导　　师： 金伟波

学科专业： 0710

授予学位： 硕士

作　　者： ;

机构地区： 西北农林科技大学

摘　　要： microRNA（miRNA）是一类长度约为22个碱基的单链小分子RNA，它广泛存在于动植物细胞内，主要通过与mRNA序列互补结合，从而在转录或转录后水平调控目标基因的表达。近年来，虽然植物miRNA不断被发现，但主要集中在已经全基因组测序的模式植物拟南芥和水稻中，而在其他植物中发现的比较少。 番茄作为一种重要的经济作物，在世界各地被广泛种植。目前miRBase官网收录的拟南芥miRNA达到337条，而番茄miRNA仅42条。可见，仍有大量的番茄miRNA需要发掘。灰霉菌（Botrytis cinerea）是一类真菌病原菌，它能够感染几乎所有的蔬菜和水果作物，给农业生产带来了巨大的危害。由于其宿主广泛等特点，灰霉菌被当作模式生物来研究真菌致病机理。目前已报道了一些植物miRNA抵御逆境胁迫的相关研究，但对番茄miRNA抵抗灰霉菌的研究相对较少。 本研究运用下一代高通量测序技术和生物信息学分析方法，结合目前流行的miRNA预测算法，试图克服当前miRNA分析程序运算时间长、对计算机硬件要求高的不足，开发一款能够利用极低的计算机资源在较短时间内完成植物miRNA的识别和差异分析的工具。 通过对主流miRNA分析工具与我们开发的工具进行对比，使用效率较高的工具对灰霉菌处理和对照的两个番茄小RNA测序数据进行miRNA识别和差异表达分析，对具有表达差异的miRNA进行靶基因预测，然后对这些靶标进行功能注释，进而试图寻找与抗灰霉病相关的miRNA。 研究结果有： 1.开发了一款植物miRNA识别与定量的工具（miR-island），它能够克服传统miRNA分析工具运算时间长、计算机硬件要求高的不足，能够利用普通的办公电脑在较短的时间内完成植物miRNA的识别和差异分析工作。通过与miRDeep-P和ShortStack比较，miR-island表现出了明显的优势。 2.利用miR-island程序包对高通量测序得到的灰霉菌胁迫处理组及其对照组miRNA进行预测和分析，共有145条miRNA表达，其中41条为番茄已知miRNA，104条为番茄新发现的miRNA。 3.通过对两个测序样品中表达的miRNA进行表达量统计和差异分析，结果表明，在灰霉菌胁迫下，处理组和对照组样品中共有32条非冗余序列（代表47条miRNA）差异表达，其中9条非冗余序列（代表13条miRNA）为番茄已知miRNA，23条非冗余序列（代表34条miRNA）为番茄新发现miRNA。上述9条非冗余序列均呈现表达上调，而另外23条非冗余序列中，有10条非冗余序列（代表12条miRNA）呈现表达下调，剩余的13条非冗余序列（代表22条miRNA）呈现表达上调。 4.结合降解组测序数据用CleaveLand4对上述具有表达差异的miRNA进行靶标预测，只有9条差异表达的非冗余序列共找到24条潜在的靶标。其中，sly-miR482b调控的四条靶基因是植物抗病蛋白mRNA，这表明sly-miR482b在灰霉菌胁迫应答中起着重要的作用。 MicroRNAs /(miRNAs/) are single-stranded RNA molecules of about22nucleotides inlength that widely exist in plant and animal cells. MiRNAs function as transcriptional orpost-transcriptional regulators through base pairing with target mRNAs. In recent years, a plenty of miRNAs have been identified in many plant species, andmainly in the whole genome sequencing model plant such as Arabidopsis and rice. However,few have been discovered in other species. As an important economic crop, tomato is widely cultivated all over the world. There arecurrently377Arabidopsis miRNAs and713rice miRNAs in miRBase, but only42miRNAsare found in tomato. Visibly, there are still a lot of tomato miRNAs need to be discovered.Botrytis cinerea is a worldwide severe crop disease, and it mainly causes the rotten fruit, fruityield and quality of tomato. Some studies of plant miRNAs resistant adversity stress havebeen reported, but relatively few have been of tomato miRNAs resistant botrytis cinerea.This study uses the next generation of high-throughput sequencing and bioinformaticsanalysis method, combined with the current popular miRNAs prediction algorithm, aiming atovercoming the current miRNAs analysis programs' drawback such as long operation timeand high requirements for computer hardware, and developing a plant miRNAs annotationand qualification tool that perform plant miRNAs identification and qualification in arelatively short time by a computer with general hardware. Through analysis of the mainstream of miRNAs tools compared with our developmenttools, the tool with high efficiency was selected to perform the identification and qualificationof tomato small RNA libraries. The targets of the differentially expressed miRNAs werepredicted with the help of degradome sequencing library. Finally, the target mRNAs weresent to functional annotation using blast2go, and botrytis cinerea responsive miRNAs wereanalyzed. The results are as follow: 1. We developed a plant miRNAs annotation and qualification tool, miR-island, and itovercome current miRNAs analysis programs' drawback such as long operation time and highrequirements for computer hardware, and perform the identification and qualification of smallRNA libraries with a general office computer in a relatively short time. MiR-island shows asignificant advantage when comparing with miRDeep-P or ShortStack. 2. By analyzing tomato small RNA libraries in two conditions using miR-island package,a total of145miRNAs were expressed. Among that,41are tomato known miRNAs inmiRBase, and104are tomato newly discovered miRNAs. 3. By annotation and qualification of two small RNA sequencing libraries, the resultshowed that under botrytis cinerea stress treatment, a total of32non-redundant sequencesrepresented for47miRNAs are differentially expressed. Among them,9non-redundantsequences represented for13tomato known miRNAs and23non-redundant sequencesrepresented for34tomato newly discovered miRNAs. The9non-redundant sequences/(represented for13tomato known miRNAs/) are up-regulated, while among the left23non-redundant sequences /(represented for34newly discovered miRNAs/),10non-redundantsequences /(represented for12miRNAs/) are down-regulated, and the left13non-redundantsequences /(represented for22miRNAs/) are up-regulated. 4. The differentially expressed miRNAs were performed for targets annotation with thehelp of degradome sequencing data using CleaveLand4. There were9differentially expressednon-redundant sequences that have a total of24potential target mRNAs. The function of the4target mRNA of sly-miR482b are all disease resistant protein and it implied thatsly-miR482b plays an important role in responding botrytis cinerea stress treatment.

关 键 词： 番茄 灰霉菌处理 差异表达

分 类 号： [S436.412.1]

领　　域： [农业科学] [农业科学]