CN111718937A - 梨果实抗衰老microRNA及其应用 - Google Patents
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Abstract
本发明公开了梨果实抗衰老microRNA及其应用。本发明获得了一种分离自‘丰水梨’在高温诱导下产生的具有调控果实衰老的microRNA(Novel_115)，其前体核苷酸序列为SEQ ID NO.2所示。通过农杆菌介导转化方法将Novel_115瞬时转化成熟梨，经生物学功能验证，表明本发明克隆的Novel_115基因具有延缓果实衰老的功能。Novel_115的发现，为调控果实衰老的分子育种提供新的基因资源。由于Novel_115基因具有同时调控多个基因的优点，为分子育种提供更高效地途径。
Description
技术领域
本发明属于植物基因工程领域，涉及梨Novel_115及其应用，具体涉及从‘丰水梨’中分离、克隆得到一个高温诱导产生的调控果实衰老的microRNA(Novel_115)及其应用。
背景技术
水果含有各种生物活性物质(包括植物化学物质、维生素、矿物质和膳食纤维)，能够降低患慢性病的风险，有助于身体健康，因此是人类饮食的重要组成部分(Liu 2013；Slavin and Lioyd 2012)。在中国，梨的产量约为1950万(粮农统计数据库2016)，大量梨不能及时出售。梨属植物种植区夏季温度高(>35℃)，采收期相对集中，难贮藏，易失去商品价值。在采后贮藏过程中，果实品质会随着新陈代谢而逐渐降低，这一过程称为衰老。在果实衰老过程中，贮藏的果实发生了一系列生理生化反应，引起了果实质地、风味、色泽、生化成分发生显著变化(Giovannoni 2001；Lurie and Crisosto 2005；Paliyath et al.2008；Tian et al.2013)。目前，人们已经开发了几种延缓果实衰老的技术，如温度、1-甲基环丙烯(1-mcp)、钙、气调、水杨酸、ATP酶和草酸(Aghdam et al.2012；Asghari and Aghdam2010；Massolo et al.2011； Zhang et al.,2011；Huang et al.2013；Wang et al.2013)。在这些技术中，温度处理易于操作，因而被广泛应用于延缓果实衰老。
microRNAs是18-30个核苷酸左右的非编码RNA(Morin et al.2008)，参与到很多植物生理生化反应中，其鉴定和挖掘的研究报道较多，大部分集中在拟南芥、水稻等模式植物 (Liu et al.2017；Ori et al.2007；Yoon et al.2010；Zhan et al.2012)。梨是世界上最受欢迎的水果之一，然而，人们对不同温度诱导下miRNA调控果实衰老的分子机制知之甚少。本发明以“丰水梨”为试材，对11组果实在代谢产物、mRNAs和miRNAs中进行测序。结合组学内的差异分析和多组学之间的关联研究，得到通过高温诱导产生的调节果实衰老的Novel_115，探讨了miRNAs对果实衰老的不同分子调节途径。利用同源克隆技术，克隆得到梨Novel_115的前体序列，构建过量表达载体，通过农杆菌介导法瞬时转化成熟梨果实使其过量表达，证明其可能参与调节梨果实衰老，从而扩展了梨果实衰老的调控途径。另外，克隆梨中调控果实衰老进程的基因很大程度上提高的作物改良和育种的效率，优化采后处理，对农业生产也具有非常重要的理论和实践意义。
发明内容
本发明的目的是提供一种高温诱导产生的调控梨果实衰老的microRNA(Novel_115)及其应用。
本发明的另一目的是提供上述microRNA的应用。
本发明的目的可通过以下技术方案实现：
一种分离自‘丰水’梨的调控果实衰老的microRNA(Novel_115)，其核苷酸序列如SEQ ID No.1所示，包含21nt的核苷酸序列。
上述microRNA(Novel_115)的前体基因，其核苷酸序列如SEQ ID No.2所示，包含226nt的前体核苷酸序列。
含有上述Novel_115前体基因的重组表达载体。该重组表达载体是以pSAK277为出发载体，将上述的Novel_115前体基因插入Hind III和Xba I位点之间所得。
含有上述Novel_115前体基因的宿主菌。
克隆上述Novel_115前体基因cDNA序列的引物对，该引物对的上游引物Novel_115-F1 序列如SEQ ID No.3所示，下游引物Novel_115-R1序列如SEQ ID No.4所示。
本发明所述的microRNA(Novel_115)在延缓果实衰老中的应用。
本发明所述的Novel_115前体基因在延缓果实衰老中的应用。
本发明所述的重组表达载体或宿主菌在延缓果实衰老中的应用。
与现有技术相比，本发明具有以下优点和效果：
1、Novel_115的发现，为延缓果实衰老的分子育种提供新的基因资源，为实施绿色农业提供新的遗传资源，该遗传资源的开发利用有利于降低农业成本和实现环境友好。
2、通过农杆菌介导遗传转化方法将miRNA瞬时转化成熟梨果实，经生物学功能验证，表明本发明克隆的Novel_115具有同时抑制多个基因表达的功能，从而延缓梨果实衰老的功能。Novel_115基因这种能同时调控多个基因的优点，为分子育种提供更高效地途径。
附图说明
图1为本发明Novel_115前体序列形成的stem-loop结构图。
图2为RT-qPCR检测的Novel_115的表达谱与Novel_115转录组数据的表达谱相关性分析。
图3为本发明pSAK277-Novel_115载体结构示意图。
图4为本发明瞬时转化梨果实表型分析。
其中：OE：Novel_115过量表达瞬时转化；control：pSAK277(空载)瞬时转化。
图5为本发明Novel_115及协作基因在瞬时转化梨果实中基因表达量分析。
具体实施方式
以下结合具体实施例对本发明做出详细的描述。根据以下的描述和这些实施例，本领域技术人员可以确定本发明的基本特征，并且在不偏离本发明精神和范围的情况下，可以对本发明做出各种改变和修改，以使其适用各种用途和条件。
实施例1不同温度贮藏下果实衰老过程中Novel_115的表达模式分析
将“丰水梨”分成三组。三组分别在40±2℃(high-temperature；HT)；25±1℃(room- temperature；RT)；和4±1℃(low-temperature；LT)下贮藏，各为120、180和300个。当 30％以上的果实腐烂时，剩下的果实被认为是衰老的果实。从预处理到室温衰老，每隔5天取样一次，然后每隔20天取样一次，直到低温老化。每6个重复至少取3个果实，去除果皮后，将果肉切成小块混合在一起。将混合样品立即冷冻在液氮中，并在-80℃下保存至使用，进一步RNA-seq分析。
实施例2 qRT-qPCR检测Novel_115表达谱与转录组组数据的相关性。
通过对RNA-seq数据进行分析，表明Novel_115在高温诱导果实衰老中起重要作用。miRNA抽提使用miRcute多糖多酚植物miRNA提取分离试剂盒(购自天根生化科技(北京)有限公司，按照该试剂盒提供的操作说明书操作)。第一链cDNA的合成用miRcute增强型miRNA cDNA第一条链合成试剂盒(购自天根生化科技(北京)有限公司，按照该试剂盒提供的操作说明书操作)。RT-qPCR分析所用Novel_115特异性正向引物序列是为SEQ ID No.5。内参基因正向引物序列为SEQ ID No.6。RT-qPCR使用miRcute增强型miRNA荧光定量检测试剂盒(购自天根生化科技(北京)有限公司，按照该试剂盒提供的操作说明书操作)。Real-timePCR所用仪器为Roche480定量PCR仪，反应体系为:2x miRcute Plus miRNA PlusPremix(SYBR&ROX)10uL，引物(10uM)各0.4uL，2ul cDNA，7.2uL PCR级别的水。反应条件为95℃变性15min；94℃预变性20s，60℃退火34s，72℃延伸10s重复45个循环；熔解曲线分析65℃到95℃，每5s升高1℃。荧光定量PCR分析结果如图2所示，结果表明，RT-qPCR检测的Novel_115的表达谱与转录组组数据的表达谱呈正相关 (p<0.05),microRNAome数据有效。
实施例3 Novel_115分离克隆
从‘丰水梨’叶片中抽提DNA，用于扩增Novel_115前体序列。DNA抽提使用植物基因组提取试剂盒(购自成都福际，按照该试剂盒提供的操作说明书操作)。扩增基因引物对为SEQ ID No.3(上游引物Novel_115-F1)和SEQ ID No.4(下游引物Novel_115-R1)。高保真DNA聚合酶
Super-Fidelity DNA Polymerase(P505-d1)购自诺唯赞生物科技公司。扩增的反应体系为50uL中包括200ng cDNA，2×Phanta Max Buffer 25uL，10mM dNTP 1uL，Phanta Max Super-Fidelity DNA Polymerase(1U/ul)1uL，10uM 2uL上述引物，加ddH2O至50uL。PCR反应在eppendorf扩增仪上按以下程序完成:95℃，预变性3分钟，95℃变性30秒，60℃退火30秒，72℃延伸40秒，35个热循环，72℃延伸10分钟， 4℃保存。产生一条单一PCR条带产物。
PCR产物经1％的琼脂糖凝胶电泳检测后，用AxyGEN小量胶回收试剂盒(购自爱思进生物技术杭州有限公司，中国)回收DNA片段，步骤参照使用说明。pSAk277载体的双酶切体系总体积为50uL,其中含有经过质粒提取获得的pSAK277载体DNA 4uL，10x K Buffer(购自NEB公司)5u L，Hind III 1u L，Xba I 1u L此及水39uL。于37℃酶切3小时后回收。回收纯化的DNA溶液与双酶切(Hind III/Xba I)的线性pSAK277载体进行连接反应，重组酶
II One Step Cloning Kit(货号：C112-01)购自诺唯赞生物科技公司，并按说明书步骤操作。连接反应体系总体积是10uL，其中包括2uL的5×CE II Buffer，50～200ng线性化克隆载体，50～200ng插入片段扩增产物，1uL
II。 37℃连接30min。待反应完成后，立即将反应放置于冰水浴中冷却5min，反应产物可直接进行转化。转化采用热击法(参照《分子克隆实验手册》第三版，科学出版社，2002)转化大肠杆菌DH5α，在含有50mg/L壮观霉素的LB固体平板中筛选阳性克隆，挑取5个阳性克隆测序(由生工生物工程(上海)股份有限公司完成)。测序结果表明，Novel_115前体序列全长为226nt，其核苷酸序列为SEQ ID NO.2所示(microRNA Novel_115的核苷酸序列如SEQ ID NO.1所示)。BLAST的结果表明该基因与Novel_115的序列相似度达到 99％，用在线软件RNA fold web server(http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/ RNAfold.cgi)预测二级结构，发现能够形成茎环结构(图1)。构建重组载体命名为pSAK277- Novel_115(如图3所示)；应用冻融法将上述重组载体导入到农杆菌GV3101中。
实施例4农杆菌介导Novel_115重组载体的瞬时转化
选取成熟期的‘砀山酥梨’为试验材料，采用农杆菌介导的转化将重组载体注射进入梨果肉，30℃避光密闭贮藏。通过观察注射区域横截面，与空载对照比较，在相同贮藏时间内，注射pSAK277区域的梨果肉比注射过表达Novel_115的先腐烂(图4)。结果表明，过表达Novel_115能够延缓梨果实衰老。
实施例5 Novel_115及其关联基因在瞬时转化梨果实中的基因表达量
选取成熟期的‘砀山酥梨’为试验材料，采用农杆菌介导的转化将重组载体注射进入梨果肉，每个处理至少4个生物学重复，30℃避光密闭贮藏。注射10d后取果肉标本，提取总RNA及miRNA进行RT-qPCR分析。总RNA抽提使用植物总RNA提取试剂盒(多糖多酚植物含量高的样本)(购自成都福际，按照该试剂盒提供的操作说明书操作)。第一链cDNA的合成用Trans一步法去基因组反转录试剂盒(购自全式金(TransGen)，按照该试剂盒提供的操作说明书操作)。RT-qPCR使用LC480SYBR Green Mix(购自都莱生物，按照该试剂盒提供的操作说明书操作)。Real-timePCR所用仪器为Roche480定量PCR仪，反应体系为:2x SYBR GreenMaster Mix10uL，上下游引物(10uM)0.4uL，2ul cDNA，7.2uL PCR 级别的水。反应条件为95℃预变性5min；95℃变性5s，65℃退火30s，72℃延伸10s重复 45个循环；熔解曲线分析65℃到95℃，每5s升高1℃。miRNA的抽提、反转录及RT- qPCR参考实施例2。荧光定量PCR分析结果如图4所示，目的基因在梨果实中的表达量高于空载对照，可以看出，目的基因确实已经成功瞬时转化到梨果实中。同时4个基因 (Pbr028015.1、Pbr008253.1、Pbr009691.1和Pbr014878.1)在果实中的表达高于对照，这与Novel_115和这四个基因之间的正相关关系是一致的(图5)。而两个基因(Pbr005846.1 和Pbr032596.1)在果实中的表达低于对照，并且Pbr002517.1在受侵染果实和对照之间的表达水平相似，这表明这三个基因在表达上与Novel_115无关。这些结果表明，Novel_115 通过影响Pbr028015.1、Pbr008253.1、Pbr009691.1和Pbr014878.1的表达而参与了高温诱导的果实衰老。
表1 Novel_115与其关联基因的相关性
表2用于RT-qPCR检测的引物
主要参考文献
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序列表
<110> 南京农业大学
<120> 梨果实抗衰老microRNA及其应用
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Claims (9)
1.一种调控果实衰老的microRNA(Novel_115)，其核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述microRNA的前体基因，其核苷酸序列如SEQ ID No.2所示。
3.含有权利要求2所述前体基因的重组表达载体。
4.根据权利要求3所述的重组表达载体，其特征在于，该重组表达载体是以pSAK277为出发载体，将权利要求2所述的前体基因插入Hind III和Xba I位点之间所得。
5.含有权利要求2所述前体基因的宿主菌。
6.克隆权利要求2所述前体基因cDNA序列的引物对，其特征在于，该引物对的上游引物Novel_115-F1序列如SEQ ID No.3所示，下游引物Novel_115-R1序列如SEQ ID No.4所示。
7.权利要求1所述的microRNA(Novel_115)在延缓果实衰老中的应用。
8.权利要求2所述的前体基因在延缓果实衰老中的应用。
9.权利要求3所述的重组表达载体在延缓果实衰老中的应用。
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| CN102168106A (zh) * | 2010-12-20 | 2011-08-31 | 南京农业大学 | 一种控制植物体内ala合成、促进生长并提高抗逆性的转基因方法 |
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