CN117448349B - 猕猴桃细菌性溃疡病感病基因AcMIF2-1及其应用 - Google Patents
猕猴桃细菌性溃疡病感病基因AcMIF2-1及其应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,公开了猕猴桃细菌性溃疡病感病基因AcMIF2‑1及其应用。所述AcMIF2‑1基因的核苷酸序列如SEQ ID NO:1所示,其编码猕猴桃巨噬细胞迁移抑制因子AcMIF2‑1。沉默AcMIF2‑1基因的猕猴桃植株对细菌性溃疡病的抗性提升,过表达AcMIF2‑1基因的猕猴桃植株对细菌性溃疡病的抗性降低,由此获知AcMIF2‑1基因在猕猴桃抗细菌性溃疡病免疫反应中起负调控作用。基于此,可将该基因用于猕猴桃培育,获取具有抗细菌性溃疡病的猕猴桃品种。本发明为猕猴桃细菌性溃疡病的安全、高效防治奠定了理论基础,具有重要意义。
Description
技术领域
本发明属于基因工程技术领域,涉及猕猴桃细菌性溃疡病感病基因AcMIF2-1及其应用。
背景技术
由丁香假单胞菌猕猴桃致病变种(Pseudomonas syringaepv.actinidiae,Psa)引起的猕猴桃细菌性溃疡病是猕猴桃产业最具毁灭性的病害。该病害在适合的温度和湿度条件下可以迅速感染整个果园,导致大面积猕猴桃死亡。该病害发展迅速、危害严重、防治困难。病害症状主要包括树干和枝蔓溃疡(皮层、维管组织褐变、坏死,伤口处多见白色至红褐色脓液)、叶片坏死病斑(典型病斑呈暗褐色,不规则且具有黄色晕圈)和“花腐”(花蕾受害,不能开放;萼片形成坏死斑)。该病害在全球猕猴桃栽培区均有发生,严重制约了猕猴桃产业的健康发展。
巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)主要在人类和动物以及一些单细胞病原体如疟原虫和利什曼病属中被研究。MIF以其作为非典型细胞因子/化学因子调节先天免疫的作用而闻名。MIF的失调与急性和慢性炎症性疾病有关,如败血症休克、类风湿性关节炎和动脉硬化,以及自身免疫性疾病和癌症。已报道多种脊椎动物感染的寄生虫能够分泌MIF蛋白,包括线虫如钩虫,原虫如利什曼病、疟原虫和蜱虫。一些研究提供证据表明,寄生虫的MIFs参与了宿主的免疫调节。大多数脊椎动物与寄生虫之间的相互作用过程复杂,其干扰宿主免疫***的分子机制仍有待阐明,但钩虫可能通过宿主的内源性MIF相关信号通路促进寄生。同样,蚜虫的MIFs释放到植物宿主体内已被证明可以调节植物免疫。植物寄生线虫Meloidogyne incognita分泌MIF蛋白,其中MiMIF-2与拟南芥的两个附件蛋白相互作用,抑制植物免疫反应并促进寄生。MIF类蛋白在大多数真核生物中是保守的,在一些类群中有新功能化的迹象。然而目前对于猕猴桃植株中MIF蛋白的相关研究十分匮乏,其与病原之间的相互作用对植物抗病性的影响仍不清楚。
基于此,猕猴桃植株中MIF的鉴定以及研究MIF编码基因表达水平对猕猴桃抗病性的影响,将对猕猴桃抗细菌性溃疡病品种的培育具有重要意义。
发明内容
基于猕猴桃植株中MIF蛋白对猕猴桃抗病性的影响尚缺乏研究,本发明从基因工程角度出发,研究MIF蛋白的编码基因表达水平对猕猴桃抗病性的影响,这将对抗病猕猴桃品种的培育具有重要的贡献价值。具体地,本发明采用如下技术方案。
本发明提供AcMIF2-1基因在猕猴桃抗细菌性溃疡病中的应用,所述AcMIF2-1基因在猕猴桃抗细菌性溃疡病免疫反应中起负调控作用;所述AcMIF2-1基因的核苷酸序列如SEQ ID NO:1所示。
进一步,在上述应用中,所述AcMIF2-1基因编码猕猴桃巨噬细胞迁移抑制因子AcMIF2-1,所述猕猴桃巨噬细胞迁移抑制因子AcMIF2-1的氨基酸序列如SEQ ID NO:2所示。
进一步,在上述应用中,沉默所述AcMIF2-1基因,或沉默所述AcMIF2-1基因的特异性片段,提高猕猴桃植株对细菌性溃疡病的抗性;用于沉默所述AcMIF2-1基因的特异性片段的核苷酸序列如SEQ ID NO:3所示。
进一步,在上述应用中,过表达所述AcMIF2-1基因,降低猕猴桃植株对细菌性溃疡病的抗性。
本发明还提供一种猕猴桃巨噬细胞迁移抑制因子AcMIF2-1在猕猴桃抗细菌性溃疡病中的应用,所述猕猴桃巨噬细胞迁移抑制因子AcMIF2-1由所述AcMIF2-1基因编码,其氨基酸序列如SEQ ID NO:2所示。
本发明还请求保护一段多核苷酸序列,所述多核苷酸序列用于特异性沉默所述AcMIF2-1基因,所述多核苷酸序列如SEQ ID NO:3所示。
本发明还提供一种猕猴桃抗细菌性溃疡病品种的培育方法,所述方法是在猕猴桃植株中沉默所述AcMIF2-1基因,或沉默所述AcMIF2-1基因的特异性片段。
与现有技术相比,本发明“猕猴桃细菌性溃疡病感病基因AcMIF2-1及其应用”具有以下有益效果:
本发明揭示了一个猕猴桃植株中可以响应Psa侵染的基因AcMIF2-1,该基因与猕猴桃-Psa互作过程中的抗病性相关,且在猕猴桃抗细菌性溃疡病免疫反应中具有负调控作用。
本发明明确了AcMIF2-1基因的功能:瞬时过表达AcMIF2-1基因后对Psa表现出感病,病斑面积、病原菌生物量显著增加;沉默AcMIF2-1基因后对Psa表现出抗病,病斑面积、病原菌生物量显著降低,由此获知所述AcMIF2-1基因是一个感病基因。
基于AcMIF2-1基因在猕猴桃植株对细菌性溃疡病免疫反应中起负调控作用,可以将AcMIF2-1基因用于创制猕猴桃抗细菌性溃疡病品种。具体方法是:在猕猴桃植株中沉默AcMIF2-1基因,沉默所述AcMIF2-1基因的方法不限于农杆菌介导法。
本发明所述AcMIF2-1基因功能的明确为猕猴桃抗溃疡病品种培育提供新方向,也为猕猴桃溃疡病安全持续防控提供重要的理论依据。
附图说明
图1为在“红阳”猕猴桃被Psa侵染后,AcMIF2-1基因在不同时间的相对表达量柱状图。
图2为沉默猕猴桃AcMIF2-1基因沉默效率检测结果。“TRV2-GFP”表示携带绿色荧光蛋白的TRV载体,作为对照组。
图3为pBin-eGFP和pBin-eGFP-AcMIF2-1瞬时过表达时猕猴桃叶片的蛋白表达Western blot检测。“pBin-eGFP”表示仅携带绿色荧光蛋白的pBin-eGFP载体,作为对照组。“pBin-eGFP-AcMIF2-1”表示过表达AcMIF2-1基因,作为实验组。
图4为沉默AcMIF2-1基因后猕猴桃叶片发病症状(A图)以及Psa生物量(B图)。“TRV2-GFP”表示仅携带绿色荧光蛋白的pTRV2载体,作为对照组。“TRV2-AcMIF2-1”表示沉默AcMIF2-1基因,作为实验组。
图5为pBin-eGFP和pBin-eGFP-AcMIF2-1瞬时过表达猕猴桃叶片接种Psa后5天的发病症状(A图)以及Psa生物量(B图)。“pBin-eGFP”表示仅携带绿色荧光蛋白的pBin-eGFP载体,作为对照组。“pBin-eGFP-AcMIF2-1”表示过表达AcMIF2-1基因,作为实验组。
具体实施方式
下面结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
以下实施例中涉及的pTRV1、pTRV2-GFP、pBin-eGFP载体,猕猴桃感病品种“红阳”、猕猴桃抗病品种“海沃德”,以及猕猴桃细菌性溃疡病的病原菌丁香假单胞菌猕猴桃致病变种(Pseudomonas syringaepv. actinidae,Psa)均由西北农林科技大学提供。
实施例1
本实施例提供AcMIF2-1基因的克隆。
基于猕猴桃基因组V3.0获取AcMIF2-1基因的核苷酸序列如SEQ ID NO:1所示,AcMIF2-1基因编码的氨基酸序列如SEQ ID NO:2所示。
使用RNA试剂盒(北京华越洋生物科技有限公司,货号0416-50)提取“红阳”品种的猕猴桃叶片RNA,使用反转录试剂盒(ThermoFisher Scientific,货号K1162)获得cNDA。以cDNA为模板,对AcMIF2-1基因的全长序列设计引物并进行PCR扩增。
用于PCR扩增的引物序列:
上游引物:(5′-GATAGCCGGTACCCCCATGGCTTCAGAATCAGAATCAGC-3′);
下游引物:(5′-CATCGTATGGGTACCCAAGCTCTGGTTCATGATCATGTTC-3′)。
PCR扩增体系包括:2×Phanta Max Master Mix 25μL、模板cDNA 1μL,加水至50μL。
PCR扩增程序:95℃预变性3min;95℃变性30s,57℃退火30s,72℃延伸30s,循环35次;最后72℃延伸5min。
琼脂糖凝胶上进行电泳分离。用Magen胶回收试剂盒HiPure Gel Pure DNA MiniKit对PCR产物进行纯化。
实施例2
本实施例提供实时荧光定量PCR(qRT-PCR)分析AcMIF2-1基因受Psa诱导后的表达情况。
取2年生“红阳”猕猴桃品种植株的健康叶片若干,叶片应大小均一、长势一致。用自来水冲洗干净,0.6%次氯酸钠表面消毒10min,无菌水漂洗3次至无刺鼻气味,最后使用无菌滤纸吸干叶表残余水分。使用无菌打孔器(φ=11mm)制备叶盘,叶盘置于装有60mL的侵染液浓度为1×104CFU/mL的Psa-M228菌液的100mL离心管中,每管装30~50个叶盘。0.1MPa真空泵渗透至叶背面浸湿达90%以上(剔除未达侵染要求的叶盘),用无菌滤纸吸干叶盘表面水分,叶正面朝下紧贴于0.5%~0.8%水琼脂平板上,28℃培养箱培养。
取侵染后0h、6h、12h、24h、48h的叶盘,每组3个重复。提取RNA反转录后进行qRT-PCR定量分析,试验结果如图1所示。图1显示了猕猴桃在受Psa诱导时AcMIF2-1基因的相对表达量柱状图,由该图可知,AcMIF2-1基因在感病品种中受Psa侵染后显著上调表达。
实施例3
本实施例提供AcMIF2-1基因瞬时过表达载体的构建。
基于猕猴桃基因组V3.0获取AcMIF2-1基因的核苷酸序列如SEQ ID NO:1所示,AcMIF2-1基因编码的氨基酸序列如SEQ ID NO:2所示。
使用RNA试剂盒(北京华越洋生物科技有限公司,货号0416-50)提取“红阳”品种的猕猴桃叶片RNA,使用反转录试剂盒(ThermoFisher Scientific,货号K1162)获得cNDA。以cDNA为模板,对AcMIF2-1基因的全长序列设计引物并进行PCR扩增。
用于PCR扩增的引物序列:
上游引物:(5′-GATAGCCGGTACCCCCATGGCTTCAGAATCAGAATCAGC-3′);
下游引物:(5′-CATCGTATGGGTACCCAAGCTCTGGTTCATGATCATGTTC-3′)。
PCR扩增体系包括:2×Phanta Max Master Mix 25μL、模板cDNA 1μL,加水至50μL。
PCR扩增程序:95℃预变性3min;95℃变性30s,57℃退火30s,72℃延伸30s,循环35次;最后72℃延伸5min。
琼脂糖凝胶进行电泳分离,用Magen胶回收试剂盒HiPure Gel Pure DNA MiniKit对PCR产物进行纯化。
纯化产物根据ClonExpress II One Step Cloning Kit(Vazyme)按照试剂盒说明书的方法,将AcMIF2-1基因连接到KpnI/BamHI酶切的pBin-eGFP载体上得到重组的pBin-eGFP-AcMIF2-1质粒。
质粒转化大肠杆菌DH5α感受态细胞,涂LB平板(含卡那霉素50μg/mL),37℃培养16h后进行菌落PCR验证,挑取三个克隆按照质粒提取试剂盒(Takara)操作提取质粒并测序(上海生工生物工程公司),序列如SEQ ID NO:4所示。将测序正确的质粒热击转化到农杆菌GV3101中,涂LB平板(含卡那霉素50μg/mL、利福平50μg/mL),28℃培养48h后进行菌落PCR验证,挑取正确克隆进行后续实验。
实施例4
本实施例提供AcMIF2-1基因沉默载体的构建。
提取猕猴桃总RNA,反转录合成cDNA。根据AcMIF2-1基因的序列(SEQ ID NO:1)设计引物,扩增部分片段用于AcMIF2-1基因沉默载体的构建。
对AcMIF2-1基因的部分片段进行PCR扩增,获得长度为345bp的核苷酸序列(SEQID NO:3),即所述AcMIF2-1基因的特异性片段。
用于扩增如SEQ ID NO:3所示的核苷酸序列的引物:
上游引物:(5′-GTGAGCTCGGTACCGGATCCATGCCTTGCCTGAACCTGTC-3′);
下游引物:(5′-TGAGTAAGGTTACCGAATTCGAAGGTGGATCCATTCCATC-3′)。
PCR扩增程序:95℃预变性2min;95℃变性15s,55℃退火15s,72℃延伸10s,循环35次;最后72℃延伸5min。
PCR产物在1%琼脂糖凝胶进行电泳分析,并用Magen胶回收试剂盒HiPure GelPure DNA Mini Kit对PCR产物进行纯化。纯化后的PCR产物根据ClonExpress II One StepCloning Kit(Vazyme)的操作方法,连接到BamHI/EcoRI酶切的pTRV2载体上,得到能够沉默AcMIF2-1基因的重组质粒pTRV2-AcMIF2-1。将该重组质粒转化大肠杆菌DH5α感受态细胞,涂LB平板(含卡那霉素50μg/mL),37℃培养16h后进行菌落PCR验证,挑取三个克隆,按照质粒提取试剂盒(Takara)操作,提取TRV2-AcMIF2-1质粒,序列如SEQ ID NO:4。将测序正确的质粒热击转化到农杆菌GV3101中,涂LB平板(含卡那霉素50μg/mL、利福平50μg/mL),28℃培养48h后菌落PCR验证,挑取正确克隆进行后续实验。
实施例5
本实施例提供在猕猴桃中沉默和过表达AcMIF2-1基因的情况。
1)农杆菌培养
接种前2天,将含有不同质粒TRV1(TRV1包括编码RNA依赖的RNA聚合酶、运动蛋白和16kd蛋白的基因,是VIGS体系的辅助病毒载体,它能够增强TRV2的复制和传播)、TRV2-GFP(仅携带绿色荧光蛋白的TRV2载体)、TRV2-AcMIF2-1(含有能够沉默AcMIF2-1基因的TRV2载体)、pBin-eGFP(仅携带绿色荧光蛋白的pBin-eGFP载体)、pBin-eGFP-AcMIF2-1(含有能够过表达AcMIF2-1基因的pBin-eGFP载体)的GV3101农杆菌接种于含50μg/mL卡那霉素、50μg/mL利福平的LB培养液中,28℃、220rpm摇床培养至OD600值为0.5。然后使用10mMMgCl2缓冲液清洗3次,再使用现配的农杆菌侵染液MMA(含0.2mM AS(乙酰丁香酮)、10mMMgCl2、10mM MES,pH值5.6)重悬。将pTRV1:pTRV2-GFP(v/v=1:1)、pTRV1:pTRV2-AcMIF2-1(v/v=1:1)、pBin-eGFP、pBin-eGFP-AcMIF2-1用锡箔纸包裹置于室温避光处理3h后备用。
2)叶盘真空渗透法
取本实验室种植的2年生感病品种“红阳”和抗病品种“海沃德”猕猴桃健康叶片若干,叶片应大小均一、长势一致。用自来水冲洗干净,0.6%的次氯酸钠表面消毒10min,无菌水漂洗3次,至无刺鼻气味,最后使用无菌滤纸吸干叶表残余水分。
使用无菌打孔器(φ=11mm)制备叶盘,叶盘置于装有60mL的不同农杆菌组合的侵染液的100mL离心管中,每管装30~50个叶盘。基因沉默选用“红阳”品种的叶片为实验材料,其中pTRV1:pTRV2-GFP为对照。基因过表达选用“海沃德”品种的叶片为实验材料,其中pBin-eGFP为对照。0.1MPa真空泵渗透至叶背面浸湿达90%以上(剔除未达侵染要求的叶盘),用无菌滤纸吸干叶盘表面水分,叶正面朝下紧贴于0.5%~0.8%水琼脂平板上,于28℃培养箱培养。
3)沉默效率检测
分别提取pTRV1:pTRV2-GFP(v/v=1:1)、pTRV1:pTRV2-AcMIF2-1(v/v=1:1)侵染的“红阳”猕猴桃叶片总RNA,以RNA反转录获得的cDNA为模板,每个处理分别取三个生物学重复,三个试验重复。以Actin为内参基因,采用qRT-PCR对AcMIF2-1基因的表达量进行检测。所用的引物为:
Q-AcMIF2-1-F:(5′-ATGCCTTGCCTGAACCTGTC-3′);
Q-AcMIF2-1-R:(5′-GAAGGTGGATCCATTCCATC-3′)。
在冰上依次加入2×ChamQ SYBR qPCR Master Mix 10μL、Q-AcMIF2-1-F 0.4μL、Q-AcMIF2-1-R 0.4μL、Template DNA/cDNA 2μL、ddH2O 7.2μL,建立总计20μL的qRT-PCR体系并进行扩增,扩增反应程序见表1。
表1.qRT-PCR扩增程序
结果表明pTRV1:pTRV2-AcMIF2-1在侵染5d后达到沉默要求,与对照pTRV1:pTRV2-GFP相比,沉默效率为64%(见图2)。
4)过表达AcMIF2-1-eGFP蛋白Western blot检测
收集农杆菌感染两天后的猕猴桃叶片进行蛋白Western blot检测。将收集的猕猴桃叶片液氮速冻后研磨,加入蛋白提取液(组分为:150mM NaCl、50mM Tris-HCl,pH7.5、10mM ethylenediaminetetraacetic acid、1.0%(v/v)NP-40,1mM phenylmethylsulfonylfluoride,1.0%(v/v)protease inhibitor cocktail)混匀置于冰上30min。18000×g离心收集上清液80μL加入5倍蛋白上样缓冲液20μL混合均匀后沸水水浴10min。取20μL样品进行SDS-PAGE,120V电泳1.5h。
反应结束后将蛋白样品转到PVDF膜上,用5% TBST牛奶孵育封膜。加入1:5000稀释的GFP一抗(Abmart)孵育2h后用TBST洗膜5次,每次5min;随后加入1:10000稀释的鼠抗(Abmart)孵育30min后用TBST洗膜洗3次,每次5min,扫膜拍照,Western blot结果见图3。Anti-GFP为GFP标签蛋白的特异性抗体。Anti-Actin为内参蛋白Actin的特异性抗体。图3中显示,通过GFP抗体Anti-GFP检测到了eGFP(25kDa)和eGFP-AcMIF2-1(35kDa)表明AcMIF2-1过表达成功。
5)猕猴桃溃疡病菌的侵染
明确AcMIF2-1基因达到沉默或过表达条件后,采用叶盘真空渗透法接种猕猴桃溃疡病原菌Psa-M228菌株(1×104CFU/mL)置于0.8%水琼脂平板上,放入人工气候箱培养,5d后观察Psa-M228侵染情况并统计病斑面积和病原菌生物量。结果表明沉默AcMIF2-1基因后叶片发病情况明显减轻(见图4中的图A),病原菌生物量较TRV2-GFP对照组相比明显下降,仅有对照组生物量0.6倍(见图4中的图B)。
而过表达AcMIF2-1基因后叶片发病情况与对照组相比明显更严重(见图5中的图A),病斑面积较pBin-eGFP对照组大约提高了2倍,病原菌生物量明显升高,约为对照组生物量1.6倍(见图5中的图B)。
综合以上实验结果表明,AcMIF2-1基因受Psa侵染后显著上调表达,与品种的感病性紧密相关。分别沉默和过表达AcMIF2-1基因后接种Psa,表型观察、病原菌生物量统计结果均表明该基因是一个感病基因。AcMIF2-1基因功能的明确为猕猴桃抗溃疡病品种培育提供新方向,也为猕猴桃溃疡病安全持续防控提供重要的理论依据。
以上所述的实施例只是本发明的一部分实施例,而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。在依据本发明构思的条件下本领域普通技术人员进行的相关推演和替换,在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
Claims (2)
1.AcMIF2-1基因在猕猴桃抗细菌性溃疡病中的应用,其特征在于,所述AcMIF2-1基因在猕猴桃抗细菌性溃疡病免疫反应中起负调控作用;
所述AcMIF2-1基因的核苷酸序列如SEQ ID NO:1所示;
沉默所述AcMIF2-1基因,或沉默所述AcMIF2-1基因的特异性片段,提高猕猴桃植株对细菌性溃疡病的抗性;
所述AcMIF2-1基因的特异性片段的核苷酸序列如SEQ ID NO:3所示。
2.一种猕猴桃抗细菌性溃疡病品种的培育方法,其特征在于,在猕猴桃植株中沉默AcMIF2-1基因,或沉默AcMIF2-1基因的特异性片段;
所述AcMIF2-1基因的核苷酸序列如SEQ ID NO:1所示;
所述AcMIF2-1基因的特异性片段的核苷酸序列如SEQ ID NO:3所示。
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