大豆逆境胁迫诱导的miRNA鉴定及功能分析

发布时间：2018-01-15 12:18

本文关键词：大豆逆境胁迫诱导的miRNA鉴定及功能分析　出处：《中国农业科学院》2015年博士论文　论文类型：学位论文

【摘要】：micro RNA是一类的非编码的单链小分子RNA,普遍存在于植物基因组中并参与转录后基因调控。mi RNA已被证明参与了植物抵抗环境胁迫(如干旱、高盐,低温和高温等)的过程。近年来,对mi RNA在各作物逆境胁迫中的作用进行了大量研究。在环境胁迫下,许多mi RNA已经在拟南芥和水稻中证明存在表达差异,并且其中的一部分更是鉴定了功能,而在大豆中却少有mi RNA涉及这方面的研究。虽然mi RNA仅代表转录本相对丰度的,但在不同物种、不同组织和不同环境胁迫条件下mi RNA的表达水平差异却很大。本研究中我们通过Northern印记杂交分析证明了如gma-mi R166a、gma-mi R168a、gma-mi R398a和gma-mi R1508a等mi RNA会被干旱、高盐、低温胁迫诱导表达。表明这些mi RNA可能是大豆幼苗逆境胁迫的应答mi RNA。因为大豆的生物多样性十分丰富,因此我们深入的研究了各种胁迫条件下不同基因型大豆幼苗中gma-mi R166a的表达。mi R166是一个即存在于单子叶植物又存在于双子叶植物中的保守mi RNA,相关研究表明它们主要和植物的发育相关,目前还没有其与非生物胁迫相关的研究结果。本研究采用5'RACE方法从15个与胁迫相关的预测靶基因中鉴定了gma-mi R166切割Glyma09g02750和Glyma07g01950两个基因,其中Glyma09g02750(命名为Gm PHB)属于HD-Zip III转录因子家族,与拟南芥PHABULOSA(At2g34710)基因有78%的相似度。实时定量PCR结果表明,各胁迫条件下Gm PHB与gma-mi R166a在不同胁迫时间的表达量呈显著负相关关系,这为进一步研究mi R166在大豆逆境胁迫中的作用提供了科学依据。此外,在拟南芥中过表达gma-MIR166a,即抑制了At PHB的表达,又增强了拟南芥的耐盐能力,这意味着gma-MIR166a可能在大豆耐盐能力方面起到了积极的调节作用。近年来,随着公共基因组数据库的使用,分子标记已经开始从基于PCR的劳动密集型标记向基于基因分型和基因功能的标记转变,还常被RNA信息相结合。然而,目前为止基于mi RNA的分子标记还仅在水稻、烟草和棉花中开发出来。本研究,通过对1669份来自中国、韩国、日本和俄罗斯的栽培大豆和一年生野生大豆中的一个特殊的具有遗传变异的单克隆位点的分析,开发了第一个大豆功能mi RNA分子标记,gma-mi R1511-In Del。结果表明,gma-mi R1511位点在栽培大豆中都有存在,而在一年生野生大豆中却存在3种等位变异。基于该结果,我们认为In Del标记可用于遗传变异的评估。gma-mi R1511-In Del标记数据和地理信息相结合进行分析发现,来自黄河流域的一年生野生大豆具有较高的遗传多样性,这为一年生野生大豆的基因多样性和大豆的起源学说提供了更多的分子证据。另外,gma-mi R1511和其靶基因Gm RPL4a在胁迫处理下的表达也存在负相关关系,这表明gma-mi R1511可能具有调节大豆发育的功能。gma-mi R1511及其靶基因目前还未在其它豆科植物中发现,因此,gma-mi R1511-In Del是可用于研究大豆遗传多样性、种质的基因分型和进化的大豆特有的分子标记。这种通过mi RNA序列定位其前体区域序列来开发分子标记的方法亦是首次报道。
[Abstract]:Micro RNA is a small single stranded non encoding RNA class, commonly exist in plant genome and involved in regulating gene transcription of.Mi RNA have been shown to be involved in plant resistance to environmental stress (such as drought, high salt, low temperature and high temperature) of the process. In recent years, done a lot of research in the MI RNA as a matter of stress in the role. Under the environmental stress, many mi RNA have proved that there were differential expression in Arabidopsis and rice, and a part of them is more functional identification in soybean and little mi RNA involved in the research. Although the MI RNA represents the relative abundance of transcripts. But in different species, different organizations and different environmental stress difference in MI expression under the condition of RNA is great. In this study we analyzed Northern blot proved that such as gma-mi R166a, gma-mi R168a, gma-mi R398a and gma-mi R1508a mi RNA Will be drought, high salt, low temperature stress induced expression. Suggest that these mi RNA may be the soybean seedling stress response of MI RNA. because soybean biological diversity is very rich, so we studied the different genotypes of Soybean Seedlings under the stress of gma-mi in the expression of R166a.Mi R166 is a mi that is conservative RNA in monocotyledonous plants also exist in dicotyledonous plants, related research shows that they mainly related to plant development, yet with the abiotic stress related research results. This research uses the 5'RACE method to predict the target gene from 15 stress-related identified gma-mi R166 cut Glyma09g02750 and Glyma07g01950 two the Glyma09g02750 gene (named Gm PHB) belongs to HD-Zip III family of transcription factors, and Arabidopsis PHABULOSA (At2g34710) 78% similarity gene. The results of real-time PCR Show that the stress condition Gm PHB and gma-mi R166a showed a significant negative correlation in the expression of different stress time, which provides a scientific basis for further research of MI R166 in soybean stress effect. Moreover, overexpression of gma-MIR166a in Arabidopsis, which inhibited the expression of At PHB, and enhanced the Arabidopsis salt tolerance, which means that gma-MIR166a may be the salt tolerance in soybean has played a positive role. In recent years, with the use of public genome database, molecular marker has begun to change from the marker gene classification and gene function based on labor intensive marker based on PCR, RNA is often combined with information. However, now mi RNA based on molecular markers also developed only in rice, tobacco and cotton. In this study, based on the 1669 from China, South Korea, Japan and Russia and cultivated soybean Analysis of Annual Wild Soybean in a special genetic variation with monoclonal sites, developed the first soybean Mi molecular markers RNA, gma-mi and R1511-In Del. results showed that gma-mi R1511 loci exist in cultivated soybean, but there are 3 alleles in wild soybean. Based on the results. We think that In Del markers can be used for the evaluation of.Gma-mi R1511-In genetic variation of Del marker data and geographic information combination analysis, genetic diversity from the Yellow River basin annual wild soybean has the high performance, the annual wild soybean genetic diversity and soybean origin theory provides a more molecular evidence. In addition, there are also a negative correlation between the expression of gma-mi R1511 and its target gene Gm RPL4a in stress treatments, suggesting that gma-mi R1511 may regulate the function of soybean growth.G Ma-mi R1511 and its target gene has not yet been found in other leguminous plants, therefore, gma-mi R1511-In Del can be used for research on soybean genetic diversity, molecular marker gene specific soybean germplasm classification and evolution. This method of locating the precursor region sequences by Mi RNA sequence to the development of molecular markers is also reported for the first time.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q943.2

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