油菜（Brassica napus）miRNA的鉴定及Bna-miR1140表达调控机制研究
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摘要
miRNAs(microRNA)是一类在进化上保守的长约21nt的非编码单链RNA，在基因表达调控的网络中处于核心位置，高效的调控其靶基因的翻译或表达，在生物体生长发育、抵抗生物或者非生物胁迫以及在生物体代谢途径中起着非常重要的作用。在短短的10年时间内，植物miRNAs的研究取得了突飞猛进的成果，成为一个新的研究领域。油菜(rape or rapeseed)作为世界性的重要油料作物，研究其miRNA所参与的遗传控制机理，对于培育理想油菜品种，增加油菜产量和降低油菜生产成本具有重要意义。
鉴于目前对油菜miRNAs及其靶基因的研究报道还很少，且sanger数据库中可参考的miRNAs信息也仅仅48条，本研究对甘蓝型油菜(Brassica napus)栽培种Westar材料进行小RNA测序及降解组分析，鉴定新的油菜miRNAs，寻找已知miRNAs和新发现的miRNAs的靶基因，并对芸薹属独特的miRNA家族Bna-miR1140进行表达调控机制研究，以期为油菜品种改良和油菜miRNAs调控机制的研究提供理论依据。
主要结果如下：
1.鉴定获得了41个新的保守油菜miRNAs以及67个芸薹属特有的新候选miRNAs，其中包括20个miRNAs*序列。利用实时荧光定量RT-PCR技术验证了测序结果。并将新发现的miRNAs提交miRBase数据库。
2.依据NCBI中现有的油菜EST和基因组数据，利用targetfinder软件预测了油菜miRNAs靶基因，分别获得保守的miRNAs和特异的新候选miRNAs靶向的基因113个和84个。其中Bna-miR1140的潜在靶基因为一种糖基转移酶（glycosyltransferase）和响应调节因子（Arabidopsis response regulator ARR8-likeprotein）。
3.克隆得到了油菜Bna-miR1140基因前体197bp的片段，构建了植物过表达载体35S::Bna-miR1140，通过农杆菌介导法浸染油菜子叶柄转化甘蓝型油菜栽培种Westar。获得了14个PCR阳性植株，其中有5株株型特异，表现双主序表型，分枝数明显增多，其他9株阳性株均与野生型油菜表型一致，推测可能与表达强度有关。调查了株型特异的5个株系T1代表型分离情况，经卡方检验发现其中有4个株系的株型变异遗传符合3:1的孟德尔分离规律。初步推测油菜Bna-miR1140可能参与调控了油菜的分枝发育。
4.分离得到了油菜Bna-miR1140基因上游1351bp的序列，利用plantCARE在线软件对启动子序列的顺式作用元件进行在线分析，该启动子含有与转录必需的RNA聚合酶结合的TATA盒以及在调控基因转录效率中发挥重要作用的CAAT盒，并进一步构建了miR1140pro::GUS植物表达载体。通过农杆菌介导法转化油菜，得到5株PCR阳性植株，对转基因植株的根、茎、叶、叶腋(茎尖分生组织，SAM)、叶柄、花、种荚等组织和器官进行GUS染色分析，结果表明GUS仅在叶柄及叶腋中表达，这与Bna-miR1140体内表达的结果一致，进一步佐证了Bnas-miR1140与调控油菜的分枝发育相关。
综上所述，本研究采用深度测序和降解组分析技术鉴定了一批油菜的miRNA，并对其靶基因进行了分析和预测。对油菜特有的小RNABna-miR1140进行了较深入的研究，转基因结果初步表明miR1140参与了油菜分枝的发育。
MicroRNAs (miRNAs) are non-coding RNAs of approximately21nucleotides that havebeen identified as important regulators of gene expression in both animals and plants. In plants,miRNAs not only post-transcriptionally regulate their targets but also interact with each other inregulatory networks to affect many aspects of development, such as developmental timing,senescence, leaf morphogenesis, reproductive development, and modulation of root architecture.miRNAs are also reported to be involved in plant responses to biotic and environmental stresses.miRNAs are in the core position of the gene expression regulation network, and effectively inhibitits target genes encoding the proteins, or inhibit the expression of target genes via otheradjustment mechanism, resulting in the phenomenon of gene silencing. In the past decades, theplant miRNA research has been made great progress and Significant achievements.As adicotyledons, oilseed rape (Brassica Napus) is one of the most important oil crops,its miRNAhas also become a hot research field of molecular biology today. The studies about miRNAgenetic control mechanism involved in the regulation of rapeseed development can improve thetheoretical basis of the developmental biology of higher plant. Meanwhile, the clone andidentification of miRNA gene involved in the regulation rapeseed development, combined withmolecular breeding means to foster the ideal rape varieties for increasing yield of rapeseed andreducing the rapeseed production costs are very practical significance.
So far, the reports about studies of rapeseed miRNAs and its targets are much less, andmiRBase currently lists48miRNAs forming17miRNA families in Brassica napus. Here, wedescribe the high-throughput sequencing analysis of sRNAs from a cultivated variety of B. napus,cv Westar, using the Illumina Solexa platform. The sRNAs library was prepared for Solexasequencing from greenhouse cultivated rape plants, new miRNAs and target genes of knownmiRNAs and newly discovered miRNAs have been identificated. In order to provide a theoreticalbasis for rape breed improvement and rape miRNAs regulatory mechanism, the expressionregulation mechanism for the Bna-miR1140of Brassica unique miRNA families has also beenstudied. The methods are as follows, Leaves, petiole, stalk, roots and shoot apices from onemonth-old seedlings were collected and total RNA from different tissues was extracted usingTrizol (Invitrogen). sRNA and degradome cDNA libraries for Solexa sequencing were constructedfollowing the Illumina protocol. The purified cDNA library was sequenced on an IlluminaGAIIx(Biological Information Technology Co., Ltd., Hangzhou LC). Bioinformatic analysis ofthe sequencing data has been carried to identify known and new miRNAs and its target genes;Stem–loop RT primers, universal reverse primer and miRNA-specific forward primers for20sequenced miRNA sequences were designed according to Varkonyi-Gasic to validate and measurethe levels of B. napus miRNA by qRT-PCR. The primers for the precursor of Bna-miR1140 sequence were designed on the basis of the miRNAbase and its full-length was obtained by PCRamplification of Rape genome database, Then the35S:: Bna-miR1140plants over expressionvector constructed infected the cotyledons of wild-type rapeseed varieties by the Agrobacteriummediated. The phenotype of positive plants with the Bna-miR1140transformed was analyzed; Thesequence of the Bna-miR1140upstream1.5kb was analyzed via plantCARE software, finding thekey elements of promoter, such as the TATA-box, G-box et al., and then design primers for it andamplify the1.5kb fragment from rapeseed genomic DNA and constructed miR1140pro::GUSplant expression vector. The tissue of positive lines with miR1140pro::GUS transformed wasanalyzed by GUS staining to analysis the Bna-miR1140expression specificity.
Through the experimental analysis, the results as follows:
1. Here,41new conserved and67brassica-specific candidate miRNAs, including20miRNA*sequences were firstly identified. The sequencing results were further confirmed using stem-loopquantitative RT-PCR. The data will be updated to incorporate future miRBase updates. Ourapproach leads to the prediction of several conserved and specific brassica miRNA targets in theavailable EST and genomic databases.113mRNA targets of conserved brassica miRNAs and84mRNA targets of new brassica-specific miRNAs were identified. Validated miRNA targets in B.napus are potentially involved in diverse biological processes, including phase transitions,flowering, hormone signaling, photosynthesis, metabolism and biotic and abiotic stress resistance.Our findings will be a useful resource toward tracing the evolution of small RNA-based regulationin Brassica napus and related species. Most importantly, this study will serve as a foundation forfuture research into the functional roles of miRNAs and their target genes in this important oilcrop.
2. The precursor of Bna-miR1140gene cloned from Rape is197bp, and the plant overexpression vector35S::Bna-miR1140was constructed successfully. After disseminating thepetiole of rapeseed cultivars Westar via agrobacterium mediated transformation method. At Last,14T0generation Rape positive strains of over expressed35S::Bna-miR1140have been identifiedby the PCR method. There were5strains performed specific plant type, they had double maininflorescence and significantly increased the number of branches, nine positive strains consistentwith the wild-type rapeseed phenotype. After investigating the phenotype separation of five T1generation lines with specific plant type, and, of which there are four strains, their plant typevariation genetic followed3:1Mendel segregation law by the chi-square test. As for the otherpositive strains of over-expressed the35S::Bna-miR1140were not performed variation, they maynot express the35S::Bna-miR1140or transformed copy numbers are few, the real reasons must befurther validated by Northern blotting and other molecular experimental means in future. By phenotypic analysis, initially speculated the Bna-miR1140may participate in the regulation ofbranching and development of oilseed rape.
3. The1351bp fragment upstream of Bna-miR1140gene has been isolated, and the cis-actingelements of the promoter sequence were analyzed by plantCARE online software, the promotercontains TATA boxes binding RNA polymerase and CAAT boxes which are necessary fortranscription, because it play important role in the regulation of gene transcription efficiency. ThemiR1140pro::GUS plant expression vector was further constructed. Similarly, five positive T0rape strains transformed miR1140pro:: GUS were got, which have been transformed byagrobacterium-mediated infection rapeseed petiole of cultivars Westar. The GUS staining analysisshowed that the1.5kb region of upstream miR1140precursor sequence have promoter function bystaining each tissue of positive rapeseed strains, which able to drive the expression of GUS in rape.The miR1140pro::GUS transgenic rapeseed roots, stems, leaves, leaf axillary (shoot apexpoints Health organization, SAM), petiole, flower, pods and other tissue were carried GUSstaining analysis, the results showed that GUS only expressed in petioles and leaf axillary, thatconsistent with the results of the Bna-miR1140vivo expression, which is further corroborated theBna-miR1140regulation rape branching and development.
Summing up the results of the study, we found that the results of the Bna-miR1140vivoexpression and miR1140pro:: GUS expression patterns are consistent, proving Bna-miR1140play a role in rape branching development process; By solexa sequencing technology, many newconservative and specific miRNAs have been identified, finding that the target gene ofBna-miR1140are glycosyltransferase and the response to the adjustment factors.
鉴于目前对油菜miRNAs及其靶基因的研究报道还很少，且sanger数据库中可参考的miRNAs信息也仅仅48条，本研究对甘蓝型油菜(Brassica napus)栽培种Westar材料进行小RNA测序及降解组分析，鉴定新的油菜miRNAs，寻找已知miRNAs和新发现的miRNAs的靶基因，并对芸薹属独特的miRNA家族Bna-miR1140进行表达调控机制研究，以期为油菜品种改良和油菜miRNAs调控机制的研究提供理论依据。
主要结果如下：
1.鉴定获得了41个新的保守油菜miRNAs以及67个芸薹属特有的新候选miRNAs，其中包括20个miRNAs*序列。利用实时荧光定量RT-PCR技术验证了测序结果。并将新发现的miRNAs提交miRBase数据库。
2.依据NCBI中现有的油菜EST和基因组数据，利用targetfinder软件预测了油菜miRNAs靶基因，分别获得保守的miRNAs和特异的新候选miRNAs靶向的基因113个和84个。其中Bna-miR1140的潜在靶基因为一种糖基转移酶（glycosyltransferase）和响应调节因子（Arabidopsis response regulator ARR8-likeprotein）。
3.克隆得到了油菜Bna-miR1140基因前体197bp的片段，构建了植物过表达载体35S::Bna-miR1140，通过农杆菌介导法浸染油菜子叶柄转化甘蓝型油菜栽培种Westar。获得了14个PCR阳性植株，其中有5株株型特异，表现双主序表型，分枝数明显增多，其他9株阳性株均与野生型油菜表型一致，推测可能与表达强度有关。调查了株型特异的5个株系T1代表型分离情况，经卡方检验发现其中有4个株系的株型变异遗传符合3:1的孟德尔分离规律。初步推测油菜Bna-miR1140可能参与调控了油菜的分枝发育。
4.分离得到了油菜Bna-miR1140基因上游1351bp的序列，利用plantCARE在线软件对启动子序列的顺式作用元件进行在线分析，该启动子含有与转录必需的RNA聚合酶结合的TATA盒以及在调控基因转录效率中发挥重要作用的CAAT盒，并进一步构建了miR1140pro::GUS植物表达载体。通过农杆菌介导法转化油菜，得到5株PCR阳性植株，对转基因植株的根、茎、叶、叶腋(茎尖分生组织，SAM)、叶柄、花、种荚等组织和器官进行GUS染色分析，结果表明GUS仅在叶柄及叶腋中表达，这与Bna-miR1140体内表达的结果一致，进一步佐证了Bnas-miR1140与调控油菜的分枝发育相关。
综上所述，本研究采用深度测序和降解组分析技术鉴定了一批油菜的miRNA，并对其靶基因进行了分析和预测。对油菜特有的小RNABna-miR1140进行了较深入的研究，转基因结果初步表明miR1140参与了油菜分枝的发育。
MicroRNAs (miRNAs) are non-coding RNAs of approximately21nucleotides that havebeen identified as important regulators of gene expression in both animals and plants. In plants,miRNAs not only post-transcriptionally regulate their targets but also interact with each other inregulatory networks to affect many aspects of development, such as developmental timing,senescence, leaf morphogenesis, reproductive development, and modulation of root architecture.miRNAs are also reported to be involved in plant responses to biotic and environmental stresses.miRNAs are in the core position of the gene expression regulation network, and effectively inhibitits target genes encoding the proteins, or inhibit the expression of target genes via otheradjustment mechanism, resulting in the phenomenon of gene silencing. In the past decades, theplant miRNA research has been made great progress and Significant achievements.As adicotyledons, oilseed rape (Brassica Napus) is one of the most important oil crops,its miRNAhas also become a hot research field of molecular biology today. The studies about miRNAgenetic control mechanism involved in the regulation of rapeseed development can improve thetheoretical basis of the developmental biology of higher plant. Meanwhile, the clone andidentification of miRNA gene involved in the regulation rapeseed development, combined withmolecular breeding means to foster the ideal rape varieties for increasing yield of rapeseed andreducing the rapeseed production costs are very practical significance.
So far, the reports about studies of rapeseed miRNAs and its targets are much less, andmiRBase currently lists48miRNAs forming17miRNA families in Brassica napus. Here, wedescribe the high-throughput sequencing analysis of sRNAs from a cultivated variety of B. napus,cv Westar, using the Illumina Solexa platform. The sRNAs library was prepared for Solexasequencing from greenhouse cultivated rape plants, new miRNAs and target genes of knownmiRNAs and newly discovered miRNAs have been identificated. In order to provide a theoreticalbasis for rape breed improvement and rape miRNAs regulatory mechanism, the expressionregulation mechanism for the Bna-miR1140of Brassica unique miRNA families has also beenstudied. The methods are as follows, Leaves, petiole, stalk, roots and shoot apices from onemonth-old seedlings were collected and total RNA from different tissues was extracted usingTrizol (Invitrogen). sRNA and degradome cDNA libraries for Solexa sequencing were constructedfollowing the Illumina protocol. The purified cDNA library was sequenced on an IlluminaGAIIx(Biological Information Technology Co., Ltd., Hangzhou LC). Bioinformatic analysis ofthe sequencing data has been carried to identify known and new miRNAs and its target genes;Stem–loop RT primers, universal reverse primer and miRNA-specific forward primers for20sequenced miRNA sequences were designed according to Varkonyi-Gasic to validate and measurethe levels of B. napus miRNA by qRT-PCR. The primers for the precursor of Bna-miR1140 sequence were designed on the basis of the miRNAbase and its full-length was obtained by PCRamplification of Rape genome database, Then the35S:: Bna-miR1140plants over expressionvector constructed infected the cotyledons of wild-type rapeseed varieties by the Agrobacteriummediated. The phenotype of positive plants with the Bna-miR1140transformed was analyzed; Thesequence of the Bna-miR1140upstream1.5kb was analyzed via plantCARE software, finding thekey elements of promoter, such as the TATA-box, G-box et al., and then design primers for it andamplify the1.5kb fragment from rapeseed genomic DNA and constructed miR1140pro::GUSplant expression vector. The tissue of positive lines with miR1140pro::GUS transformed wasanalyzed by GUS staining to analysis the Bna-miR1140expression specificity.
Through the experimental analysis, the results as follows:
1. Here,41new conserved and67brassica-specific candidate miRNAs, including20miRNA*sequences were firstly identified. The sequencing results were further confirmed using stem-loopquantitative RT-PCR. The data will be updated to incorporate future miRBase updates. Ourapproach leads to the prediction of several conserved and specific brassica miRNA targets in theavailable EST and genomic databases.113mRNA targets of conserved brassica miRNAs and84mRNA targets of new brassica-specific miRNAs were identified. Validated miRNA targets in B.napus are potentially involved in diverse biological processes, including phase transitions,flowering, hormone signaling, photosynthesis, metabolism and biotic and abiotic stress resistance.Our findings will be a useful resource toward tracing the evolution of small RNA-based regulationin Brassica napus and related species. Most importantly, this study will serve as a foundation forfuture research into the functional roles of miRNAs and their target genes in this important oilcrop.
2. The precursor of Bna-miR1140gene cloned from Rape is197bp, and the plant overexpression vector35S::Bna-miR1140was constructed successfully. After disseminating thepetiole of rapeseed cultivars Westar via agrobacterium mediated transformation method. At Last,14T0generation Rape positive strains of over expressed35S::Bna-miR1140have been identifiedby the PCR method. There were5strains performed specific plant type, they had double maininflorescence and significantly increased the number of branches, nine positive strains consistentwith the wild-type rapeseed phenotype. After investigating the phenotype separation of five T1generation lines with specific plant type, and, of which there are four strains, their plant typevariation genetic followed3:1Mendel segregation law by the chi-square test. As for the otherpositive strains of over-expressed the35S::Bna-miR1140were not performed variation, they maynot express the35S::Bna-miR1140or transformed copy numbers are few, the real reasons must befurther validated by Northern blotting and other molecular experimental means in future. By phenotypic analysis, initially speculated the Bna-miR1140may participate in the regulation ofbranching and development of oilseed rape.
3. The1351bp fragment upstream of Bna-miR1140gene has been isolated, and the cis-actingelements of the promoter sequence were analyzed by plantCARE online software, the promotercontains TATA boxes binding RNA polymerase and CAAT boxes which are necessary fortranscription, because it play important role in the regulation of gene transcription efficiency. ThemiR1140pro::GUS plant expression vector was further constructed. Similarly, five positive T0rape strains transformed miR1140pro:: GUS were got, which have been transformed byagrobacterium-mediated infection rapeseed petiole of cultivars Westar. The GUS staining analysisshowed that the1.5kb region of upstream miR1140precursor sequence have promoter function bystaining each tissue of positive rapeseed strains, which able to drive the expression of GUS in rape.The miR1140pro::GUS transgenic rapeseed roots, stems, leaves, leaf axillary (shoot apexpoints Health organization, SAM), petiole, flower, pods and other tissue were carried GUSstaining analysis, the results showed that GUS only expressed in petioles and leaf axillary, thatconsistent with the results of the Bna-miR1140vivo expression, which is further corroborated theBna-miR1140regulation rape branching and development.
Summing up the results of the study, we found that the results of the Bna-miR1140vivoexpression and miR1140pro:: GUS expression patterns are consistent, proving Bna-miR1140play a role in rape branching development process; By solexa sequencing technology, many newconservative and specific miRNAs have been identified, finding that the target gene ofBna-miR1140are glycosyltransferase and the response to the adjustment factors.
引文
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