CN117363626A - 一种柑橘CsGATA17基因及其提高柑橘溃疡病抗性的方法 - Google Patents
一种柑橘CsGATA17基因及其提高柑橘溃疡病抗性的方法 Download PDFInfo
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Abstract
本发明公开了一种柑橘CsGATA17基因及其提高柑橘溃疡病抗性的方法,涉及分子生物学技术领域,柑橘CsGATA17基因的核苷酸序列为SEQ ID NO:1所示;方法具体包括以下步骤:(1)克隆柑橘CsGATA17编码基因的RNAi片段;(2)构建干扰载体;(3)干扰载体转化柑橘,得到转基因植株。通过将一个CsGATA17编码基因的干扰载体整合到柑橘中,降低CsGATA17的转录水平,显著提高柑橘对溃疡病的抗性,并且不影响转基因植株的表型,在柑橘抗溃疡病育种中具有重大的应用价值,可以作为候选基因同多个溃疡病抗、感病基因进行溃疡病抗性育种。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及一种柑橘CsGATA17基因及其提高柑橘溃疡病抗性的方法。
背景技术
柑橘溃疡病(Citrus bacterial canker,CBC)是由地毯草黄单胞杆菌柑橘亚种(Xanthomonas citri subsp.citri,Xcc)引起的细菌性病害,其危害当前大多数主栽柑橘品种。因此,加强对柑橘溃疡病防控研究是柑橘产业发展的迫切需求。柑橘溃疡病的传统防控手段,例如病树焚烧和农药使用等需要投入大量人力、物力和财力,且会造成巨大环境危害。因此,柑橘溃疡病防治更多寄希望于培育抗病新种质。
分子育种可定向、高效培育抗病新种质,因此目前得到了快速发展和广泛应用。近年来,通过生物技术手段已获得一些抗溃疡病的柑橘资源,例如转柞蚕抗菌肽D基因的锦橙、新会橙、脐橙株系;过表达CsBZIP40的晚锦橙株系;基因定点编辑柑橘溃疡病感病基因CsLOB1启动子获得的对柑橘溃疡病抗性提高的植株等。但优质候选基因仍然匮乏,且功能和作用机制研究不深,因此,亟待有针对性地挖掘更多与柑橘溃疡病紧密相关的基因,深入解析其功能和机制,用于抗溃疡病分子育种。
转录因子GATA是一组参与植物生长、发育和代谢的转录调节因子,因其能够与靶基因启动子上的W-GATA-R(W=T/A,R=G/A)序列结合而得名(Shi et al.,2022)。近年来,人们对植物GATA的调控和生物效应的认识有了很大的提高。植物中的GATA家族成员参与光形态生长、叶绿体发育、叶绿素生物合成、根叶和花发育以及气孔形成和光合作用的调节等(Schwechheimer et al.,2022)。例如,在杨树中,PdGATA19可以直接激活PdNRT2.4b基因(一种高亲和力硝酸盐转运蛋白)的启动子,显著增加杨树在低硝酸盐供应水平下的硝酸盐吸收,在调节和提高氮利用效率和生长方面发挥重要作用(An et al.,2020)。小麦的TaGATA1可以通过直接调节TaABI5的表达来增强小麦种子的休眠能力,从而提高穗发芽抗性(Wei et al.,2023)。GATA家族成员在植物非生物胁迫中也起着重要作用。例如,OsGATA16通过抑制一些与寒冷相关的基因,例如OsWRKY45-1、OsSRFP1、OsCYL4和OsMYB30来作为耐寒性的活性调节因子,提高水稻幼苗的耐寒性(Zhang et al.,2021)。GATA家族成员在植物生物胁迫中也起着至关重要的作用。例如,TaGATA1转录激活因子通过结合和激活一些茉莉酸信号介导的防御基因,正向调控小麦抗纹枯病的防御反应(Liu et al.,2020)。虽然GATA转录因子调控在植物病害抗性中已有部分应用,但在柑橘溃疡病领域尚未有相关研究报道。
发明内容
本发明所要解决的技术问题是目前的柑橘溃疡病抗性不足的问题,目的在于提供一种柑橘CsGATA17基因及其提高柑橘溃疡病抗性的方法,解决了目前的柑橘溃疡病抗性不足的问题。
本发明通过下述技术方案实现:
一方面,本申请提供一种柑橘CsGATA17基因,所述柑橘CsGATA17基因的核苷酸序列如SEQ ID NO:1所示。
另一方面,本申请提供一种柑橘CsGATA17基因提高柑橘溃疡病抗性的方法,降低柑橘属植物中CsGATA17的转录水平,所述柑橘CsGATA17基因的核苷酸序列如SEQ ID NO:1所示。
本发明将一个CsGATA17编码基因的干扰载体整合到柑橘中,降低CsGATA17的转录水平,显著提高柑橘对溃疡病的抗性,并且不影响转基因植株的表型,在柑橘抗溃疡病育种中具有重大的应用价值,可以作为候选基因同多个溃疡病抗、感病基因进行溃疡病抗性育种。
进一步的,采用RNA干扰的方式降低柑橘中CsGATA17基因的转录水平。
进一步的,柑橘CsGATA17基因提高柑橘溃疡病抗性的方法包括以下步骤:
步骤1:克隆柑橘CsGATA17编码基因的RNAi片段;
步骤2:构建干扰载体;
步骤3:干扰载体转化柑橘,得到转基因植株。
本发明从柑橘感染溃疡病前后差异表达的基因中获得了一个在抗病品种中受柑橘溃疡病菌诱导下调,在感病品种中上调的转录因子CsGATA17,构建CsGATA17编码基因的干扰载体,通过根癌农杆菌介导转化柑橘后对柑橘溃疡病表现出明显抗性。
进一步的,步骤1中,所述RNAi片段核苷酸序列如SEQ ID NO:2所示。
进一步的,步骤1中的克隆方法为:提取柑橘总RNA,然后反转录为cDNA,最后采用高保真酶PCR扩增得到CsGATA17编码基因的RNAi片段。
进一步的,克隆CsGATA17编码基因的RNAi片段所采用的引物为CsGATA17-RNAi-F和CsGATA17-RNAi-R;CsGATA17-RNAi-F的核苷酸序列如SEQ ID NO:3;CsGATA17-RNAi-R的核苷酸序列如SEQ ID NO:4所示。
进一步的,步骤2中干扰载体的构建方法为:步骤1中获得的PCR产物分两组,第一组用SwaⅠ和AscⅠ双酶切,第二组用BamHⅠ和XbaⅠ双酶切,酶切回收的两组片段同时连接到pUC-RNAi载体,得到的中间载体pUC-RNAi-CsGATA17和pLGNe超量表达载体用KpnⅠ和SalⅠ分别双酶切,将含有RNAi片段的酶切产物连接到pLGNe载体上,构建出最终的干扰载体pLGNe-CsGATA17-RNAi。
进一步的,步骤3中,干扰载体转化柑橘的方法为:干扰载体通过电击法转化根癌农杆菌,再用根癌农杆菌介导转化柑橘外植体,遗传转化后的外植体细胞再经离体培养、染色鉴定、嫁接后得到转基因植株。
进一步的,步骤3中得到转基因植株后,对转基因植株进行抗性评价,判定CsGATA17干扰提高了柑橘溃疡病抗性。
进一步的,对转基因植株进行抗性评价前,通过PCR验证转基因植株,采用的引物为ID-CsGATA17-F和ID-CsGATA17-R;ID-CsGATA17-F的核苷酸序列如SEQ ID NO:5所示,ID-CsGATA17-R的核苷酸序列如SEQ ID NO:6所示。
进一步的,PCR验证后,用qRT-PCR验证转基因植株中CsGATA17的转录水平,采用的引物为RT-CsGATA17-F和RT-CsGATA17-R;RT-CsGATA17-F的核苷酸序列如SEQ ID NO:7所示,RT-CsGATA17-R的核苷酸序列如SEQ ID NO:8所示。
本发明与现有技术相比,具有如下的优点和有益效果:
(1)本发明将一个CsGATA17编码基因的干扰载体整合到柑橘中,降低CsGATA17的转录水平,显著提高柑橘对溃疡病的抗性,并且不影响转基因植株的表型,在柑橘抗溃疡病育种中具有重大的应用价值,可以作为候选基因同多个溃疡病抗、感病基因进行溃疡病抗性育种;
(2)本发明从柑橘感染溃疡病前后差异表达的基因中获得了一个在抗病品种中受柑橘溃疡病菌诱导下调,在感病品种中上调的转录因子CsGATA17,构建CsGATA17编码基因的干扰载体,通过根癌农杆菌介导转化柑橘后对柑橘溃疡病表现出明显抗性;
(3)本发明通过克隆柑橘CsGATA17编码基因的RNAi片段,构建干扰载体,然后转化柑橘,得到的转基因植株溃疡病病斑面积可最大降低至现有柑橘的54%,病情指数可最大降低至现有柑橘的48%,能够显著的减轻溃疡病的发病程度,而且没有影响柑橘表型,因此得出CsGATA17干扰可大大提高柑橘对溃疡病的抗性。相比基因编辑技术对柑橘基因进行沉默,RNAi技术更稳定,获得基因沉默植株的几率更大,而且适用于柑橘这种高杂合度的物种,是一种有潜力的提高柑橘溃疡病抗性的生物工程技术,对柑橘抗溃疡病分子育种有较大价值。
附图说明
为了更清楚地说明本发明示例性实施方式的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。在附图中:
图1为本发明CsGATA17的结构域示意图;
图2为本发明柑橘CsGATA17的Xcc诱导表达图:数据柱上的小写字母表示差异显著性(P<0.05);
图3为本发明CsGATA17的亚细胞定位图;
图4为本发明实施流程图;
图5为本发明CsGATA17干扰载体构建流程图:GUS:NPTII,β-葡萄糖酸苷酶基因;CaMV 35S,来源于花椰菜花叶病毒的植物组成性启动子;NOS,冠瘿碱合成酶基因终止子;
图6为本发明柑橘遗传转化流程图;
图7为本发明转基因植株GUS染色图:WT表示野生型对照;R1、R2表示两个转基因植株,下同;
图8为本发明转基因植株PCR检测图;
图9为本发明转基因植株qRT-PCR检测图;
图10为本发明转基因植株表型图;
图11为本发明转基因柑橘叶片接种Xcc后的发病情况;
图12为本发明转基因柑橘叶片接种Xcc后病斑大小统计图;
图13为本发明转基因柑橘叶片接种Xcc后病情指数统计图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
在以下描述中,为了提供对本发明的透彻理解阐述了大量特定细节。然而,对于本领域普通技术人员显而易见的是:不必采用这些特定细节来实行本发明。在其他实施例中,为了避免混淆本发明,未具体描述公知材料或方法。
在整个说明书中,对“一个实施例”、“实施例”、“一个示例”或“示例”的提及意味着:结合该实施例或示例描述的特定特征、结构或特性被包含在本发明至少一个实施例中。因此,在整个说明书的各个地方出现的短语“一个实施例”、“实施例”、“一个示例”或“示例”不一定都指同一实施例或示例。此外,可以以任何适当的组合和/或子组合将特定的特征、结构或特性组合在一个或多个实施例或示例中。此外,本领域普通技术人员应当理解,在此提供的示图都是为了说明的目的,并且示图不一定是按比例绘制的。这里使用的术语“和/或”包括一个或多个相关列出的项目的任何和所有组合。
实施例1
柑橘CsGATA17的信息学与表达分析
1.柑橘CsGATA17的生物信息学分析
CsGATA17的位于5号染色体上,基因全长为1 747 bp,含有2个外显子。使用Pfam软件对CsGATA17的结构进行分析,发现CsGATA17编码321个氨基酸,含有一个由287个氨基酸残基组成的GATA DNA-binding domian蛋白结构域(如图1所示)。
CsGATA17基因的核苷酸序列SEQ ID NO:1为:
ATGGACTTTTGCCGGAATGTGGCTGTTTCCGGTGACCAGTACCAGCAAGACCAAGTCCTCTCGCCTTCATCTATCCCTCCCTCTTCCTCCTCCAATCTTGCTCTTGCTGACCCTCTCGATGACCTTTTCCCTGCTCATACCACTGAAGTGGATGTTAGCTTGGAATGGCTTTCAATATTTGTAGAGGACTGTTTGTCGAGTTCAGGAATTTGTCTCCCAGCTTCTGAATTGCCCACCAAAAATAATGCAGCTGCTACAACTGCAGCTCCGTCGCCAAAACCCTTGCAGCAGCAGCAGCAGCAAAAAGAAAGTACCACCACCACCCCAAGCCCGCCACCTTCCCTGGAAAAATTTGTTGTTCCGGGCAAGGCAAGAAGCAAAAGAAAGAGAGCCTCAAGCACTGCTAAATTAACTCAAACAAGCACGTCATTATCATCGTTAACAACTGGTTGTTGGACGACCACTCATAACAACCACCCGGCAGATACCCAACTCTTTCACTCCGACGACCCTCCTCTGCTTCAAGTTCAACAGGCCTTCTGGCTTGCTGATAGTCAACTCATTTTCCCCAAAAAAGAAACCACCAACACCAACACTATCAACACCAACAGCAACAAGAAAGCGAAAGCTAATGATGGTGATGAGGAGGAGGAGGAGGCAAAAGAAGAAGAAACAGAAATTGGCAAGGAAGTTGAAGTTGTCCAGCAGCAGCAGCAGCAGCAGCAGCAAGGAAGGAGGTGCAGTCATTGTCTATCACAAAGGACGCCACAGTGGAGGGCGGGACCATTGGGTCCAAAGACTCTGTGTAATGCATGTGGGGTGAGGTACAAGTCTGGGAGGTTACTGCCTGAGTATAGGCCGGCCAAGAGCCCCACTTTTGTTAGCTACTTGCACTCCAATTCTCACAAGAAAGTCTTGGAGATGAGAATGGCTCTGATGCCTTCTCCTTCTTCACTTCCCAAGTAA。
2.柑橘CsGATA17的Xcc诱导表达
为验证CsGATA17与Xcc侵染之间的关系,通过在抗病品种四季橘和感病品种晚锦橙中注射柑橘Xcc,以CsGATA17特异性区域设计qRT-PCR实时引物RT-CsGATA17-F和RT-CsGATA17-R,分别如SEQ ID NO:7和SEQ ID NO:8所示核苷酸序列。对CsGATA17受Xcc侵染的诱导表达特性进行分析发现:晚锦橙中,CsGATA17的表达量在病菌感染期间表达量明显上升。与之相反,四季橘中,CsGATA17在病菌感染期间表达量总体呈下降趋势(如图2所示)。上述结果表明受Xcc诱导后,CsGATA17在感病品种晚锦橙和抗病品种四季橘中有不同表达特性,推测CsGATA17可能是一个溃疡病感病基因,其表达水平与溃疡病抗性呈负相关。
其中,RT-CsGATA17-F的核苷酸序列SEQ ID NO:7为:
GAAACCACCAACACCAACACT。
RT-CsGATA17-F的核苷酸序列SEQ ID NO:8为:
CCTCCTCCTCCTCATCACCA。
3.柑橘CsGATA17的亚细胞定位
为确定CsGATA17在细胞内的定位,我们构建CsGATA17与报告基因绿色荧光蛋白基因EGFP融合表达载体并转化拟南芥原生质体,在激光共聚焦扫描显微镜下观察其荧光表达部位。在目标蛋白荧光通道观察到CsGATA17主要定位在细胞核,少量在细胞膜,而对照组中各区域均能观察到荧光,叶绿体荧光通道的红色荧光排除了叶绿体自发荧光对目的蛋白定位的干扰,明场和叠加图进一步明确CsGATA17主要定位在细胞核(如图3所示)。定位在细胞核是CsGATA17发挥其转录因子调控作用的前提。
实施例2
具体实施例流程如图4所示。
1.CsGATA17编码基因的RNAi片段克隆
用RNA提取试剂盒(艾德莱,CAT:RN09)提取晚锦橙的RNA。使用RecombinantDNaseI(TAKARA)合成cDNA。使用引物CsGATA17-RNAi-F和CsGATA17-RNAi-R克隆CsGATA17编码基因的RNAi片段,长度为378bp,序列如SEQ ID NO:2所示核苷酸序列,所采用的引物如SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列。PCR采用PrimeSTAR master mix(TAKARA)。扩增体系:10X PCR mix:2.5μL;引物CsGATA17-RNAi-F(5μmol·L-1):1μL;引物CsGATA17-RNAi-R(5μmol·L-1):1μL;cDNA约60ng;加ddH2O至25μL。扩增程序:98℃,5min;98℃,30s,56℃,30s,72℃,1.5min,35个循环;72℃延伸10min。紫外灯下,用洁净的刀片切下含有目的片段的琼脂糖凝胶块,利用DNA凝胶回收试剂盒(艾德莱)回收RNAi片段。
其中,RNAi片段的核苷酸序列SEQ ID NO:2为:
TGGCTTGCTGATAGTCAACTCATTTTCCCCAAAAAAGAAACCACCAACACCAACGCTATCAACACCAACAGCAACAAGAAAGCGAAAGCTAATGATGGTGATGAGGAGGAGGAGGCAAAAGAAGAAGAAACAGAAATTGGCAAGGAAGTTGAAGTTGTCCAGCAGCAGCAGCAGCAGCAAGGAAGGAGGTGCAGTCATTGTCTATCACAAAGGACGCCACAGTGGAGGGCGGGACCATTGGGTCCAAAGACTCTGTGTAATGCATGTGGGGTGAGGTACAAGTCTGGGAGGTTACTGCCTGAGTATAGGCCGGCCAAGAGCCCTACTTTTGTTAGCTACTTGCACTCCAATTCTCACAAGAAAGTCTTGGAGATGAGA。
引物CsGATA17-RNAi-F的核苷酸序列SEQ ID NO:3为:
TGGCTTGCTGATAGTCAACTC。
引物CsGATA17-RNAi-R的核苷酸序列SEQ ID NO:4为:
TCTCATCTCCAAGACTTTCTTGTG。
2.CsGATA17干扰载体构建并转化根癌农杆菌
根据图5所示的流程构建干扰载体,具体操作如下:CsGATA17编码基因的RNAi片段的PCR产物回收后分两组,第一组用SwaⅠ和AscⅠ双酶切,第二组用BamHⅠ和XbaⅠ双酶切,酶切回收的两组片段同时连接到pUC-RNAi载体(Functional analysis of citrus AP2transcription factors identified CsAP2-09 involved in citrus canker diseaseresponse and tolerance;柑橘溃疡病相关转录因子CsBZIP40的功能研究,贾瑞瑞,西南大学;CsWAKL08,a pathogen-induced wall-associated receptor-like kinase in sweetorange,confers resistance to citrus bacterial canker via ROS control and JAsignaling),得到的中间载体pUC-RNAi-CsGATA17和pLGNe超量表达载体用KpnⅠ和SalⅠ分别双酶切,将含有RNAi片段的酶切产物连接到pLGNe载体(柑橘超量表达CsNBS-LRR通过SA信号途径增强对溃疡病抗性;柑橘溃疡病相关基因CsPGIP的克隆与表达;柑橘响应溃疡病菌转录因子基因CsAP2-09的克隆与功能分析;Functional analysis of citrus AP2transcription factors identified CsAP2-09 involved in citrus canker diseaseresponse and tolerance;柑橘溃疡病相关转录因子CsBZIP40的功能研究,贾瑞瑞,西南大学;CsWAKL08,a pathogen-induced wall-associated receptor-like kinase in sweetorange,confers resistance to citrus bacterial canker via ROS control and JAsignaling),构建出最终的干扰载体pLGNe-CsGATA17-RNAi。载体构建所用限制性内切酶购自(THERMO)公司,按照使用说明操作。
用电激法将构建的干扰导入根癌农杆菌EHA105,具体操作如下:预先取冻存的EHA105根癌农杆菌感受态细胞(50μL),于冰上融化;加入2μL所构建的干扰载体于感受态细胞中,吹打混匀后,冰上放置5min。加入1mL LB液体培养基至电击杯中,移液枪吹打混匀,移至无菌离心管中,260r·min-1,28℃摇床振荡培养40min;10000r·min-1将菌液离心1min,弃上清液(剩约100μL重悬菌体),重悬后用移液枪打到LK固体培养基上,涂布均匀,28℃倒置暗培养2天。菌斑长出后,挑取单菌落至LK液体培养基中,恒温摇床上(28℃)振荡过夜。
3.遗传转化柑橘(晚锦橙)
根据图6所示的流程进行柑橘遗传转化,具体操作如下:
柑橘实生苗上胚轴的获得:取新鲜柑橘(晚锦橙)洗净,用70%酒精表面消毒,在无菌的条件下取出种子,剥掉种皮,接种在种子萌发培养基上萌发,28℃下暗培养3周,然后在16h光照/8h黑暗的光周期下培养1周。无菌条件下取萌发幼苗上胚轴切成1cm左右的茎段,用于根癌农杆菌的遗传转化。
根癌农杆菌的制备:用于转染的根癌农杆菌菌液(含CsGATA17干扰载体)加入80%的无菌甘油保存于-70℃的超低温培养箱中。转染前,在含50mg·L-1卡那霉素的LB固体培养基上划线培养。挑根癌农杆菌单菌落,接种于25mL含有相同抗生素的LB液体培养基中,28℃震荡培养过夜。次日,测浓度后将菌液稀释成OD600=0.1的菌液进行二摇,3h后,待菌液处于对数生长期(OD600=0.5)时,于5000r·min-1离心10min,弃上清,用PH=5.4的MS液体培养基重悬后用于转染。
柑橘上胚轴茎段的转化:将切成1cm左右的柑橘(晚锦橙)上胚轴茎段在根癌农杆菌中浸泡10min,期间轻微晃动。取出茎段后将表面的菌液吸干;将茎段转移到共培养培养基中,26℃暗培养3d。共培养完成后,将上胚轴转移到筛选培养基中,28℃暗培养7d,外植体在28℃、16h光照/8h黑暗培养,每两周继代一次。待幼苗长到1cm以上时,将GUS染色初筛阳性芽切下后嫁接到无菌试管晚锦橙苗,在成苗培养基中进行培养;待幼苗长到5cm左右时将其嫁接到枳实生苗上,在温室中进行培养。
用到的培养基如下:
种子萌发培养基:MS+30g·L-1蔗糖+2.5g·L-1Gelrite,PH=5.8。
共培养培养基:MS+2mg·L-1 BA+0.5mg·L-1 IAA+1mg·L-1 2,4–D+100μmol AS+30g·L-1蔗糖+2.5g·L-1Gelrite,PH=5.8。
筛选培养基:MS+2mg·L-1 BA+0.5mg·L-1 IAA+500mg·L-1Cef+50mg·L-1 Kan+30g·L-1蔗糖+2.5g·L-1Gelrite,PH 5.8。
成苗培养基:MS+30g·L-1蔗糖,PH=5.8。
4.转基因植株验证
转基因植株使用GUS染色、PCR检测、qRT-PCR方式进行验证,具体方法如下:
GUS染色鉴定:将转基因植株叶尖切下,进行GUS组织化学染色,阳性植株叶尖边缘显蓝色,野生型植株叶尖不显色(如图7所示)。
PCR检测:取转基因植株叶片100mg,使用艾德莱公司试剂盒(CAT:DN15)提取基因组DNA,PCR检测CsGATA17干扰片段的整合。PCR反应条件:94℃3min;94℃30s,58℃30s,72℃30s,30次循环;72℃10min。检测引物为ID-CsGATA17-F(SEQ ID NO:5)和ID-CsGATA17-R(SEQ ID NO:6)。阳性植株可以得到约525bp的扩增片段,而对照植株无扩增(如图8所示)。
qRT-PCR分析:提取转基因柑橘叶片总RNA并反转录合成cDNA。目的基因的检测引物为RT-CsGATA17-F(SEQ ID NO:7)和RT-CsGATA17-R(SEQ ID NO:8)。反应体积20μL,反应条件:95℃3min,94℃10s;56℃10s,72℃10s,40次循环;72℃10min。实验重复三次。采用2-△△Ct法计算转基因植株中CsGATA17基因的相对表达量:定义水处理的样本为参照因子,即其CsGATA17的表达水平为1,计算转基因柑橘中相对参照因子基因表达的倍数2-△△Ct,为其相对表达量。结果显示,CsGATA17基因转录水平在转基因植株中相比野生型植株被很大程度的降低,表达量最高下调至野生型对照的39%(如图9所示)。
5.转基因植株表型观察
观察转基因植株表型,并未发现明显差异。干扰CsGATA17基因并未对植株的表型和发育产生明显影响(如图10所示)。
6.转基因植株的抗性评价
采用离体针刺法对转基因植株进行溃疡病抗性评价,具体操作如下:采集成熟叶片清洗后用75%的酒精消毒后置于超纯水中冲洗;以叶脉为中心进行针刺;用移液器点样溃疡病菌液,每针孔点样1μL(1X105CFU·mL-1)。于28℃恒温光照培养箱中培养(16h光照/8h黑暗)。叶片点菌后培养10天拍照,用Image J V1.47软件统计病斑大小。根据病情指数公式计算病情指数。按照病斑面积将病情分为0-7级,以字母R表示病斑面积0级(R≤0.5mm2),1级(0.5mm2<R≤1.0mm2),2级(1.0mm2<R≤1.5mm2),3级(1.5mm2<R≤2.0mm2),4级(2.0mm2<R≤2.5mm2),5级(2.5mm2<R≤3.0mm2),6级(3.0mm2<R≤3.5mm2),7级(R>3.5mm2)。根据公式计算发病程度:DI=100×Σ【各级病斑数×相应级数值】/(病斑总数×最大级数)。接种溃疡病菌10天后,发现转基因植株和野生型对照的发病症状差异较大(如图11所示),转基因植株叶片上的病斑面积(如图12所示)和病情指数(如图13所示)均明显小于野生型对照,病斑面积最多降为野生型柑橘叶片的54%,病情指数为野生型的48%。所以,CsGATA17干扰可大大提高柑橘对溃疡病的抗性。
由此可见,CsGATA17的干扰可很大程度上降低溃疡病的病斑面积,减轻溃疡病的发病程度。该基因可以独立用作抗病分子育种,也可以与其他的抗病或感病基因一起进行柑橘抗溃疡病分子育种。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (12)
1.一种柑橘CsGATA17基因,其特征在于,所述柑橘CsGATA17基因的核苷酸序列如SEQID NO:1所示。
2.一种柑橘CsGATA17基因提高柑橘溃疡病抗性的方法,其特征在于,降低柑橘属植物中CsGATA17的转录水平,所述柑橘CsGATA17基因的核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求2所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,采用RNA干扰的方式降低柑橘中CsGATA17基因的转录水平。
4.根据权利要求2所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,具体方法包括以下步骤:
步骤1:克隆柑橘CsGATA17编码基因的RNAi片段;
步骤2:构建干扰载体;
步骤3:干扰载体转化柑橘,得到转基因植株。
5.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,步骤1中,所述RNAi片段的核苷酸序列如SEQ ID NO:2所示。
6.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,步骤1中的克隆方法为:提取柑橘总RNA,然后反转录为cDNA,最后采用高保真酶PCR扩增得到CsGATA17编码基因的RNAi片段。
7.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,克隆CsGATA17编码基因的RNAi片段所采用的引物为CsGATA17-RNAi-F和CsGATA17-RNAi-R;CsGATA17-RNAi-F的核苷酸序列如SEQ ID NO:3所示;CsGATA17-RNAi-R的核苷酸序列如SEQ ID NO:4所示。
8.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,步骤2中干扰载体的构建方法为:步骤1中获得的PCR产物分两组,第一组用SwaⅠ和AscⅠ双酶切,第二组用BamHⅠ和XbaⅠ双酶切,酶切回收的两组片段同时连接到pUC-RNAi载体,得到的中间载体pUC-RNAi-CsGATA17和pLGNe超量表达载体用KpnⅠ和SalⅠ分别双酶切,将含有RNAi片段的酶切产物连接到pLGNe载体上,构建出最终的干扰载体pLGNe-CsGATA17-RNAi。
9.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,步骤3中,干扰载体转化柑橘的方法为:干扰载体通过电击法转化根癌农杆菌,再用根癌农杆菌介导转化柑橘外植体,遗传转化后的外植体细胞再经离体培养、染色鉴定、嫁接后得到转基因植株。
10.根据权利要求4所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,步骤3中得到转基因植株后,对转基因植株进行抗性评价,判定CsGATA17干扰提高了柑橘溃疡病抗性。
11.根据权利要求10所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,对转基因植株进行抗性评价前,通过PCR验证转基因植株,采用的引物为ID-CsGATA17-F和ID-CsGATA17-R;ID-CsGATA17-F的核苷酸序列如SEQ ID NO:5所示,ID-CsGATA17-R的核苷酸序列如SEQ ID NO:6所示。
12.根据权利要求11所述的一种柑橘CsGATA17基因增强柑橘溃疡病抗性的方法,其特征在于,PCR验证后,用qRT-PCR验证转基因植株中CsGATA17的转录水平,采用的引物为RT-CsGATA17-F和RT-CsGATA17-R;RT-CsGATA17-F的核苷酸序列如SEQ ID NO:7所示,RT-CsGATA17-R的核苷酸序列如SEQ ID NO:8所示。
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