‘金冠’苹果miRNA克隆分析及Md-miRLn11靶向调控Md-NBS基因表达影响斑点落叶病抗性研究
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摘要
MicroRNA (miRNA)作为非编码小RNA在植物生长发育各个阶段都起到重要作用。RNA前体(primary RNA)经核醣核酸聚合酶Ⅱ(RNA polym erase Ⅱ)转录后依靠自身的序列特点折叠形成颈环二级结构,再由DCL1酶切割形成21-24nt长度的成熟miRNA。目前在权威的miRBase数据库中已经有52个物种的4053条：miRNA序列注册(miRBaese Release18),它们分别属于不同的miRNA家族,在植物的生长发育、植物抗逆等方面起到重要作用。miRNA的功能多样性以及可以在砧木和接穗之间传递的特点使得人们猜测它在以嫁接为主要繁殖方式的果树中起到重要的调节作用。然而目前有“世界第一水果”之称的苹果有关miRNA的报道极少,特别是苹果miRNA与抗病之间的研究仍是空白。在当前真菌病害严重威胁苹果产业发展的情况下,研究苹果miRNA与抗病性的关系具有非常重要的理论与应用价值。本研究以‘金冠’苹果为试材,利用各种技术手段,克隆了miRNA及其前体,分析了miRNA作用,明确了Md-miRLn11靶向NBS类蛋白基因调控真菌病害-斑点落叶病,主要研究结果如下：
1.利用目前常用的高通量测序技术和生物信息学预测技术,结合‘金冠’苹果基因组信息、miRBase和PMRB (plant miRNA database)数据库中的已知miRNA信息全面分析预测苹果miRNA。通过以上两种方法并根据序列的二级结构特点、自由能大小等,最终共筛选得到146条可能的苹果miRNA前体序列,将这些miRNA前体序列定位在‘金冠’苹果基因组中,发现苹果基因组中miRNA呈现一种集中成束出现的规律,同一家族的miRNA经常在基因组中2-3M的区域内出现。
2.根据苹果基因组信息设计特异性引物克隆这146个miRNA前体,结果显示克隆结果与预测序列基本相符(个别碱基与基因组中序列不同,现已经多次测序确定)。同时利用茎环引物反转录的方法在‘金冠’苹果中克隆这146条miRNA前体产生的成熟miRNA,共获得11个新miRNA,22个保守miRNA,非保守miRNA8个。推测未克隆到的miRNA可能在苹果生长发育的其他时期表达,或者在逆境条件下表达。
3.提取‘金冠’苹果叶片、韧皮部、花、果肉、果皮小分子RNA,利用real-timePCR的方法分析这41个成熟miRNA在这些组织中的表达量。结果显示这些miRNA在各组织中的表达量大不相同,各个组织中miRNA的整体表达水平也不相同。总体分析,在叶片和花中miRNA总体表达量较小,而在果实中总体表达量较高。由此推测在苹果果实的发育过程中参与各种代谢的miRNA种类更多,苹果果实发育过程相对于叶片和花发育过程可能更复杂。
4.根据苹果基因组信息,运用SOPE软件将146个‘金冠’苹果pri-miRNA可形成的成熟miRNA在基因组的靶位点做出预测。发现几乎所有miRNA都对应多个对应的靶位点,个别miRNA未找到基因组中的靶位点。并根据http://sma-tools.cmp.uea.ac.uk/软件预测已知miRNA的功能,根据http://linux1.softberry.com/berry.phtml软件预测新miRNA的功能。针对能够预测到功能的新miRNA,利用miRNA靶基因5'RACE的方法鉴定miRNA与靶基因之间的切割关系,最终证明Md-miRLn3、Md-miRLn4、Md-miRLn11可以分别切割其对应的靶基因,对苹果各个miRNA功能的预测可以应用在今后的实验中。在预测中发现Md-miRLn11靶向一个NBS类抗病蛋白基因,可能与植物抗真菌病相关。
5.根据Md-miRLn11可靶向切割Md-NBS基因的实验基础,对40个抗感苹果斑点落叶病品种的Md-miRLn11与Md-NBS基因表达进行分析。Real-time PCR的结果显示在感病品种中Md-miRLn11的表达量显著高于抗病品种,而抗病品种中Md-NBS基因的表达量明显高于感病品种,二者之间存在一定的对应关系。由以上两实验初步证明Md-miRLn11可靶向控制Md-NBS基因的表达。
6.进一步分析Md-miRLn11与抗病基因Md-NBS之间的关系发现,抗性苹果品种‘鸡冠’叶片注射Md-miRLn11超表达载体后接种斑点落叶病菌,96.5%的株系都表现出不同程度的斑点落叶病表型；在感病品种‘富士’中注射Md-NBS基因超表达载体,53.9%的株系表现出斑点落叶病抗性,仍有29.5%的植株表现出较严重的感病表型。实验结果表明在苹果中Md-miRLn11可以通过调控Md-NBS基因的表达改变植株对斑点落叶病的抗性,即超表达Md-miRLn11降低Md-NBS基因表达量导致斑点落叶病抗性降低；超表达Md-NBS基因可在一定程度上增强斑点落叶病抗性。
综上所述,本研究在‘金冠’苹果中克隆获得了146个miRNA,11个为其他物种中未见报到的新microRNA,其中,Md-miRLn11靶向一个NBS-LRR类蛋白基因,Md-miRLn11通过调控Md-NBS基因表达影响真菌病害-苹果斑点落叶病抗性。
As an important plant non-coding small RNA, microRNA plays an important role in various stages of plant growth process. In plants, the precursors (primary RNA) transcription by RNA polymerase Ⅱ (RNA polymerase Ⅱ), and rely on their own sequence features and the secondary structure of the stem-loop to produce mature microRNA. Then cut by the DCL1enzyme to form the21-24nt length of small RNA, which regulates a variety of plant physiological metabolism, resistance and grow of plant (Chen,2004, Zhu et al2008). The miRBase database showed4053miRNA sequences which from52species registered (miRBaese Release18), they belong to different microRNA family, play an important role in plant growth and development or stresses. MiRNA functional diversity and can be passed between the rootstock and scion features make it save the species reproduction quality in grafted fruit trees obviously. However, at present the apple miRNA rarely reported, especially in the apple between miRNA and disease research is still blank. The research of relationship between miRNA and disease resistance has a very important value of theory and application in fruit. In this study, Apple'Golden Delicious'as test material, the miRNA and their precursors were cloned by various techniques. The function analyze showed that Md-miRLn11targeted an NBS class protein gene and regulated leaf spot disease. The mainly results are as follows:
1. The high-throughput sequencing and bioinformatics technology was combined and used to prediction miRNAs in apple'Golden Delicious'. And than compared with the apples genomic and the miRBase and PMRB (plant miRNA database) database. Through the above two methods and in accordance to the RNA secondary structural characteristics, size, free energy and so on,146possible miRNA precursors were found. These pre-miRNA sequences were located on apple genome and it shows a focused bundle phenomenon, the same family miRNA often apperes at a2-3M position in the genome.
2. According to the apple genomic information specific primers designed and than146miRNA precursors the cloned. The results showed that clone basically consistent with the predicted sequence (individual nucleotide sequences were different with the genome sequence, we revised them according to the several sequencing results). Than the Stem-loop primer method was used to clone mature miRNA of the146miRNA precursors. The resuled showed a total of11new miRNA,22conserved miRNA, and8non-conserved miRNA. Speculated that miRNA which can not be cloned may express in other periods of growth and development of apple or under stress conditions.
3. Small RNA was extracted from apple'Golden Delicious' leaf, phloem, flower, fruit pulp, peel for the real-time PCR assay to analyze those41mature miRNA expression in those tissues. The results showed that these miRNA expression in those tissues very differently. Comprehensive analysis, there is smaller amount of miRNA expression in the leaves and flowers, and larger amount of miRNA expression in the fruit. This result indicated that more miRNA involved in the process of apple fruit development and little miRNA related to the process of leaf and flower development.
4. According to the apple genomic information, the SOPE Software was used to compare146mature miRNA formed from pri-miRNA to the apple genom, than their target sites were predicted at located in the genome. We found that almost all miRNA target not noly one target sites, individual is not found miRNA target site in the genome. And according to the miRNA prediction software of http://srna-tools.cmp.uea.ac.uk/to pridict the known conserved miRNA function, according to the miRNA prediction software of http://linux1.softberry.com/berry.phtml to pridict the new miRNA function. In able to predict functions for new miRNA, the method of5'RACE was used to identified the miRNAcutting position in the target gene. Ultimately proved Md-miRLn3, Md-miRLn4, Md-miRLn11can cut their respectively corresponding target genes, this indicat that each predicted miRNA function can be applied in future experiments. In one of them, Md-miRLn11, targeted a NBS class resistance gene, may be associated with plant disease related.
5. According to the result of Md-miRLn11can cut Md-NBS gene, the Md-miRLn11and Md-NBS gene expression were analysised in40anti-susceptible apple varieties. Real-time PCR results showed that the Md-miRLn11expression was significantly higher in susceptible varieties than that in resistant varieties, and Md-NBS gene expression was significantly higher in resistant varieties than in the susceptible varieties. Thus these two preliminary experiments prove Md-miRLn11can control Md-NBS target gene expression.
6. Further analysis of the Md-miRLn11and its target gene Md-NBS found that, when infiltrated Md-miRLn11in resistant varieties apple'JiGuan'then inoculated leaf spot pathogen,96.5%of the plants showed different degrees of leaf spot disease phenotype. When infiltrated Md-NBS in susceptible apple 'Fuji' then inoculated leaf spot pathogen,53.9%of the plants showed resistance to leaf spot disease. There is29.5%of the plants still showed disease phenotype. Experimental results show that the apple Md-miRLn11can regulate Md-NBS gene expression and changes plant resistance against leaf spot disease, overexpression Md-miRLn11reduce Md-NBS gene expression causes leaf spot resistance reduced; Overexpressing Md-NBS gene expression can enhance rsistance to leaf spot resistance in a certain extent.
In summary,146microRNA precursors were cloned from apple (Golden Delicious) by RNA sequencing and computational analysis, and found11to be novel.. Among them, Md-miRLnll targeted the Md-NBS gene (an NBS-LRR protein), and decreases resistance to Alternaria leaf spot.
1.利用目前常用的高通量测序技术和生物信息学预测技术,结合‘金冠’苹果基因组信息、miRBase和PMRB (plant miRNA database)数据库中的已知miRNA信息全面分析预测苹果miRNA。通过以上两种方法并根据序列的二级结构特点、自由能大小等,最终共筛选得到146条可能的苹果miRNA前体序列,将这些miRNA前体序列定位在‘金冠’苹果基因组中,发现苹果基因组中miRNA呈现一种集中成束出现的规律,同一家族的miRNA经常在基因组中2-3M的区域内出现。
2.根据苹果基因组信息设计特异性引物克隆这146个miRNA前体,结果显示克隆结果与预测序列基本相符(个别碱基与基因组中序列不同,现已经多次测序确定)。同时利用茎环引物反转录的方法在‘金冠’苹果中克隆这146条miRNA前体产生的成熟miRNA,共获得11个新miRNA,22个保守miRNA,非保守miRNA8个。推测未克隆到的miRNA可能在苹果生长发育的其他时期表达,或者在逆境条件下表达。
3.提取‘金冠’苹果叶片、韧皮部、花、果肉、果皮小分子RNA,利用real-timePCR的方法分析这41个成熟miRNA在这些组织中的表达量。结果显示这些miRNA在各组织中的表达量大不相同,各个组织中miRNA的整体表达水平也不相同。总体分析,在叶片和花中miRNA总体表达量较小,而在果实中总体表达量较高。由此推测在苹果果实的发育过程中参与各种代谢的miRNA种类更多,苹果果实发育过程相对于叶片和花发育过程可能更复杂。
4.根据苹果基因组信息,运用SOPE软件将146个‘金冠’苹果pri-miRNA可形成的成熟miRNA在基因组的靶位点做出预测。发现几乎所有miRNA都对应多个对应的靶位点,个别miRNA未找到基因组中的靶位点。并根据http://sma-tools.cmp.uea.ac.uk/软件预测已知miRNA的功能,根据http://linux1.softberry.com/berry.phtml软件预测新miRNA的功能。针对能够预测到功能的新miRNA,利用miRNA靶基因5'RACE的方法鉴定miRNA与靶基因之间的切割关系,最终证明Md-miRLn3、Md-miRLn4、Md-miRLn11可以分别切割其对应的靶基因,对苹果各个miRNA功能的预测可以应用在今后的实验中。在预测中发现Md-miRLn11靶向一个NBS类抗病蛋白基因,可能与植物抗真菌病相关。
5.根据Md-miRLn11可靶向切割Md-NBS基因的实验基础,对40个抗感苹果斑点落叶病品种的Md-miRLn11与Md-NBS基因表达进行分析。Real-time PCR的结果显示在感病品种中Md-miRLn11的表达量显著高于抗病品种,而抗病品种中Md-NBS基因的表达量明显高于感病品种,二者之间存在一定的对应关系。由以上两实验初步证明Md-miRLn11可靶向控制Md-NBS基因的表达。
6.进一步分析Md-miRLn11与抗病基因Md-NBS之间的关系发现,抗性苹果品种‘鸡冠’叶片注射Md-miRLn11超表达载体后接种斑点落叶病菌,96.5%的株系都表现出不同程度的斑点落叶病表型；在感病品种‘富士’中注射Md-NBS基因超表达载体,53.9%的株系表现出斑点落叶病抗性,仍有29.5%的植株表现出较严重的感病表型。实验结果表明在苹果中Md-miRLn11可以通过调控Md-NBS基因的表达改变植株对斑点落叶病的抗性,即超表达Md-miRLn11降低Md-NBS基因表达量导致斑点落叶病抗性降低；超表达Md-NBS基因可在一定程度上增强斑点落叶病抗性。
综上所述,本研究在‘金冠’苹果中克隆获得了146个miRNA,11个为其他物种中未见报到的新microRNA,其中,Md-miRLn11靶向一个NBS-LRR类蛋白基因,Md-miRLn11通过调控Md-NBS基因表达影响真菌病害-苹果斑点落叶病抗性。
As an important plant non-coding small RNA, microRNA plays an important role in various stages of plant growth process. In plants, the precursors (primary RNA) transcription by RNA polymerase Ⅱ (RNA polymerase Ⅱ), and rely on their own sequence features and the secondary structure of the stem-loop to produce mature microRNA. Then cut by the DCL1enzyme to form the21-24nt length of small RNA, which regulates a variety of plant physiological metabolism, resistance and grow of plant (Chen,2004, Zhu et al2008). The miRBase database showed4053miRNA sequences which from52species registered (miRBaese Release18), they belong to different microRNA family, play an important role in plant growth and development or stresses. MiRNA functional diversity and can be passed between the rootstock and scion features make it save the species reproduction quality in grafted fruit trees obviously. However, at present the apple miRNA rarely reported, especially in the apple between miRNA and disease research is still blank. The research of relationship between miRNA and disease resistance has a very important value of theory and application in fruit. In this study, Apple'Golden Delicious'as test material, the miRNA and their precursors were cloned by various techniques. The function analyze showed that Md-miRLn11targeted an NBS class protein gene and regulated leaf spot disease. The mainly results are as follows:
1. The high-throughput sequencing and bioinformatics technology was combined and used to prediction miRNAs in apple'Golden Delicious'. And than compared with the apples genomic and the miRBase and PMRB (plant miRNA database) database. Through the above two methods and in accordance to the RNA secondary structural characteristics, size, free energy and so on,146possible miRNA precursors were found. These pre-miRNA sequences were located on apple genome and it shows a focused bundle phenomenon, the same family miRNA often apperes at a2-3M position in the genome.
2. According to the apple genomic information specific primers designed and than146miRNA precursors the cloned. The results showed that clone basically consistent with the predicted sequence (individual nucleotide sequences were different with the genome sequence, we revised them according to the several sequencing results). Than the Stem-loop primer method was used to clone mature miRNA of the146miRNA precursors. The resuled showed a total of11new miRNA,22conserved miRNA, and8non-conserved miRNA. Speculated that miRNA which can not be cloned may express in other periods of growth and development of apple or under stress conditions.
3. Small RNA was extracted from apple'Golden Delicious' leaf, phloem, flower, fruit pulp, peel for the real-time PCR assay to analyze those41mature miRNA expression in those tissues. The results showed that these miRNA expression in those tissues very differently. Comprehensive analysis, there is smaller amount of miRNA expression in the leaves and flowers, and larger amount of miRNA expression in the fruit. This result indicated that more miRNA involved in the process of apple fruit development and little miRNA related to the process of leaf and flower development.
4. According to the apple genomic information, the SOPE Software was used to compare146mature miRNA formed from pri-miRNA to the apple genom, than their target sites were predicted at located in the genome. We found that almost all miRNA target not noly one target sites, individual is not found miRNA target site in the genome. And according to the miRNA prediction software of http://srna-tools.cmp.uea.ac.uk/to pridict the known conserved miRNA function, according to the miRNA prediction software of http://linux1.softberry.com/berry.phtml to pridict the new miRNA function. In able to predict functions for new miRNA, the method of5'RACE was used to identified the miRNAcutting position in the target gene. Ultimately proved Md-miRLn3, Md-miRLn4, Md-miRLn11can cut their respectively corresponding target genes, this indicat that each predicted miRNA function can be applied in future experiments. In one of them, Md-miRLn11, targeted a NBS class resistance gene, may be associated with plant disease related.
5. According to the result of Md-miRLn11can cut Md-NBS gene, the Md-miRLn11and Md-NBS gene expression were analysised in40anti-susceptible apple varieties. Real-time PCR results showed that the Md-miRLn11expression was significantly higher in susceptible varieties than that in resistant varieties, and Md-NBS gene expression was significantly higher in resistant varieties than in the susceptible varieties. Thus these two preliminary experiments prove Md-miRLn11can control Md-NBS target gene expression.
6. Further analysis of the Md-miRLn11and its target gene Md-NBS found that, when infiltrated Md-miRLn11in resistant varieties apple'JiGuan'then inoculated leaf spot pathogen,96.5%of the plants showed different degrees of leaf spot disease phenotype. When infiltrated Md-NBS in susceptible apple 'Fuji' then inoculated leaf spot pathogen,53.9%of the plants showed resistance to leaf spot disease. There is29.5%of the plants still showed disease phenotype. Experimental results show that the apple Md-miRLn11can regulate Md-NBS gene expression and changes plant resistance against leaf spot disease, overexpression Md-miRLn11reduce Md-NBS gene expression causes leaf spot resistance reduced; Overexpressing Md-NBS gene expression can enhance rsistance to leaf spot resistance in a certain extent.
In summary,146microRNA precursors were cloned from apple (Golden Delicious) by RNA sequencing and computational analysis, and found11to be novel.. Among them, Md-miRLnll targeted the Md-NBS gene (an NBS-LRR protein), and decreases resistance to Alternaria leaf spot.
引文
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