玉米C型细胞质雄性不育系及保持系microRNA克隆与功能分析
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摘要
microRNA(简称miRNA)作为真核生物体内的一类负调控因子,在生物体内所起的调节作用几乎涉及到所有的细胞水平。从2002年首次发现植物miRNA以来,在短短六年时间里关于植物miRNA方面的研究报道层出不穷,但是迄今为止在各物种中所鉴定的miRNA数量还远远不及理论上的饱和值,而对miRNA相关功能研究更是该领域面临的一大挑战。作为非模式植物的玉米,在miRNA方面的研究自然落后于拟南芥和水稻,目前仅仅有96条玉米miRNA序列被登陆到miRNA数据库中。因此玉米miRNA的分离鉴定及功能分析在今后的很长一段时间内依然将是该领域的一个研究热点。本研究将玉米c型细胞质雄性不育系C48-2及其保持系N48-2作为研究对象,通过组织分离克隆的方法鉴定在花药中表达的miRNA,进一步对这些miRNA的靶基因进行了预测,并结合miRNA在不育系和保持系之间的表达差异和靶基因的相关功能分析,初步推断这些miRNA与玉米C型细胞质雄性不育特性之间的关系。现将本研究的结果小结如下:
1.克隆获得了81条大小在18～26bp的小分子RNA,主要富集在20～24bp范围内,去除tRNA、rRNA以及降解序列之后,通过mfold分析序列在基因组上是否形成符合miRNA前体标准的二级结构,结果得到10条符合标准的序列。
2.这10条序列的长度在21～24nt范围内,且其中5'端第一个核苷酸为“U”的有5条,这些特点和其它已鉴定的植物miRNA一致。进一步对它们进行物种间保守性分析发现,其中6条在水稻、小麦和拟南芥中存在不同程度的保守性,总体趋势表现为在单子叶水稻和小麦中的保守性高于双子叶植物拟南芥。
3.通过加尾-引物延伸RT-PCR法,对以上10条候选miRNA在C48-2和N48-2花药中的表达进行真实性验证,结果发现它们在两个材料的花药均有表达,证明了这10个miRNA是真实存在的,将其鉴定为新的玉米miRNA,归属于10个新的miRNA家族。
4.通过miRU植物miRNA靶基因预测软件和CSRDB、NCBI数据库以及Pfam氨基酸功能域分析软件,预测到其中9个miRNA作用的10靶序列。在这些靶序列中,有3个是包括MADS-box在内的转录因子,3个属于花药代谢过程中的关键酶,分别是NADH脱氢酶、细胞色素单P450单加氧酶和二氢黄酮醇-4-还原酶,1个为22KD的α-醇溶蛋白,2个分别是某种不确定的膜内在蛋白和具备抗氧化作用的特殊分子伴侣,另外一个功能不明。在这些靶基因中,NADH脱氢酶、细胞色素P450单加氧酶和二氢黄酮醇-4-还原酶已经被证明参与花药的能量代谢和物质代谢过程,并与植物雄性不育有着紧密联系;而MADS-box基因则是近年来被受关注的一类与植物育性相关的转录因子。
5.用实时荧光定量PCR法对这10个miRNA在C48-2和N48-2不同发育时期花药中的表达量进行检测,结果表明,在四分体期,10个miRNA在两个材料之间表达量均存在显著差异,表现为在C48-2中的表达量低于N48-2。此外,其中3个miRNA的表达量在两个材料的单核期也表现出明显差异,miR-1在C48-2中的表达量低于N48-2,而miR-6和miR-10与之相反。
6.结合miRNA在不育系、保持系间的表达量和其靶基因的相关功能分析,对miRNA调控与花药发育以及玉米细胞质雄性不育之间的关系有了初步的认识,总结如下:
第一,从四分体期到单核期,可育材料中10个miRNA的下调表达可能是花药正常发育的重要条件,也可能是花药正常发育后产生的结果,而miRNA在可育材料中有规律的时序性表达,证明了miRNA参与了花药发育的调控。
第二,miR-1、miR-2和miR-6在四分体时期与单核期可育系和保持系中的表达量均存在明显差异,暗示着这3个miRNA在四分体时期和单核期都参与了雄性败育过程。而miR-3、miR-4、miR-5、miR-7、miR-8、miR-9、miR-10对雄性不育的调控在单核期的表现得更明显一些。
第三,miRNA调控可能是引起不育的原因:miRNA在不育系中的异常表达导致了花药代谢过程中的关键酶以及相关转录因子的异常表达,进一步造成花药能量代谢和物质代谢的紊乱,从而使花粉得不到正常的发育;
第四,miRNA表达的异常可能是雄性不育产生的结果:受玉米细胞质雄性不育特性的影响,参与miRNA剪切过程的相关蛋白和调控miRNA基因表达的转录因子等表达量发生变化,从而在细胞内miRNA的含量相应变化,进而使受miRNA调控的基因表达发生异常。
As a class of negative regulators in eukaryote organism,miRNA(abbreviated to "miRNA")regulations almost reach to all cellular levels of organism.It has only been about six years from the first plant miRNA was found in 2002,a great deal of research findings about plant miRNAs have been reported.However the identified plant miRNAs are still far from saturation,and understanding about miRNA founction is being faced with some formidable problem.Being a non-model plant,Zea mays fall behind Arabidopsis and Oryza sativa on miRNA research accordingly.And only 96 identified miRNAs from Zea mays were submitted to miRNA database so far.For this reason,cloning and characterization of miRNA in maize is being a hot academic domain in the future.In the research,miRNAs were abstracted and cloned from C-type cytoplasmic male sterile line 48-2 and its maintainer in maize,and target genes of miRNA were subsequently predicted.As a result,some conclussions were made concerning relationship between miRNA regulation and C-type cytoplasmic male sterility in maize by assay of miRNA content differentiation and relative function of miRNA targets.The findings of the research are as follows:
1.Eighty one sequences sized from 18 to 26nt were cloned,of which the majority has the length ranging from 20 to 24nt.The sequences,excluding tRNA, rRNA and degraded fragments,were analyzed by m-fold program for stem-loop.The results showed that ten small RNAs could form stem-loop when joined with flanking sequence in genomes.
2.The lengths of the ten sequences range from 21 to 24nt,and five of the sequences began with "U" from 5' terminatio.The trend is similar to what have been reported in other plant miRNAs.By "blast" genomes of other plants including rice, wheat and Arabidopsis,conservation of miRNA was identified among them.As a result,six miRNA sequences exhibit conservation to a large extent.On the other hand, there is a trend that these miRNAs were more conservative among maize,rice and wheat than between maize and Arabidopsis.
3.By RNA-tailing and primer-extension RT-PCR,the ten small RNAs were detected in both anthers of line C48-2 and anthers of line N48-2,which indicates their reality in maize.In according to above reasons,the ten sequences were identified to be new microRNAs in plants,and attributed respectively to 10 new miRNA families.
4.Then targets were respectively predicted for 9 of the miRNAs by a series of methods including using of miRU program,searching CSRDB and NCBI,analyzing fuctional domain of amino acid by "Pfam".Resultantly,the targets are involved in 3 such transcription factors as MADS-box,3 important enzymes ralative to anther metabolism,named respectively NADH dehydrogenase,cytochrome P450 monooxygenase and dihydroflavonol-4-reductase,1 zein-alpha,1 integral membrane protein and 1 antioxyen molecular chaperones.Among them,NADH dehydrogenase, cytochrome P450 monooxygenase and dihydroflavonol-4-reductase have been confirmed to participate in the process of energy or substance metabolism of anther, and be tightly related to plant male sterility.As to MADS-box genes,they are regarded as a class of transcription factors concorning plant male sterility,and have got close attention in recent year.
5.The contents of the ten miRNA in anthers of both C48-2 and N48-2 were assayed by real-time PCR.The result showed the miRNA contents in N48-2 was significantly larger than that in C48-2 at tetrad stage.In addition,3 of the ten miRNAs also displayed distinct expression at monocaryon stage between the two lines.Namely, the content of miR-1 in line C48-2 was smaller than that in line N48-2,but it is the adverse case for miR-6 and miR-10.
6.By analyzing both distinction of miRNA contents between sterile line C48-2 and N48-2 and function of target genes,the relationship between miRNA regulation and cytoplasmic male sterility in maize got explaination to some extent,as follows:
First,from tetrad stage to monocaryon stage,expression of the ten miRNA in line N48-2 downregulated,which is essential for fertility of line N48-2 probably.On the other hand,it may be the result of normal development for anther.By the regular downregulation of the ten miRNA in line N48-2,it can be conclude they get involved in the progress of anther development.
Second,as to miR-1,miR-2 and miR-6,their regulation is responsible for male sterility both at tetrad stage and in monocaryon period for expression disparity between the two lines is exhibited at both the two stages.With regard to miR-3, miR-4,miR-5,miR-7,miR-8,miR-9 and miR-10,pathways of C-type cytoplasmic male sterility mediated by their regulation occur dominately in tetrad period;
Third,miRNA regulation may be the reason of male sterility,namely,dramaticly abnormal contents of miRNAs in sterile line make significantly different conents of mRNA of important enzymes and transcription factors ralative to anther metabolism, lead to disorder of substance and energy of anther subsequently,and result in abnormal development of pollen at last;
Finally,inordinate of miRNAs in male sterility has the chance to be the result from male sterility.In other words,in the effect of male sterility,expression of some protein is disturbed,including enzyme and transcription factors responding for biosynthesis of miRNA.Accordingly,normal expression of miRNAs is inhibitted. Furthermore,the gene regulated by miRNAs fails to normal expression.
1.克隆获得了81条大小在18～26bp的小分子RNA,主要富集在20～24bp范围内,去除tRNA、rRNA以及降解序列之后,通过mfold分析序列在基因组上是否形成符合miRNA前体标准的二级结构,结果得到10条符合标准的序列。
2.这10条序列的长度在21～24nt范围内,且其中5'端第一个核苷酸为“U”的有5条,这些特点和其它已鉴定的植物miRNA一致。进一步对它们进行物种间保守性分析发现,其中6条在水稻、小麦和拟南芥中存在不同程度的保守性,总体趋势表现为在单子叶水稻和小麦中的保守性高于双子叶植物拟南芥。
3.通过加尾-引物延伸RT-PCR法,对以上10条候选miRNA在C48-2和N48-2花药中的表达进行真实性验证,结果发现它们在两个材料的花药均有表达,证明了这10个miRNA是真实存在的,将其鉴定为新的玉米miRNA,归属于10个新的miRNA家族。
4.通过miRU植物miRNA靶基因预测软件和CSRDB、NCBI数据库以及Pfam氨基酸功能域分析软件,预测到其中9个miRNA作用的10靶序列。在这些靶序列中,有3个是包括MADS-box在内的转录因子,3个属于花药代谢过程中的关键酶,分别是NADH脱氢酶、细胞色素单P450单加氧酶和二氢黄酮醇-4-还原酶,1个为22KD的α-醇溶蛋白,2个分别是某种不确定的膜内在蛋白和具备抗氧化作用的特殊分子伴侣,另外一个功能不明。在这些靶基因中,NADH脱氢酶、细胞色素P450单加氧酶和二氢黄酮醇-4-还原酶已经被证明参与花药的能量代谢和物质代谢过程,并与植物雄性不育有着紧密联系;而MADS-box基因则是近年来被受关注的一类与植物育性相关的转录因子。
5.用实时荧光定量PCR法对这10个miRNA在C48-2和N48-2不同发育时期花药中的表达量进行检测,结果表明,在四分体期,10个miRNA在两个材料之间表达量均存在显著差异,表现为在C48-2中的表达量低于N48-2。此外,其中3个miRNA的表达量在两个材料的单核期也表现出明显差异,miR-1在C48-2中的表达量低于N48-2,而miR-6和miR-10与之相反。
6.结合miRNA在不育系、保持系间的表达量和其靶基因的相关功能分析,对miRNA调控与花药发育以及玉米细胞质雄性不育之间的关系有了初步的认识,总结如下:
第一,从四分体期到单核期,可育材料中10个miRNA的下调表达可能是花药正常发育的重要条件,也可能是花药正常发育后产生的结果,而miRNA在可育材料中有规律的时序性表达,证明了miRNA参与了花药发育的调控。
第二,miR-1、miR-2和miR-6在四分体时期与单核期可育系和保持系中的表达量均存在明显差异,暗示着这3个miRNA在四分体时期和单核期都参与了雄性败育过程。而miR-3、miR-4、miR-5、miR-7、miR-8、miR-9、miR-10对雄性不育的调控在单核期的表现得更明显一些。
第三,miRNA调控可能是引起不育的原因:miRNA在不育系中的异常表达导致了花药代谢过程中的关键酶以及相关转录因子的异常表达,进一步造成花药能量代谢和物质代谢的紊乱,从而使花粉得不到正常的发育;
第四,miRNA表达的异常可能是雄性不育产生的结果:受玉米细胞质雄性不育特性的影响,参与miRNA剪切过程的相关蛋白和调控miRNA基因表达的转录因子等表达量发生变化,从而在细胞内miRNA的含量相应变化,进而使受miRNA调控的基因表达发生异常。
As a class of negative regulators in eukaryote organism,miRNA(abbreviated to "miRNA")regulations almost reach to all cellular levels of organism.It has only been about six years from the first plant miRNA was found in 2002,a great deal of research findings about plant miRNAs have been reported.However the identified plant miRNAs are still far from saturation,and understanding about miRNA founction is being faced with some formidable problem.Being a non-model plant,Zea mays fall behind Arabidopsis and Oryza sativa on miRNA research accordingly.And only 96 identified miRNAs from Zea mays were submitted to miRNA database so far.For this reason,cloning and characterization of miRNA in maize is being a hot academic domain in the future.In the research,miRNAs were abstracted and cloned from C-type cytoplasmic male sterile line 48-2 and its maintainer in maize,and target genes of miRNA were subsequently predicted.As a result,some conclussions were made concerning relationship between miRNA regulation and C-type cytoplasmic male sterility in maize by assay of miRNA content differentiation and relative function of miRNA targets.The findings of the research are as follows:
1.Eighty one sequences sized from 18 to 26nt were cloned,of which the majority has the length ranging from 20 to 24nt.The sequences,excluding tRNA, rRNA and degraded fragments,were analyzed by m-fold program for stem-loop.The results showed that ten small RNAs could form stem-loop when joined with flanking sequence in genomes.
2.The lengths of the ten sequences range from 21 to 24nt,and five of the sequences began with "U" from 5' terminatio.The trend is similar to what have been reported in other plant miRNAs.By "blast" genomes of other plants including rice, wheat and Arabidopsis,conservation of miRNA was identified among them.As a result,six miRNA sequences exhibit conservation to a large extent.On the other hand, there is a trend that these miRNAs were more conservative among maize,rice and wheat than between maize and Arabidopsis.
3.By RNA-tailing and primer-extension RT-PCR,the ten small RNAs were detected in both anthers of line C48-2 and anthers of line N48-2,which indicates their reality in maize.In according to above reasons,the ten sequences were identified to be new microRNAs in plants,and attributed respectively to 10 new miRNA families.
4.Then targets were respectively predicted for 9 of the miRNAs by a series of methods including using of miRU program,searching CSRDB and NCBI,analyzing fuctional domain of amino acid by "Pfam".Resultantly,the targets are involved in 3 such transcription factors as MADS-box,3 important enzymes ralative to anther metabolism,named respectively NADH dehydrogenase,cytochrome P450 monooxygenase and dihydroflavonol-4-reductase,1 zein-alpha,1 integral membrane protein and 1 antioxyen molecular chaperones.Among them,NADH dehydrogenase, cytochrome P450 monooxygenase and dihydroflavonol-4-reductase have been confirmed to participate in the process of energy or substance metabolism of anther, and be tightly related to plant male sterility.As to MADS-box genes,they are regarded as a class of transcription factors concorning plant male sterility,and have got close attention in recent year.
5.The contents of the ten miRNA in anthers of both C48-2 and N48-2 were assayed by real-time PCR.The result showed the miRNA contents in N48-2 was significantly larger than that in C48-2 at tetrad stage.In addition,3 of the ten miRNAs also displayed distinct expression at monocaryon stage between the two lines.Namely, the content of miR-1 in line C48-2 was smaller than that in line N48-2,but it is the adverse case for miR-6 and miR-10.
6.By analyzing both distinction of miRNA contents between sterile line C48-2 and N48-2 and function of target genes,the relationship between miRNA regulation and cytoplasmic male sterility in maize got explaination to some extent,as follows:
First,from tetrad stage to monocaryon stage,expression of the ten miRNA in line N48-2 downregulated,which is essential for fertility of line N48-2 probably.On the other hand,it may be the result of normal development for anther.By the regular downregulation of the ten miRNA in line N48-2,it can be conclude they get involved in the progress of anther development.
Second,as to miR-1,miR-2 and miR-6,their regulation is responsible for male sterility both at tetrad stage and in monocaryon period for expression disparity between the two lines is exhibited at both the two stages.With regard to miR-3, miR-4,miR-5,miR-7,miR-8,miR-9 and miR-10,pathways of C-type cytoplasmic male sterility mediated by their regulation occur dominately in tetrad period;
Third,miRNA regulation may be the reason of male sterility,namely,dramaticly abnormal contents of miRNAs in sterile line make significantly different conents of mRNA of important enzymes and transcription factors ralative to anther metabolism, lead to disorder of substance and energy of anther subsequently,and result in abnormal development of pollen at last;
Finally,inordinate of miRNAs in male sterility has the chance to be the result from male sterility.In other words,in the effect of male sterility,expression of some protein is disturbed,including enzyme and transcription factors responding for biosynthesis of miRNA.Accordingly,normal expression of miRNAs is inhibitted. Furthermore,the gene regulated by miRNAs fails to normal expression.
引文
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