CN115927317A - miR6478在增强植物抗病性中的用途 - Google Patents
miR6478在增强植物抗病性中的用途 Download PDFInfo
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Abstract
本发明公开了miR6478在增强植物抗病性中的用途。本发明以转miR6478的黄瓜为研究对象，从分子生物学的角度研究了miR6478对染病黄瓜受损伤程度的影响，证明了miR6478对黄瓜抗病途径的调控。本发明对揭示miR6478的功能及黄瓜新品种的培育具有重大意义。
Description
技术领域
本发明属于生物技术领域，具体涉及miR6478在增强植物抗病性中的用途。
背景技术
黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus，CGMMV)严重影响了黄瓜、西瓜和甜瓜等葫芦科作物的品质和产量。植物内源mircoRNA(miRNA)可通过调控下游靶基因参与叶片形态建成、花和果实发育、响应生物和非生物胁迫等多个生命过程。研究发现，CGMMV胁迫条件下，多个miRNA均可通过调控其靶基因参与黄瓜抗病毒反应。例如，miR159和csa-miRn6-3p可在转录后水平上调控多个与植物免疫反应有关的基因，如潜在抗病基因丝氨酸/苏氨酸蛋白激酶PBS1、植物细胞程序性死亡的正调控因子GAMYB和参与水杨酸介导的防御反应的基因H2B等。因此，筛选参与植物与病毒互作过程的关键miRNA，获得miRNA介导的抗CGMMV的转基因黄瓜，可为筛选新的黄瓜抗病毒基因提供科学依据，为综合防控黄瓜绿斑驳花叶病毒病提供新思路，进而为培育抗病毒作物奠定基础、提供指导。
发明内容
本发明的目的是提供一种与植物抗病性相关的miRNA及其应用。
本发明所提供的黄瓜内源miRNA，名称为miR6478，其核苷酸序列是SEQ ID No.1，其中，SEQ ID No.1由21个核苷酸组成。
与miR6478相关的生物材料也属于本发明的保护范围。
与miR6478相关的生物材料为短串联靶标模拟(Short tandem target mimic，STTM)序列，其编码抑制所述miRNA分子的核酸分子。
与miR6478相关的生物材料为表达盒，所述表达盒包含编码抑制所述miRNA分子的核酸分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
在本发明的一个实施方式中，编码抑制所述miRNA分子的核酸分子如SEQ IDNo.2。
与miR6478相关的生物材料为重组载体，所述重组载体包含编码抑制所述miRNA分子的核酸分子、或所述表达盒。
与miR6478相关的生物材料为重组微生物，所述重组微生物包含编码抑制所述miRNA分子的核酸分子、或所述表达盒、或所述重组载体。
与miR6478相关的生物材料为转基因植物细胞系，所述转基因植物细胞系包含编码抑制所述miRNA分子的核酸分子、或所述表达盒、或所述重组载体。
所述miRNA分子、所述短串联靶标模拟序列、所述表达盒、或所述重组载体、或所述重组微生物、或所述转基因植物细胞系可用于调控植物抗病性。
在本发明的实施例中，所述载体可为质粒、噬菌体或病毒载体。
在本发明的一个具体的实施方式中，所述重组载体可为表达抑制miR6478表达的核酸分子的表达载体。具体的，可为含有SEQ ID No.2的重组载体。所述含有SEQ ID No.2的重组载体，是指将长度为126bp的miR6478的短串联靶标模拟(Short tandem targetmimic，STTM)序列(SEQ ID No.2所示的DNA片段)通过同源重组整合到pTRV2载体中获得的载体pTRV2:STTM-miR6478。
其中，STTM是长度为48个碱基的特定序列(GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAA T)，是将两个TM(Target mimic)连接起来，在两个TM上的miRNA切割位点处，都有一个由3个碱基(cta)构成的突起结构，由于这个凸起的存在，导致miRNA能与其结合但无法真正的对其切割，进而起抑制miRNA功能的作用。因此，可利用该载体表达miRNA的靶标模拟序列来沉默植物内源miRNA。
可通过基于烟草脆裂病毒(Tobacco rattle virus，TRV)的miRNA沉默(Virus-based MicroRNA Silencing，VbMS)技术在黄瓜中瞬时沉默miR6478。所述重组载体为pTRV2:STTM-miR6478载体，pTRV2:STTM-miR6478载体是将SEQ ID No.2所示片段导入pTRV2载体获得的重组载体。
本发明提供了一种培育抗病性增强的转基因植物的方法，包括沉默受体植物中的所述miRNA分子，获得抗病性高于所述受体植物的转基因植物。
具体的，可采用VbMS技术在黄瓜中瞬时沉默miR6478。在本发明的实施例中，是通过用含有pTRV1的重组微生物和含有pTRV2:STTM-miR6478载体的重组微生物共同转染所述受体植物获得。
所述培育抗病性增强的转基因植物的方法中，通过使用所述重组微生物向所述向受体植物中导入所述表达miRNA分子的核酸分子、所述表达盒、或所述重组载体。
在本发明的实施方式中，所述重组微生物可为酵母、细菌、藻或真菌。所述细菌可为农杆菌；所述农杆菌具体可为GV3101农杆菌。
在本发明的具体实施例中，所述培育抗病性增强的转基因植物的方法中，所述重组微生物为含有pTRV1和pTRV2:STTM-miR6478的GV3101农杆菌。
上述方法中，所述转基因植物的抗病性高于所述受体植物体现在如下A1)—A2)中的全部或部分：
A1)所述转基因植物中CGMMV RNA的积累量低于所述受体植物；
A2)所述转基因植物叶片上的褪绿、黄化斑点少于所述受体植物。
在本发明的实施例中，所述转基因植物细胞系不包括植物的繁殖材料。
在本发明的实施例中，所述调控植物抗病性为提高植物抗病性，具体体现在如下B1)—B2)中的全部或部分：
B1)当植物中miR6478的表达量降低时，所述植物中CGMMV RNA的积累量减少；
B2)当植物中miR6478的表达量降低时，所述植物叶片上的褪绿、黄化斑点减少。
上述方法中，所述植物为双子叶植物；所述双子叶植物可为黄瓜；所述黄瓜具体可为新泰密刺黄瓜。
上述方法中，所述转基因植物理解为不仅包含将所述基因转化目的植物得到的第一代转基因植物，也包括其子代。对于转基因植物，可以在该物种中繁殖该基因，也可用常规育种技术将该基因转移进入相同物种的其它品种，特别包括商业品种中。所述转基因植物包括种子、完整植株和细胞。
本发明采用生物信息学预测、分子克隆技术、农杆菌介导的转化、基于烟草脆裂病毒的miRNA沉默(Virus-based MicroRNA Silencing，VbMS)、实时荧光定量PCR等多种生物学手段，以沉默miR6478的黄瓜为研究对象，以转入空载体pTRV1+pTRV2的黄瓜为对照，首次沉默miR6478，通过测定CGMMV接种后病毒RNA积累量，观察病害发生后叶片上的褪绿、黄化斑点，从分子生物学角度研究了miR6478对染病植物受损伤程度的影响。证明了与对照组植物相比，沉默miR6478的转基因植物的抗病性高于对照组植物，说明miR6478是与植物抗病性相关的微小RNA，可用于调控目的植物的抗病性。
附图说明
图1为黄瓜miR6478的短串联靶标模拟序列示意图
图2为沉默miR6478黄瓜中miR6478的定量分析
图3为沉默miR6478黄瓜中CGMMV RNA积累量的分析
图2-图3中TRV:00代表空白对照组(使用pTRV1+pTRV2混合菌液，按照1:1比例混合后浸润黄瓜幼苗)
图2-图3中TS代表瞬时沉默(Transient silencing)
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的农杆菌感受态细胞GV3101可从上海唯地生物技术有限公司购买。
下述实施例中的黄瓜自交系“新泰密刺”购自山东省新泰市新泰密刺黄瓜原种场。
下述实施例中的载体pTRV1、pTRV2可从湖南丰晖生物科技有限公司购买。
本发明中的miR6478成熟序列为序列表中SEQ ID No.1所示的RNA分子。
本发明中的用于沉默miR6478的核酸序列为序列表中SEQ ID No.2所示的DNA分子。
本发明以转miR6478的黄瓜为研究对象，以野生型新泰密刺黄瓜为对照，通过检测挑战接种黄瓜绿斑驳花叶病毒的受体植物中病毒RNA的积累量，从分子生物学的角度研究了miR6478对染病植物受损伤程度的影响，证明了miR6478对植物抗病途径的调控。本发明对揭示miR6478的抗病功能及抗病黄瓜品种的培育具有重大意义，有助于丰富蔬菜育种资源。
实施例1、miR6478在调控黄瓜抗病性中的应用
一、沉默miR6478对黄瓜抗病性的影响
设计并合成miR6478的短串联靶标模拟(Short tandem targets mimic，STTM)序列，采用基于烟草脆裂病毒(Tobacco rattle virus，TRV)的miRNA沉默技术(Virus-basedMicroRNA Silencing，VbMS)，在黄瓜幼苗中沉默miR6478，试验流程见图2。
1、沉默miR6478载体构建和黄瓜获得
(1)沉默载体pTRV2:STTM-miR6478的构建和转化农杆菌
人工合成miR6478的STTM片段(SEQ ID No.2)，使用InFusion HD Cloning Kit(购自Takara公司)将上述目的片段克隆入pTRV2载体(购自湖南丰晖生物科技有限公司)，获得pTRV2:STTM-miR6478载体，取2.5μL In-Fusion反应液将pTRV2:STTM-miR6478载体转化入大肠杆菌感受态细胞Stellar(购自北京全式金生物技术有限公司)；将测序分析鉴定为阳性的pTRV1(购自湖南丰晖生物科技有限公司)、pTRV2:STTM-miR6478的质粒DNA分别转入农杆菌GV3101感受态细胞(购自上海唯地生物技术有限公司)中，挑取单菌落进行菌落PCR验证，提取质粒DNA进行测序。
(2)沉默miR6478黄瓜的获得
采用基于病毒的miRNA沉默(Virus-based MicroRNA Silencing，VbMS)技术在黄瓜中瞬时沉默miR6478。具体步骤如下：
①新泰密刺黄瓜种子经催芽后，播种于塑料花盆，放置于植物生长培养箱生长。
②当黄瓜幼苗第一片真叶生长点露出时(约5-7天)，在黑暗条件下，用刀片轻轻造成微小伤口，用浸润液复苏的含有pTRV1和pTRV2:STTM-miR6478载体的农杆菌(按照1:1的比例混合)浸湿棉花，并放置在微伤口处；使用pTRV1+pTRV2混合菌液(按照1:1的比例混合)处理作为空白对照；以转入pTRV1和pTRV2:PDS的农杆菌(按照1:1的比例混合)作为沉默指示对照组，其中pTRV2:PDS(PDS为八氢番茄红素去饱和酶，Phytoene desaturase，登录号ABE99707)具体为将PDS的片段插入pTRV2载体的CP和Rz之间，替换原有的MCS，进而获得pTRV2:PDS。每间隔2h将棉花团用相应菌液再次润湿，重复3次，浸润完成后移去带有菌液的棉花团，将黄瓜幼苗置于植物生长培养箱中，在16h光照，8h黑暗条件下生长，并在生长期间按需进行浇水。
2、沉默miR6478对黄瓜抗病性的影响
(1)黄瓜幼苗处理后生长约10天，当第一片真叶展开时，摩擦接种CGMMV。黄瓜植株生长第15-20天，对植株上第2片以及第3片叶片进行采样，提取总RNA，采用实时荧光定量PCR方法分别检测miR6478的表达水平以及CGMMV病毒RNA积累情况。
(2)分别采用miR6478、CGMMV的特异性引物进行qRT-PCR检测，并采用
法对miR6478的及CGMMV外壳蛋白基因的相对表达量进行计算，分析表达情况。miR6478的上游引物序列为5’-CCGACCTTAGCTCAGTTGGTG-3’，下游通用引物由miRcute增强型miRNA荧光定量检测试剂盒(SYBR)(FP411，购自天根生化科技(北京)有限公司)提供；内参基因EF-1a的上游引物序列为5’-ACTGGTGGTTTTGAGGCTGGT-3’，下游引物序列为5’-CTTGGAGTATTTGGGTGTGGT-3’；内参基因Ubiquitin的上游引物序列为5’-CTAATGGGGAGTGGGGAAGTA-3’，下游引物序列为5’-GTCTGGATGGACAATGTTGAT-3’；CGMMV外壳蛋白基因的qPCR引物序列为上游5’-ACAGCCGCTAGGGCTGAGATA-3’，下游5’-CCAATGAGCAAACCGTTCGAT-3’。
结果显示：在转染含pTRV1的农杆菌和含pTRV2:STTM-miR6478的农杆菌获得的沉默miR6478的黄瓜中(图2、图3中的TS-miR6478)，相对于转空载体(pTRV1+pTRV2)对照(图2、图3中的TRV:00)，miR6478的表达水平降低至0.004(图2)，CGMMV RNA的积累量降低至0.076(图3)。沉默miR6478的黄瓜叶片上的褪绿、黄化斑点少于转空载体对照。
综上所述，与对照组植物相比，沉默miR6478的转基因植物的抗病性高于对照组植物，miR6478是与植物抗病性相关的微小RNA，可用于调控目的植物的抗病性。
Claims (9)
1.一种miRNA分子，其特征在于，所述miRNA分子的成熟序列为序列表中的SEQ IDNo.1。
2.一种短串联靶标模拟序列，其特征在于，所述短串联靶标模拟序列编码抑制权利要求1中所述miRNA分子的核酸分子，其核苷酸序列为序列表中的SEQ ID No.2。
3.一种表达盒，其特征在于，所述表达盒包含权利要求2所述短串联靶标模拟序列。
4.一种重组载体，其特征在于，所述重组载体包含权利要求2所述短串联靶标模拟序列或权利要求3所述表达盒。
5.一种重组微生物，其特征在于，所述重组微生物包含权利要求2所述短串联靶标模拟序列、或权利要求3所述表达盒、或权利要求4所述重组载体。
6.一种转基因植物细胞系，其特征在于，所述转基因植物细胞系包含权利要求2所述短串联靶标模拟序列、或权利要求3所述表达盒、或权利要求4所述重组载体、或权利要求5所述重组微生物。
7.权利要求1所述miRNA分子、或权利要求2所述短串联靶标模拟序列、或权利要求3所述表达盒、或权利要求4所述重组载体、或权利要求5所述重组微生物、或权利要求6所述转基因植物细胞系在调控植物抗病性中的应用。
8.一种培育抗病性增强的转基因植物的方法，其特征在于，通过沉默受体植物中的权利要求1所述miRNA分子获得抗病性高于所述受体植物的转基因植物；
优选的，所述植物为双子叶植物。
9.根据权利要求8所述方法，其特征在于，通过利用权利要求2所述短串联靶标模拟序列采用基于烟草脆裂病毒的miRNA沉默技术在黄瓜中瞬时沉默权利要求1所述miRNA分子。
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