大麦微小RNA的克隆和鉴定
摘要
大麦是全世界排名第五的粮食作物，在全球各种环境条件下均广泛种植，其产品用于动物饲料、麦芽酿造及人类直接食用。此外，由于前人积累了大麦丰富的遗传信息资源，大麦被认为是禾本科的模式物种，大麦正成为小麦遗传育种研究的参考和重要的基因来源。
微小RNA（miRNAs）是一类在转录后水平调控各种生物代谢过程的内源性RNA，参与生物的生长发育和各种代谢活动，并参与植物对外界环境胁迫的应答和病害防御。在过去数年间，许多主要农作物的miRNA都被鉴定出来。然而，大麦的miRNA的信息却非常有限。
在本研究中，我们选用栽培麦芽大麦品种Clipper，提取其不同发育阶段根、茎、叶、穗等组织的总RNA，分离构建小RNA文库，采用第二代测序技术，即Solexa测序技术获得大麦的sRNA的序列数据库，通过生物信息学技术系统地鉴定大麦保守的miRNA以及特异的miRNA，并对结果进行了验证，取得主要结果如下：
（1）通过测序得到10,495,264个高质量的sRNA读数，其中大于18nt的共有4,045,224条sRNA，共9,540,562个读数，经过比对基因组，这其中包含核糖体RN（ArRNA）有83,300条序列，1,006,189读数，占到sRNA总数的10.55%；重复序列（repeat）23,642条序列，共90,970读数，占sRNA总数的0.95%；小核RNA（small nuclear RNA，snRNA）3,020条序列，15,180读数，占到sRNA的0.16%；核仁小RNA（small Nucleolus RNA，snoRNA）1,872条序列，6,711个读数，占总数的0.07%；转运RNA（transfer ribonucleicacid，tRNA）16,957条序列，共1,154,444读数，占12.10%；未注释（unannatation）的sRNA3,916,433条序列，7,267,068读数，占到总数的76.17%。
（2）对未注释的3,914,563条sRNA，比对miRBase18.0中4742条植物miRNA，有3282条序列比对上211个miRNA，隶属于88个家族，共543,016个读数；其中126个miRNA，隶属于58个家族，共323,131个读数，则是我们首次报道。
（3）运用华大基因公司（BGI）开发的Mireap软件对所有的大麦基因组序列和非编码表达序列标签（non coding EST）等的二级结构进行比对分析，依据Dicer酶切位点，最小自由能等参数和错配数目等标准，我们鉴定出133个大麦新的miRNA，隶属于50个家族，此外，我们还发现15个miRNA*。
（4）茎环qRT-PCR技术被用来验证克隆和生物信息分析结果，无论保守的还是大麦特异的6个miRNA都有不同水平的表达；3个保守的miRNA的表达水平ΔCT值为-0.2，远远高于3个特异miRNA的ΔCT值（4.8）。这一结果与高通量分析的结果一致，证明了分析结果的准确性。
（5）使用psRNATarget工具对鉴定出的miRNA的靶基因位点进行预测，获得29个miRNA的靶基因，包括GAMyb，核转录等转录因子，参与丝氨酸/苏氨酸激酶蛋白质合成的酶类，抗病相关蛋白10，抗性蛋白调控相关基因，淀粉分支酶（SBE）和种子软化蛋白等也被预测到是miRNA的作用目标。
综上，本研究工作首次系统的分析了大麦小RNA，揭示了所有的344个miRNAs，这些结果显著增加了大麦的miRNA数据库的数量。将有助于揭示miRNA在大麦基因表达调控的机制，为大麦以及小麦的遗传改良提供理论依据。
Barley（Hordeum vulgare L.）is one of the principal cereal crops in the world cultivatedin all temperate areas ranking fifth in world crop production. It’s used for animal feed,brewing malts, and human consumption. It has been considered as a model species for geneticanalysis thanks to its widely available genetic information. Therefore, barley can be thereference of wheat genetics and breeding and the source of specific useful genes
McroRNAs（miRNAs）are small class of endogenous RNAs that regulates the geneexpression which is involved in various biological and metabolic processes and stressresisitance at the post-transcriptional level in both plants and animals. Many major crops havebeen traced for miRNAs in past few years, yet miRNAs of barley（Hordeum vulgare L.）arestill questioned.
In this study, we identified small RNA transcriptome of barley to uncover novel andspecial microRNAs, sequenced using Solexa sequencing method. Millions reads wereobtained from short RNA libraries generated from pooled total RNA extracted from barley（cv.Clipper）roots, stems, leaves and spikes, after bioinformatics analysis, We identified theconserved and barley-specific miRNA. The main results are as follows.
First,10,367,915high quality reads（9,540,562clean reads）are obtained by Solexasequencing method， including4,045,224unique sRNA which the length more than18Nucleotide acid（nt）. After blast with genome sequence, in small RNA dataset there are83,300unique Ribosomal RNA accounted1,006,189reads, nd23,642unique repeataccounted90,970reads and3,020small nuclear RNA accounted15,180reads,1,872smallNucleolus RNA accounted6,711reads and16,957transfer ribonucleic acid accounted1,154,444reads. In addition,3,916,433unique unannatation sRNAs have7,267,068reads,accounted76.17percent of total sRNA.
Secondly, the unannatation small RNA sequences were used to do a Blastn searchagainst the miRNA databas（emiRBase18.0）in order to identify conserved miRNAs in barley.4742sequence were successful matched the previous211miRNAs, belonging to88families,as a total of543,016reads, of which126miRNA belonging to58families is first founded in barley by us.
The third, the genome sequence and all non coding EST were analysis by softwareMireap developed by BGI, corresponding to Criteria of secondary structure, Dicer enzymesites and the minimum free energy, mismatch number and other parameters.133miRNAwere identified that can be divided into50families. All133novel miRNAs are considered tobe species-specific because no homologs were found in other species. In addition,15miRNA*is the first reported by us.
The fourth, part of miRNA were validated by stem-loop qRT-PCR. The qRT-PCRresults demonstrate that all tested miRNAs are expressed in barey. However, the expressionlevels of the different miRNAs varied, the average expression level of conserved miRNA isΔCT-0.2, which is much higher than the novel miRNA（ΔCT4.8）. This result is the sameas the results of our high throughput analysis.
At last, to deepen the knowledge about the functions of the newly identifiedspecies-specific as well as conserved barley miRNAs, putative targets of these miRNAs werepredicted. In total, target genes of fourteen conserved and twelve novel barley miRNAfamilies were predicted. Transcription factors, including GAMyb transcription factor, nucleartranscription factor NAC were predicted to be potential targets of barley miRNAs.Furthermore, genes directly involved in protein synthesis, e.g., Serine/threonine kinase-likeprotein and protein synthesis inhibitor were also the targets of barley miRNAs.
In summary, our work provided a first systematic analysis of barley transcriptome touncover miRNAs and significantly increases number of novel miRNA in barley. These resultsreveal the miRNA regulation of gene expression and provide theoretical basis for barleygenetic improvement, even for the wheat.
微小RNA（miRNAs）是一类在转录后水平调控各种生物代谢过程的内源性RNA，参与生物的生长发育和各种代谢活动，并参与植物对外界环境胁迫的应答和病害防御。在过去数年间，许多主要农作物的miRNA都被鉴定出来。然而，大麦的miRNA的信息却非常有限。
在本研究中，我们选用栽培麦芽大麦品种Clipper，提取其不同发育阶段根、茎、叶、穗等组织的总RNA，分离构建小RNA文库，采用第二代测序技术，即Solexa测序技术获得大麦的sRNA的序列数据库，通过生物信息学技术系统地鉴定大麦保守的miRNA以及特异的miRNA，并对结果进行了验证，取得主要结果如下：
（1）通过测序得到10,495,264个高质量的sRNA读数，其中大于18nt的共有4,045,224条sRNA，共9,540,562个读数，经过比对基因组，这其中包含核糖体RN（ArRNA）有83,300条序列，1,006,189读数，占到sRNA总数的10.55%；重复序列（repeat）23,642条序列，共90,970读数，占sRNA总数的0.95%；小核RNA（small nuclear RNA，snRNA）3,020条序列，15,180读数，占到sRNA的0.16%；核仁小RNA（small Nucleolus RNA，snoRNA）1,872条序列，6,711个读数，占总数的0.07%；转运RNA（transfer ribonucleicacid，tRNA）16,957条序列，共1,154,444读数，占12.10%；未注释（unannatation）的sRNA3,916,433条序列，7,267,068读数，占到总数的76.17%。
（2）对未注释的3,914,563条sRNA，比对miRBase18.0中4742条植物miRNA，有3282条序列比对上211个miRNA，隶属于88个家族，共543,016个读数；其中126个miRNA，隶属于58个家族，共323,131个读数，则是我们首次报道。
（3）运用华大基因公司（BGI）开发的Mireap软件对所有的大麦基因组序列和非编码表达序列标签（non coding EST）等的二级结构进行比对分析，依据Dicer酶切位点，最小自由能等参数和错配数目等标准，我们鉴定出133个大麦新的miRNA，隶属于50个家族，此外，我们还发现15个miRNA*。
（4）茎环qRT-PCR技术被用来验证克隆和生物信息分析结果，无论保守的还是大麦特异的6个miRNA都有不同水平的表达；3个保守的miRNA的表达水平ΔCT值为-0.2，远远高于3个特异miRNA的ΔCT值（4.8）。这一结果与高通量分析的结果一致，证明了分析结果的准确性。
（5）使用psRNATarget工具对鉴定出的miRNA的靶基因位点进行预测，获得29个miRNA的靶基因，包括GAMyb，核转录等转录因子，参与丝氨酸/苏氨酸激酶蛋白质合成的酶类，抗病相关蛋白10，抗性蛋白调控相关基因，淀粉分支酶（SBE）和种子软化蛋白等也被预测到是miRNA的作用目标。
综上，本研究工作首次系统的分析了大麦小RNA，揭示了所有的344个miRNAs，这些结果显著增加了大麦的miRNA数据库的数量。将有助于揭示miRNA在大麦基因表达调控的机制，为大麦以及小麦的遗传改良提供理论依据。
Barley（Hordeum vulgare L.）is one of the principal cereal crops in the world cultivatedin all temperate areas ranking fifth in world crop production. It’s used for animal feed,brewing malts, and human consumption. It has been considered as a model species for geneticanalysis thanks to its widely available genetic information. Therefore, barley can be thereference of wheat genetics and breeding and the source of specific useful genes
McroRNAs（miRNAs）are small class of endogenous RNAs that regulates the geneexpression which is involved in various biological and metabolic processes and stressresisitance at the post-transcriptional level in both plants and animals. Many major crops havebeen traced for miRNAs in past few years, yet miRNAs of barley（Hordeum vulgare L.）arestill questioned.
In this study, we identified small RNA transcriptome of barley to uncover novel andspecial microRNAs, sequenced using Solexa sequencing method. Millions reads wereobtained from short RNA libraries generated from pooled total RNA extracted from barley（cv.Clipper）roots, stems, leaves and spikes, after bioinformatics analysis, We identified theconserved and barley-specific miRNA. The main results are as follows.
First,10,367,915high quality reads（9,540,562clean reads）are obtained by Solexasequencing method， including4,045,224unique sRNA which the length more than18Nucleotide acid（nt）. After blast with genome sequence, in small RNA dataset there are83,300unique Ribosomal RNA accounted1,006,189reads, nd23,642unique repeataccounted90,970reads and3,020small nuclear RNA accounted15,180reads,1,872smallNucleolus RNA accounted6,711reads and16,957transfer ribonucleic acid accounted1,154,444reads. In addition,3,916,433unique unannatation sRNAs have7,267,068reads,accounted76.17percent of total sRNA.
Secondly, the unannatation small RNA sequences were used to do a Blastn searchagainst the miRNA databas（emiRBase18.0）in order to identify conserved miRNAs in barley.4742sequence were successful matched the previous211miRNAs, belonging to88families,as a total of543,016reads, of which126miRNA belonging to58families is first founded in barley by us.
The third, the genome sequence and all non coding EST were analysis by softwareMireap developed by BGI, corresponding to Criteria of secondary structure, Dicer enzymesites and the minimum free energy, mismatch number and other parameters.133miRNAwere identified that can be divided into50families. All133novel miRNAs are considered tobe species-specific because no homologs were found in other species. In addition,15miRNA*is the first reported by us.
The fourth, part of miRNA were validated by stem-loop qRT-PCR. The qRT-PCRresults demonstrate that all tested miRNAs are expressed in barey. However, the expressionlevels of the different miRNAs varied, the average expression level of conserved miRNA isΔCT-0.2, which is much higher than the novel miRNA（ΔCT4.8）. This result is the sameas the results of our high throughput analysis.
At last, to deepen the knowledge about the functions of the newly identifiedspecies-specific as well as conserved barley miRNAs, putative targets of these miRNAs werepredicted. In total, target genes of fourteen conserved and twelve novel barley miRNAfamilies were predicted. Transcription factors, including GAMyb transcription factor, nucleartranscription factor NAC were predicted to be potential targets of barley miRNAs.Furthermore, genes directly involved in protein synthesis, e.g., Serine/threonine kinase-likeprotein and protein synthesis inhibitor were also the targets of barley miRNAs.
In summary, our work provided a first systematic analysis of barley transcriptome touncover miRNAs and significantly increases number of novel miRNA in barley. These resultsreveal the miRNA regulation of gene expression and provide theoretical basis for barleygenetic improvement, even for the wheat.
引文
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