马铃薯microRNA及其靶基因的预测与功能分析
摘要
MicroRNAs (miRNAs)是一类约21-23 nt的非编码单链小RNA分子。已有研究表明,miRNA所调节的靶mRNA在很多植物的生物合成过程中起到重要作用,参与了根、茎、叶、花的形态发生、信号转导及不同环境的胁迫等。近年来,通过克隆手段和生物信息学方法,在植物上已鉴定出上百种miRNA和它的靶序列,但是主要集中在拟南芥、玉米等一些模式植物中,而且很多miRNA的功能尚不清楚。
马铃薯(Solanum tuberosum L.)是世界上主要粮食和蔬菜兼用作物之一。目前通过计算机预测法已获得了80多条马铃薯的miRNAs及其可能调控的靶基因。但与其他生物相比,目前已鉴定的马铃薯miRNA基因还远未达到饱和,而且对它们的生物功能尚不清楚。本研究将利用计算机预测法获得马铃薯新的miRNA,并明确其作用的靶基因,以期有目的地利用miRNA进行马铃薯的遗传改良,从而为进一步培育优质、高效、抗逆的马铃薯新种质奠定理论基础。取得的主要研究结果如下：
1.本文根据miRNA序列在植物中的高度保守性,通过拟南芥、水稻、玉米等植物中已知的miRNA序列,与马铃薯的表达序列标签(EST)、基因组测序序列(GSS)以及核酸数据库(nr)中的序列进行比对,最后通过在线比对软件blastx去掉编码序列,共发现32条新的miRNAs,这32条miRNAs前体通过RNAfold分析均可折叠形成miRNA家族的标准二级结构。
2.利用新发现的马铃薯miRNAs通过在线软件miRU和miRU2与编码蛋白的数据库比对,通过靶基因预测,共预测到了100个靶基因,这些靶基因分别编码与植物生长发育、新陈代谢、信号转导、转录调节、胁迫反应等相关的蛋白。
3.通过对miRNA 167b和miRNA482b两条miRNAs进行RT-PCR实验,发现马铃薯miR167b在芽、茎和根组织中有稳定的表达,而在叶组织中却没有表达。miR482b在马铃薯的芽、叶、茎和根中均有表达,在芽、茎和根中表达丰度比较高,但是在叶组织中表达量非常少,反映了miRNA的组织特异性表达。
MicroRNAs (miRNAs) are a class of non-coding small RNAs with 21-23 nucleotide length. Increasing evidence has shown that miRNAs play multiple roles in biological processes, involved in the morphogenesis of root, stem, leaf and flower, signal transduction and environmental stress and so on. In recent years, hundreds of miRNA and its target sequence have been identified in plants by cloning method and bioinformatics method, but mainly in Arabidopsis thaliana, maize and some other model plants, and many of the functions of miRNA is not clear.
Potato (Solarium tuberosum L.) is one of the world's food crops and vegetables, in which more than 80 potato miRNAs and their target genes have been obtained through the computational prediction. However, in comparison with other organisms, the miRNA genes identified for potato are still far from reaching saturation, and their biological functions are unknown. In this study, new potato miRNA were obtained by computational prediction, and target genes were identified in order to conduct purposeful use of miRNA genetic improvement of potato, so as to lay the theoretical foundation for the new of further developing high-quality, efficient and stress resistance of potato germplasm. The results are as follows:
1. In the present paper, according to the conservative property of miRNAs in plant, a total of 32 novel miRNAs were identified from potato following a range of filtering criteria by using previously deposited miRNA sequences from Arabidopsis thaliana, Oryza sativa and other plant species to blast potato expressed sequence tags (EST), genomic survey sequence (GSS) and nucleotide databases, and removing the protein coding genes by on line software blastx. All the potential miRNA precursors can be folded into the typical secondary structure of miRNA family.
2. Using the miRNAs sequences,100 target genes were identified by blasting the databases of coding protein using on line software miRU and miRU2. The genes encode protein involved in growth and development, metabolism, signal transduction, transcriptional regulation and stress responses.
3. By RT-PCR experiments, the results showed that potato miR167b was found with stable expression in the bud, stem and root tissues, but did not express in the leaf tissue. MiR482b in potato bud, leaf, stem and root were expressed, which expressed in relatively high abundance in bud, stem and root, but in leaf tissue in very small amounts, which reflected the specificity expression of miRNAs in the tissue.
马铃薯(Solanum tuberosum L.)是世界上主要粮食和蔬菜兼用作物之一。目前通过计算机预测法已获得了80多条马铃薯的miRNAs及其可能调控的靶基因。但与其他生物相比,目前已鉴定的马铃薯miRNA基因还远未达到饱和,而且对它们的生物功能尚不清楚。本研究将利用计算机预测法获得马铃薯新的miRNA,并明确其作用的靶基因,以期有目的地利用miRNA进行马铃薯的遗传改良,从而为进一步培育优质、高效、抗逆的马铃薯新种质奠定理论基础。取得的主要研究结果如下：
1.本文根据miRNA序列在植物中的高度保守性,通过拟南芥、水稻、玉米等植物中已知的miRNA序列,与马铃薯的表达序列标签(EST)、基因组测序序列(GSS)以及核酸数据库(nr)中的序列进行比对,最后通过在线比对软件blastx去掉编码序列,共发现32条新的miRNAs,这32条miRNAs前体通过RNAfold分析均可折叠形成miRNA家族的标准二级结构。
2.利用新发现的马铃薯miRNAs通过在线软件miRU和miRU2与编码蛋白的数据库比对,通过靶基因预测,共预测到了100个靶基因,这些靶基因分别编码与植物生长发育、新陈代谢、信号转导、转录调节、胁迫反应等相关的蛋白。
3.通过对miRNA 167b和miRNA482b两条miRNAs进行RT-PCR实验,发现马铃薯miR167b在芽、茎和根组织中有稳定的表达,而在叶组织中却没有表达。miR482b在马铃薯的芽、叶、茎和根中均有表达,在芽、茎和根中表达丰度比较高,但是在叶组织中表达量非常少,反映了miRNA的组织特异性表达。
MicroRNAs (miRNAs) are a class of non-coding small RNAs with 21-23 nucleotide length. Increasing evidence has shown that miRNAs play multiple roles in biological processes, involved in the morphogenesis of root, stem, leaf and flower, signal transduction and environmental stress and so on. In recent years, hundreds of miRNA and its target sequence have been identified in plants by cloning method and bioinformatics method, but mainly in Arabidopsis thaliana, maize and some other model plants, and many of the functions of miRNA is not clear.
Potato (Solarium tuberosum L.) is one of the world's food crops and vegetables, in which more than 80 potato miRNAs and their target genes have been obtained through the computational prediction. However, in comparison with other organisms, the miRNA genes identified for potato are still far from reaching saturation, and their biological functions are unknown. In this study, new potato miRNA were obtained by computational prediction, and target genes were identified in order to conduct purposeful use of miRNA genetic improvement of potato, so as to lay the theoretical foundation for the new of further developing high-quality, efficient and stress resistance of potato germplasm. The results are as follows:
1. In the present paper, according to the conservative property of miRNAs in plant, a total of 32 novel miRNAs were identified from potato following a range of filtering criteria by using previously deposited miRNA sequences from Arabidopsis thaliana, Oryza sativa and other plant species to blast potato expressed sequence tags (EST), genomic survey sequence (GSS) and nucleotide databases, and removing the protein coding genes by on line software blastx. All the potential miRNA precursors can be folded into the typical secondary structure of miRNA family.
2. Using the miRNAs sequences,100 target genes were identified by blasting the databases of coding protein using on line software miRU and miRU2. The genes encode protein involved in growth and development, metabolism, signal transduction, transcriptional regulation and stress responses.
3. By RT-PCR experiments, the results showed that potato miR167b was found with stable expression in the bud, stem and root tissues, but did not express in the leaf tissue. MiR482b in potato bud, leaf, stem and root were expressed, which expressed in relatively high abundance in bud, stem and root, but in leaf tissue in very small amounts, which reflected the specificity expression of miRNAs in the tissue.
引文
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