麦中的调控作用提供参考。(3)整合mRNA和小RNA表达分析解析大麦籽粒发育大麦是研究禾本科作物籽粒发育的重要模式材料,在本研究中,我们结合转录组和小RNA高通量测序,比较分析大麦籽粒发育过程中基因和小RNAs的动态表达变化,包括籽粒发育4个重要阶段:早期物质存储前期准备阶段(stage01:0-5DPA,开花后0-5天),晚期物质存储前期准备阶段或过渡阶段(stage02:6-10DPA)、早期物质存储阶段(stage03:11-15DPA)和levelsoff阶段(stage04:16-20DPA)。转录组数据分析发现,在大麦籽粒发育过程中,初级和次级代谢相关基因表达活性均发生了显著变化,并且转录活性变化与生理变化相一致,即随着大麦籽粒的成熟,基因表达活性逐渐降低,变化最剧烈的时间出现在晚期物质存储前期准备阶段(stage02)向早期物质存储阶段(stage03)过渡时,并且ABA和糖信号转导在阶段转换中起着重要作用。另外,籽粒自身光合作用相关基因表达最活跃时期出现在stage01和stage02,且主要参与脂肪代谢和核苷酸代谢,而非碳水化合物合成。整合小RNAs数据分析发现,绝大部分miRNAs表达最活跃时期出现在基因表达最不活跃(stage04)和最活跃时期(stage01),而siRNAs主要在基因最不活跃时期(stage03和stage04)表达活性最高,表明在大麦籽粒发育过程中,miRNAs可能主要扮演着“regulator”的角色,其通过介导基因表达调控参与各种新陈代谢过程,而siRNAs主要扮演着“silencer”的角色,主要负责抑制基因活性,直至种子休眠。此外,我们鉴定到8908基因与大麦籽粒发育相关,包括298个时期特异性表达和8610个显著差异表达基因,其中,分别有131和1695个基因受miRNAs和siRNAs调控。本研究可为解析小RNAs介导的基因表达调控在大麦籽粒发育过程中的作用提供重要信息,也为大麦及其它禾本科作物籽粒相关性状遗传改良提供参考。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S512.3
，

本文编号：1708909