耐旱野生大豆MicroRNA的鉴定与表达分析
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摘要
小RNA(MicroRNA,miRNA)在转录后基因沉默过程中扮演重要角色,它可以介导具有同源序列的靶基因miRNA裂解或抑制其翻译。野生大豆(Glycine soja)是栽培大豆改良的重要遗传资源,对其开展miRNA的鉴定与功能研究,将进一步促进野生大豆资源在栽培大豆育种中的利用。本研究利用小分子RNA文库串联测序、Solexa高通量测序等技术,对抗旱野生大豆资源S101中的小分子RNA进行分离、鉴定,从中获得与抗旱相关的miRNA;结合靶基因裂解位点验证实验,通过构建miRNA表达载体、转化拟南芥,对miRNA的功能进行初步研究。
本研究在野生大豆miRNA的分离、鉴定与功能分析等方面获得了大量重要信息,进一步拓宽了对植物miRNA的理解和认识。详细研究结果如下:
1、小分子RNA文库串联测序数据分析
通过构建小分子RNA文库并串联测序,共获得2,880条小分子RNA序列,序列分布图显示19~24 nt的序列为1,347条(1,022条单一序列),占46.8%;21nt小分子RNA表达丰度最高,占334条(223条单一序列)。
①与已知miRNA(miRBase_mature, Release 11.0)同源比对发现,有15条小RNA序列与植物已知miRNA一致或同源(≤2bp mismatches),其中14条序列与栽培大豆miRNA同源。它们分别隶属于8个保守家族:miR156、miR159、miR160、miR167、miR168、miR171、miR319和miR396。除了miR171家族以外,其他7个保守家族均已在大豆中报道;
②与栽培大豆EST数据库分析发现, 15个保守miRNA候选者中有9个可以在27条大豆EST序列中找到完全匹配的位点,其中gso-miR167对应的两条前体序列与大豆已知miR167前体序列不同,但能形成特征性的发卡环结构,说明miR167可能有两个新的候选者及其前体;
③在预测分析新miRNA的过程中发现,442条候选小RNA序列中有74条可以在407条大豆EST序列中找到完全匹配的位点,其中9条小RNA序列(8个家族)对应的22条EST序列具备miRNA前体特征;
④预测发现的15个保守miRNA和9个新miRNA候选者的长度均在20-22nt之间,多数5’端为尿嘧啶,符合miRNA的典型特征。
2、小分子RNA文库高通量测序数据分析
新一代测序技术为miRNA的分离、鉴定提供了高效的分析平台。利用Solexa高通量序列测定仪,分析了野生大豆小分子RNA文库,共获得高质量序列3,161,992条(1,282,308条单一序列)。
①与大豆基因组序列信息分析表明,12,734条小RNA对应的基因组序列能通过mFold和mirCheck检测,具备miRNA的特征性的发卡环结构,由此可见miRNA群体的复杂程度。经过进一步的复杂分析和严格筛选,共得到141个保守的miRNA和171个新miRNA;
②171个新miRNA中,53个新miRNA共预测到206个靶基因,其功能涉及到植物生长发育和环境响应的各个方面;
③分析结果还发现许多值得深入研究的现象。例如:在植物中普遍存在的miR159和miR319前体中发现了串联miRNA的现象、某些保守miRNA星号链的大量存在、大豆miRNA前体的正负链匹配等。
3、miRNA功能的初步分析
对小分子RNA文库串联测序获得的8个保守和8个新的miRNA家族进行了Nothern杂交、靶基因预测、转化拟南芥等初步的功能分析。
①Nothern杂交验证表明,8个保守和8个新的miRNA家族在野生大豆正常生长和干旱胁迫情况下都有表达,但表达丰度各异。其中,4个保守miRNA和3个新miRNA在干旱胁迫前后表现为不同程度的丰度变化。miR160、miR167、miR319、miR396和gso-miR2在干旱胁迫后明显下调表达;gso-miR1和gso-miR6干旱胁迫后上调表达。针对8个新发现的miRNA家族的组织特异性分析表明,所有miRNA在正常生长的野生大豆的根、茎、叶中的表达丰度均各有特点。多数表现为高度的组织特异性表达,并且倾向于在根中相对高丰度表达。
②靶基因预测分析发现,7个新的miRNA家族对应的32个靶基因多数为功能上已注释的抗病或抗性蛋白。靶基因裂解位点验证实验表明,其中gso-miR7对应的功能未知基因TC233731和gso-miR8对应的TC225607(R 9蛋白)的裂解位点与预测一致。
③通过构建miRNA表达载体、转化拟南芥,对gso-miR5、gso-miR6、gso-miR7和gso-miR8进行了miRNA功能的初步研究,发现gso-miR8对应的T1代植株表现出早开花的突变表型。
Small RNA (MicroRNA, miRNA) plays important roles in post-transcriptional gene silence bydirecting target mRNAcleavage or translational inhibition. Wild soybean (Glycine soja) is considered tobe the most important gene sources for genetic modification of domesticated soybean. Identification andfunctional analysis of miRNAin the Glycine soja will further promote the utilization of genetic resoursesfor soybean breeding. This study used two approaches to separate and identify miRNAin accession S101of drought-resistant wild soybean: concatenating cDNA fragments for sequencing and high throughputsequencing. Combining with examinations of putative miRNA-directed cleavage products, constructingartifical pre-miRNAs and transformation of Arabidopsis thaliana, we performed preliminary functionalresearch for novel miRNAs.
This study obtained important information from separation and identification of miRNAin Glycinesoja, and broadened the current understanding and awareness of plant miRNAs. The detailed results areas follws:
1.Data analysis of small RNAlibraryfrom concatenation forsequencing
Through constructing a small RNA library and sequencing after concatenation, we obtained 2,880high quality small RNAsequences. The distribution map indicated that 1,347 (1,022 Unique) were 19-24nt in length, which composing 46.8%. The most abundant class was 21 nt, which contained 334sequences (223 unique).
①Comparing these small RNAs with the mature miRBase (release 11.0) by BLASTN, we found15 conserved miRNAs homologs (≤2bp mismatches), in which 14 were conserved with domesticatedsoybean. The 15 conserved miRNA candidates can compose 8 families: miR156, miR159, miR160,miR167, miR168, miR171, miR319 and miR396. Except miR171, all these conserved families had beenpreviouslypredicted in domesticated soybean.
②Analysed combining the domesticated soybean EST database, 9 of the 15 homologs could findperfectly matched hits on 27 ESTs. 17 ESTs were consistent with the reported soybean miRNAprecursors except 10 ESTs (corresponding to miR167 homolog), which had been classified as novel twoputative gso-miR167 precursors after secondarystructure analysis.
③For novel miRNAprediction, 74 small RNAin 442 candidates could find perfect matching hitsin 407 soybean EST sequences. As a result, 22 ESTs corresponding to 9 small RNAs (8 families) wereidentified with characteristic hairpin structures, which were deemed as novel miRNAcandidates.
④All these miRNA candidates (15 conserved and 9 novel) were 20~22 nt in length and most oftheir 5’terminal nucleotides were uridine, which was consistent with the miRNAcharacteristic.
2.Data analysis of small RNAlibraryfrom high throughput sequencing
The next-generation sequencing technology (Solexa) afforded an effective approach foridentification of miRNAs. Using the Solexa sequencing technology, we constructed a small RNAlibraryand obtained 3,161,992 high quality reads (1,282,308 Unique).
①Combining the recent soybean genome database (Glyma1), bioinformatics analyses indicated that 12,734 miRNAcandidates passed RNAfold and MIRcheck evaluation, which shown the complexityof miRNA groups. Finally, 141 conserved miRNAs comprising 34 families and 171 novel miRNAscomprising 100 families were identified in wild soybean after strict screening and manual conformed.
②Among 171 novel miRNA candidates, 206 putative target genes with various functions werepredicted for 53 miRNAs, which were involved in varied aspects of environmental responses anddevelopmental processes in plants.
③Additionally, based on the high through-put advantage, some interesting phenomena wereobserved from further analysis. Such as: tandem patterns of miRNA in miR159 and miR319 precursors,some miRNA* accumulation, and querying the qualification of some gam-miRNAs which seemed to begenerated fromdouble strands.
3. Preliminary functional analysis of miRNAs
We performed preliminary functional analysis of 8 conserved and 8 novel miRNAfamilies obtainedfrom small RNA library. Such as, Nothern blot、target prediction and transformation of Arabidopsisthaliana.
①Northern blot analysis indicated that all the 8 conserved and 8 novel miRNA families weredetectable in wild soybean seedlings under normal or drought stressed conditions. Among them, fourconserved and three novel miRNAs displayed different abundance in respond to drought stress. miR160、miR167、miR319、miR396 and gso-miR2 were down-regulated, while gso-miR1 and gso-miR6 wereup-regulated. Aiming at the 8 novel miRNA families, Northern blot analysis were also performed in inroots, stems and leaves of wild soybean. Most of them were inclined to express in roots and exhibitedhigh tissue-specific expression, which suggested that the miRNAs’biogenesis and functions might bevaried in different organs.
②Target prediction found 32 genes as putative targets for 7 novel miRNAs. Except that part ofthese targets was not annotated, most of them were resistant proteins. We examined the presence ofputative miRNA-directed cleavage products by 5’-RACE experiments. Two of these potential targetswere validated, TC233731 for gso-miR7 and TC225607 for gso-miR8. Targets of gso-miR8 were afamilyof resistant proteins in whichTC225607 coding for an R 9 protein.
③Through constructing miRNAexpression vector and transformation of Arabidopsis thaliana, thepreliminary functional research (gso-miR5、gso-miR6、gso-miR7 and gso-miR8) indicated that mosttransgenic plantlets of gso-miR8 exhibited earlyflowering mutant phenotype.
本研究在野生大豆miRNA的分离、鉴定与功能分析等方面获得了大量重要信息,进一步拓宽了对植物miRNA的理解和认识。详细研究结果如下:
1、小分子RNA文库串联测序数据分析
通过构建小分子RNA文库并串联测序,共获得2,880条小分子RNA序列,序列分布图显示19~24 nt的序列为1,347条(1,022条单一序列),占46.8%;21nt小分子RNA表达丰度最高,占334条(223条单一序列)。
①与已知miRNA(miRBase_mature, Release 11.0)同源比对发现,有15条小RNA序列与植物已知miRNA一致或同源(≤2bp mismatches),其中14条序列与栽培大豆miRNA同源。它们分别隶属于8个保守家族:miR156、miR159、miR160、miR167、miR168、miR171、miR319和miR396。除了miR171家族以外,其他7个保守家族均已在大豆中报道;
②与栽培大豆EST数据库分析发现, 15个保守miRNA候选者中有9个可以在27条大豆EST序列中找到完全匹配的位点,其中gso-miR167对应的两条前体序列与大豆已知miR167前体序列不同,但能形成特征性的发卡环结构,说明miR167可能有两个新的候选者及其前体;
③在预测分析新miRNA的过程中发现,442条候选小RNA序列中有74条可以在407条大豆EST序列中找到完全匹配的位点,其中9条小RNA序列(8个家族)对应的22条EST序列具备miRNA前体特征;
④预测发现的15个保守miRNA和9个新miRNA候选者的长度均在20-22nt之间,多数5’端为尿嘧啶,符合miRNA的典型特征。
2、小分子RNA文库高通量测序数据分析
新一代测序技术为miRNA的分离、鉴定提供了高效的分析平台。利用Solexa高通量序列测定仪,分析了野生大豆小分子RNA文库,共获得高质量序列3,161,992条(1,282,308条单一序列)。
①与大豆基因组序列信息分析表明,12,734条小RNA对应的基因组序列能通过mFold和mirCheck检测,具备miRNA的特征性的发卡环结构,由此可见miRNA群体的复杂程度。经过进一步的复杂分析和严格筛选,共得到141个保守的miRNA和171个新miRNA;
②171个新miRNA中,53个新miRNA共预测到206个靶基因,其功能涉及到植物生长发育和环境响应的各个方面;
③分析结果还发现许多值得深入研究的现象。例如:在植物中普遍存在的miR159和miR319前体中发现了串联miRNA的现象、某些保守miRNA星号链的大量存在、大豆miRNA前体的正负链匹配等。
3、miRNA功能的初步分析
对小分子RNA文库串联测序获得的8个保守和8个新的miRNA家族进行了Nothern杂交、靶基因预测、转化拟南芥等初步的功能分析。
①Nothern杂交验证表明,8个保守和8个新的miRNA家族在野生大豆正常生长和干旱胁迫情况下都有表达,但表达丰度各异。其中,4个保守miRNA和3个新miRNA在干旱胁迫前后表现为不同程度的丰度变化。miR160、miR167、miR319、miR396和gso-miR2在干旱胁迫后明显下调表达;gso-miR1和gso-miR6干旱胁迫后上调表达。针对8个新发现的miRNA家族的组织特异性分析表明,所有miRNA在正常生长的野生大豆的根、茎、叶中的表达丰度均各有特点。多数表现为高度的组织特异性表达,并且倾向于在根中相对高丰度表达。
②靶基因预测分析发现,7个新的miRNA家族对应的32个靶基因多数为功能上已注释的抗病或抗性蛋白。靶基因裂解位点验证实验表明,其中gso-miR7对应的功能未知基因TC233731和gso-miR8对应的TC225607(R 9蛋白)的裂解位点与预测一致。
③通过构建miRNA表达载体、转化拟南芥,对gso-miR5、gso-miR6、gso-miR7和gso-miR8进行了miRNA功能的初步研究,发现gso-miR8对应的T1代植株表现出早开花的突变表型。
Small RNA (MicroRNA, miRNA) plays important roles in post-transcriptional gene silence bydirecting target mRNAcleavage or translational inhibition. Wild soybean (Glycine soja) is considered tobe the most important gene sources for genetic modification of domesticated soybean. Identification andfunctional analysis of miRNAin the Glycine soja will further promote the utilization of genetic resoursesfor soybean breeding. This study used two approaches to separate and identify miRNAin accession S101of drought-resistant wild soybean: concatenating cDNA fragments for sequencing and high throughputsequencing. Combining with examinations of putative miRNA-directed cleavage products, constructingartifical pre-miRNAs and transformation of Arabidopsis thaliana, we performed preliminary functionalresearch for novel miRNAs.
This study obtained important information from separation and identification of miRNAin Glycinesoja, and broadened the current understanding and awareness of plant miRNAs. The detailed results areas follws:
1.Data analysis of small RNAlibraryfrom concatenation forsequencing
Through constructing a small RNA library and sequencing after concatenation, we obtained 2,880high quality small RNAsequences. The distribution map indicated that 1,347 (1,022 Unique) were 19-24nt in length, which composing 46.8%. The most abundant class was 21 nt, which contained 334sequences (223 unique).
①Comparing these small RNAs with the mature miRBase (release 11.0) by BLASTN, we found15 conserved miRNAs homologs (≤2bp mismatches), in which 14 were conserved with domesticatedsoybean. The 15 conserved miRNA candidates can compose 8 families: miR156, miR159, miR160,miR167, miR168, miR171, miR319 and miR396. Except miR171, all these conserved families had beenpreviouslypredicted in domesticated soybean.
②Analysed combining the domesticated soybean EST database, 9 of the 15 homologs could findperfectly matched hits on 27 ESTs. 17 ESTs were consistent with the reported soybean miRNAprecursors except 10 ESTs (corresponding to miR167 homolog), which had been classified as novel twoputative gso-miR167 precursors after secondarystructure analysis.
③For novel miRNAprediction, 74 small RNAin 442 candidates could find perfect matching hitsin 407 soybean EST sequences. As a result, 22 ESTs corresponding to 9 small RNAs (8 families) wereidentified with characteristic hairpin structures, which were deemed as novel miRNAcandidates.
④All these miRNA candidates (15 conserved and 9 novel) were 20~22 nt in length and most oftheir 5’terminal nucleotides were uridine, which was consistent with the miRNAcharacteristic.
2.Data analysis of small RNAlibraryfrom high throughput sequencing
The next-generation sequencing technology (Solexa) afforded an effective approach foridentification of miRNAs. Using the Solexa sequencing technology, we constructed a small RNAlibraryand obtained 3,161,992 high quality reads (1,282,308 Unique).
①Combining the recent soybean genome database (Glyma1), bioinformatics analyses indicated that 12,734 miRNAcandidates passed RNAfold and MIRcheck evaluation, which shown the complexityof miRNA groups. Finally, 141 conserved miRNAs comprising 34 families and 171 novel miRNAscomprising 100 families were identified in wild soybean after strict screening and manual conformed.
②Among 171 novel miRNA candidates, 206 putative target genes with various functions werepredicted for 53 miRNAs, which were involved in varied aspects of environmental responses anddevelopmental processes in plants.
③Additionally, based on the high through-put advantage, some interesting phenomena wereobserved from further analysis. Such as: tandem patterns of miRNA in miR159 and miR319 precursors,some miRNA* accumulation, and querying the qualification of some gam-miRNAs which seemed to begenerated fromdouble strands.
3. Preliminary functional analysis of miRNAs
We performed preliminary functional analysis of 8 conserved and 8 novel miRNAfamilies obtainedfrom small RNA library. Such as, Nothern blot、target prediction and transformation of Arabidopsisthaliana.
①Northern blot analysis indicated that all the 8 conserved and 8 novel miRNA families weredetectable in wild soybean seedlings under normal or drought stressed conditions. Among them, fourconserved and three novel miRNAs displayed different abundance in respond to drought stress. miR160、miR167、miR319、miR396 and gso-miR2 were down-regulated, while gso-miR1 and gso-miR6 wereup-regulated. Aiming at the 8 novel miRNA families, Northern blot analysis were also performed in inroots, stems and leaves of wild soybean. Most of them were inclined to express in roots and exhibitedhigh tissue-specific expression, which suggested that the miRNAs’biogenesis and functions might bevaried in different organs.
②Target prediction found 32 genes as putative targets for 7 novel miRNAs. Except that part ofthese targets was not annotated, most of them were resistant proteins. We examined the presence ofputative miRNA-directed cleavage products by 5’-RACE experiments. Two of these potential targetswere validated, TC233731 for gso-miR7 and TC225607 for gso-miR8. Targets of gso-miR8 were afamilyof resistant proteins in whichTC225607 coding for an R 9 protein.
③Through constructing miRNAexpression vector and transformation of Arabidopsis thaliana, thepreliminary functional research (gso-miR5、gso-miR6、gso-miR7 and gso-miR8) indicated that mosttransgenic plantlets of gso-miR8 exhibited earlyflowering mutant phenotype.
引文
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