CN111635455B - 一类植物抗病相关蛋白及其应用 - Google Patents
一类植物抗病相关蛋白及其应用 Download PDFInfo
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- CN111635455B CN111635455B CN202010431120.2A CN202010431120A CN111635455B CN 111635455 B CN111635455 B CN 111635455B CN 202010431120 A CN202010431120 A CN 202010431120A CN 111635455 B CN111635455 B CN 111635455B
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Abstract
本发明涉及植物生物技术领域,具体涉及一类植物抗病相关蛋白及其应用。本发明发现了一种植物抗病相关蛋白CCP,该蛋白属于铜伴侣蛋白,拟南芥中的CCP蛋白序列如SEQ ID NO.1所示。在植物中提高CCP蛋白的表达量能够显著提高植物对病原体的广谱抗病性,可单独或与其他植物药剂联合用于植物病害的防治,有利于提高植物的产量和质量。
Description
技术领域
本发明涉及植物生物技术领域,具体涉及具有提高植物抗病性功能的一类植物抗病相关蛋白、核酸及其应用。
背景技术
植物在生长过程中会受到很多病原体攻击,植物细胞通常具有几道屏障的防御抵抗。植物的免疫防御系统主要分为先天性免疫和获得性免疫。先天性免疫主要分为两种:PTI(PAMP-triggered immunity)和ETI(Effector triggered immunity)。当植物通过PTI免疫机制对病原物进行抵抗后,病原物为了能够更好地侵染宿主,会向植物的体内注入抑制PTI的效应因子,进一步增强病原物对宿主的侵染。植物体的免疫系统为了匹配这种进化机制,进化出了R(resistance proteins)基因,该基因能够直接或间接地识别这些效应因子,进而激发ETI免疫机制以抵御病原物的侵染。植物的获得性抗性(Systemic acquiredresistance,SAR)是一种被病原物所诱导的植物免疫机制,其不仅仅是特异性针对初侵染点的免疫作用,而是一种广谱的免疫性抗病机制。开发参与植物免疫调节,能够提高植物抗病能力的基因或蛋白对于植物病害防治具有重要意义。
发明内容
本发明的目的之一是提供一种植物抗病相关蛋白CCP及其编码基因,该蛋白属于铜伴侣蛋白。本发明的另一目的是提供该植物抗病相关蛋白的应用。
本发明通过对病毒侵染植物进行转录组测序分析,靶定了可能参与植物免疫调节的基因,通过筛选和功能验证,发现CCP蛋白(拟南芥的CCP蛋白的氨基酸序列如SEQ IDNO.1所示)能够显著提高植物的抗病性。
CCP蛋白属于铜伴侣蛋白家族。铜离子是植物正常生长发育必不可少的金属离子,是植物体内重要生理过程酶(离子运输、激素合成和体内自由基清除等重要生理)的组分。铜离子的运输、定位和功能发挥均需要铜离子结合蛋白(铜伴侣蛋白)的作用。
拟南芥(Arabidopsis thaliana)CCP的氨基酸序列如SEQ ID NO.1所示,高粱(Sorghum bicolor)CCP的氨基酸序列如SEQ ID NO.2所示,水稻(Oryza sativa IndicaGroup)CCP的氨基酸序列如SEQ ID NO.3所示,短柄草(Brachypodium distachyon)CCP的氨基酸序列如SEQ ID NO.4所示,小麦(Triticum aestivum)CCP的氨基酸序列如SEQ ID NO.5所示,大麦(Hordeum vulgare)CCP的氨基酸序列如SEQ ID NO.6所示,玉米(Zea mays)CCP的氨基酸序列如SEQ ID NO.7所示,大豆(Glycine max)CCP的氨基酸序列如SEQ ID NO.8所示,油菜(Brassica napus)CCP的氨基酸序列如SEQ ID NO.9所示,棉花(Gossypiumraimondii)CCP的氨基酸序列如SEQ ID NO.10所示,蒺藜苜蓿(Medicago truncatula)CCP的氨基酸序列如SEQ ID NO.11所示,本生烟(Nicotiana benthamiana)CCP的氨基酸序列如SEQ ID NO.12所示。
上述植物抗病相关蛋白可从各植物中分离获得。
基于本发明的上述发现,本发明具体提供如下技术方案:
第一方面,本发明提供一种植物抗病相关蛋白,其具有如SEQ ID NO.1-12任一所示的序列,或者,具有如SEQ ID NO.1-12任一所示的序列经缺失、替换或添加一个或多个氨基酸得到的具有提高植物抗病性功能的蛋白的氨基酸序列。
本发明所述的植物抗病相关蛋白CCP主要包括两个结构域:N端的CBS结构域和C端的NLS结构域,其中,CBS结构域为铜离子结合区域,是CCP蛋白进行铜离子运输的重要结构区域;NLS结构域为核定位信号区域。
本领域技术人员可通过对所述植物抗病相关蛋白的关键结构域以外的氨基酸序列进行替换、缺失或插入一个或多个氨基酸,获得具有相同功能的植物抗病相关蛋白。
本发明还提供编码所述植物抗病相关蛋白的核酸。
本发明所述的核酸可以从任何植物中分离获得,包括但不限于拟南芥、玉米、水稻、大豆等。
以拟南芥为例,所述核酸具有如下任一核苷酸序列:
(1)如SEQ ID NO.13或14所示的核苷酸序列;
(2)在严格条件下能够与(1)所述序列杂交的核酸序列;
(3)与(1)所述序列互补的核苷酸序列;
(4)在(1)所述序列的基础之上,经过一个或多个碱基的替换、插入或缺失得到的具有提高植物抗病性功能的核酸的序列。
本发明提供所述核酸的生物材料,所述生物材料为表达盒、载体、宿主细胞或转基因细胞系。
第二方面,本发明提供所述植物抗病相关蛋白或所述核酸或所述生物材料在调控植物免疫力或抗病性中的应用。
本发明提供所述植物抗病相关蛋白或所述核酸或所述生物材料在抗病植物遗传育种中的应用。
本发明提供所述植物抗病相关蛋白或所述核酸或所述生物材料在植物病害防治中的应用。
第三方面,本发明提供一种抗病转基因植物的构建方法,包括:提高植物中序列如SEQ ID NO.1-12任一所示的植物抗病相关蛋白的表达量。
本发明还提供一种防治植物病害的方法,包括:提高植物中序列如SEQ ID NO.1-12任一所示的植物抗病相关蛋白的表达量。
本发明的植物抗病相关蛋白可单独或与其他植物病害防治手段联用。
在现有的植物病害防治方法中,含有铜离子的药剂(例如:波尔多液)通过破环作物表面的病原菌结构,发挥抑制和防止水果和植物表面的病害的功能。为进一步提高植物病害防治的效果,可在提高所述植物中序列如SEQ ID NO.1-12任一所示的植物抗病相关蛋白的表达量的同时,给植物施用含铜离子的抗病药剂。采用这种联合防治的方法既可以通过提高植物自身的免疫抗性进行防治,又可以通过波尔多液的外用破环作物表面的病原体结构进行防治,达到“内外兼治”的效果,可更好地提高植物病害防治效果。
所述含铜离子的抗病药剂可为波尔多液。
本发明中,所述植物包括但不限于拟南芥、水稻、短柄草、玉米、小麦、高梁、棉花、大豆、花生、大麦、油菜、棉花、蒺藜苜蓿、本生烟、燕麦、黑麦、小米、香蕉、香瓜、苹果、黄瓜、甜菜等。
第五方面,本发明提供所述植物抗病相关蛋白的制备方法,包括:将所述植物抗病相关蛋白的编码基因导入宿主细胞中,表达所述植物抗病相关蛋白。
具体地,所述制备方法包括:
(1)扩增所述植物抗病相关蛋白的编码基因;
(2)构建携带所述植物抗病相关蛋白的编码基因的表达载体;
(3)将所述表达载体转化到宿主细胞;
(4)培养宿主细胞,获得所述植物抗病相关蛋白。
本发明的有益效果在于:本发明发现了一种植物抗病相关蛋白CCP,该蛋白为铜伴侣蛋白,在植物中提高CCP蛋白的表达量能够显著提高植物对多种病原体(假单胞菌、CMV、TuMV、BSMV等)的抵抗力,而将植物中的CCP基因敲除,则会导致植物抗病力的显著下降。CCP蛋白具有广谱的抗病性,可抵抗包括假单胞菌等病原菌以及CMV、TuMV、BSMV等植物病毒对植物的侵染以及在植物体内的增殖,有效提高植物的广谱抗病性。
附图说明
图1本发明实施例1中巢式PCR和CCP序列分析结果,其中,A为琼脂糖凝胶电泳显示3'RACE和5′RACE的结果,其中M代表琼脂糖凝胶电泳的marker;B为鉴定CCP的cDNA的核苷酸序列和氨基酸序列图。
图2为本发明实施例1中将拟南芥的CCP、ATX1、CCH和CCS进行氨基酸序列比对的结果,其中,横线标注的为保守的铜离子结合位点(CBS)。
图3为本发明实施例1中CCP蛋白序列和人的铜伴侣蛋白序列的比对,其中,黑线标注的CBS代表铜离子结合位点,NLS代表预测的核定位信号区域。
图4为本发明实施例1中CCP在不同植物中同源蛋白的系统发育树,基因的登录号或基因的位置均标注在植物物种名称的后面,CCP用加粗的字体标出,图中的标尺是0.05,Dicots代表双子叶植物,Monocots代表单子叶植物。
图5为本发明实施例2中载体pMDC32-3×Flag的载体图谱。
图6为本发明实施例2中CCP过表达植株中CCP的mRNA和蛋白水平检测结果,其中,A为RT-PCR检测CCP超表达转基因植株中CCPmRNA的积累水平,Col-0为野生型拟南芥,OE-1到OE-10为不同的CCP超表达转基因植株,Actin是内参基因;B为Western blotting检测CCP超表达转基因株系OE-CCP-3和OE-CCP-4的CCP蛋白积累水平,以Flag标签的特异性抗体进行蛋白检测,loading control是植物体内蛋白的定量。
图7为本发明实施例2中CCP敲除突变体植株的构建和鉴定结果,其中,A为构建ccp-1和ccp-2的突变体植株的靶点在CCP基因cDNA上的位置示意图,黑色框代表编码区,灰色线条代表非编区域;B为利用RT-PCR对突变体ccp-1和ccp-2的进行鉴定,其中Col-0是以野生型拟南芥为模板进行的PCR;C和D分别为ccp-1和ccp-2的测序结果,将Col-0与ccp-1和ccp-2的测序结果进行序列比对,ccp-1中的CCP基因缺失了445个核苷酸序列,ccp-2中的CCP基因插入了一个T核苷酸序列。
图8为本发明实施例3中拟南芥对丁香假单胞杆菌的抗性分析结果,其中,A为野生型拟南芥Col-0、npr1-1以及CCP过表达植株CCPOE-和CCPOE-接种缓冲液(Mock,对照)和接种Pst DC3000(infection)的72h后的表型,图中的标尺是2厘米;B为野生型拟南芥Col-0、npr1-1以及CCP敲除突变体ccp-1和ccp-2接种缓冲液(Mock,对照)和接种Pst DC3000(infection)的72h后的表型,图中的标尺是2厘米;C为PstDC3000在Col-0、npr1-1、CCPOE-3和CCPOE-4叶片上生长48h后的数量;D为Pst DC3000在Col-0、npr1-1、ccp-1和ccp-2叶片上接种48h后的数量;C和D中,Pst DC3000起始的菌落数OD600=0.0002,CFU/cm2是每cm2叶片上的菌落生长单位。
图9为本发明实施例3中拟南芥对CMV和TuMV的抗性分析,其中,A为野生型拟南芥Col-0、CCP过表达植株CCPOE-3、CCPOE-4以及CCP敲除突变体ccp-1和ccp-2接种CMV-2blm(25μg/mL)病毒粒子12d后的表型,图中的标尺是1厘米,Mock为未接种病毒的对照;B为RT-PCR检测Col-0、CCPOE-3、CCPOE-4、ccp-1和ccp-2接种CMV-2blm后样品中CCP mRNA积累量,表明CCP的转基因植株中CCP的转录水平较高,,其中Actin是内参基因;C为Western blotting检测CMV-2blm的积累量,用CMV的CP蛋白的特异性抗体及进行检测,loading control代表植物体内蛋白的定量;D为Col-0、CCPOE-3、CCPOE-4、ccp-1和ccp-2注射接种TuMV-GFP(OD600=0.5)10d(10)和13d(13)后的表型;E为RT-PCR检测Col-0、CCPOE-3、CCPOE-4、ccp-1和ccp-2接种TuMV-GFP后样品中CCP mRNA的积累量,表明CCP的转基因植株中CCP的转录水平较高。Actin是内参基因;F为Western blotting检测TuMV-GFP的积累量,用GFP蛋白的特异性抗体进行检测,loading control代表植物体内蛋白的定量。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用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。
实施例1 CCP基因的获得
通过对接种黄瓜花叶病毒(CMV)一周的拟南芥植株进行转录组测序,对转录组测序结果进行分析发现基因AT4G05030部分转录本的表达量相较于对照(MOCK)上调。利用巢式PCR进行检验,结果表明表达量上调的转录本是基因AT4G05030的一部分(图1的A)。将该基因命名为CCP,拟南芥中CCP的全长氨基酸序列如SEQ ID NO.1所示,cDNA的核苷酸序列如SEQ ID NO.13所示,基因组核苷酸序列如SEQ ID NO.14所示。根据CCP基因的序列设计合成扩增引物。
对CCP进行氨基酸序列和核苷酸序列的分析,CCP蛋白由77个氨基酸组成,CCP的cDNA含有234个核苷酸(图1的B)。通过序列分析发现CCP含有一个保守的铜离子结合区域CBS(图2)。通过与人的铜伴侣蛋白ATOX1比较发现,在CCP中还含有一个核定位信号区域NLS(图3)。通过不同植物中的CCP同源序列分析发现,CCP在很多的单子叶和双子叶中都是保守存在的(图4)。
实施例2 CCP基因的过表达以及突变体植株的构建
1、CCP基因的过表达植株的构建
(1)植物组织总RNA的提取
利用Trizol法提取植物组织总RNA,具体方法如下:采取植物的组织,用天平称取0.1g,加入液氮将植物的组织迅速研磨成粉末状,转移到2mL EP管中,加入1mL Trizol,利用振荡器将其充分混匀。向上述的溶液中加入200μL三氯甲烷,振荡20-30s,室温静置10min。4℃、12000rpm离心15min,将上清大约600μL转移到新的离心管中,加入600μL三氯甲烷,振荡混匀,室温静置10min。4℃、12000rpm离心15min,再次将上清大约500μL转移到新的离心管中,加入500μL异丙醇,混匀,室温放置30min。4℃、12000rpm离心20min,弃上清,所得的沉淀用70%冷乙醇和无水乙醇先后洗涤一次(4℃,12000rpm,5min),沉淀晾干后,加入50μL DEPC-H2O进行溶解。
(2)CCP基因扩增和真核表达载体的构建
利用步骤(1)提取的总RNA进行反转录反应,反应体系如表1所示。
表1反转录反应体系
先将总RNA、oligo dT(5’-TTTTTTTTTTTTTTTTTTTTT-3’),和DEPC-H2O加入,95℃变性5min后,置于冰上。再加入余下的试剂。PCR反应程序:42℃,60-90min;75℃,10min;25℃,5min。
以反转录得到的cDNA作为模板扩增CCP基因,使用的引物序列如下:
正向引物:5’-AGGCGCGCCATGGCCAAGAAAATCTTGAT-3’,(含AscI位点);
反向引物:5’-GACTAGTAGTGTCGTCAACATCTGTGAC-3’,(含SpeI位点)。
扩增体系:模板DNA,特异性引物各1μL(10mM),25μL高保真2×Phanta Max MasterMix,用灭菌去离子水补至50μL并混匀。
反应程序:95℃预变性5min,之后每个循环为95℃变性20s,退火30s(退火温度根据Tm值而定),72℃延伸适当时间20-30s/1000bp,25-30个循环,72℃充分延伸10min,25℃,1min。
将获得的PCR片段连接到pMDC32-3×Flag载体(Wang X,Cao X,Liu M,etal.Hsc70-2 is required for Beet black scorch virus infection throughinteraction with replication and capsid proteins.Scientific reports,2018,8(1):4526)(图5),获得一个携带CCP基因的重组载体pMDC32-CCP。
(3)拟南芥的转基因
利用农杆菌浸染法将重组载体pMDC32-CCP转化拟南芥。
农杆菌浸染液的配制如表2所示。
表2浸染液配方
农杆菌的培养与花序的浸染方法具体如下:
用枪头挑去活化的含有目的质粒的农杆菌,加入到4mL LB液体培养基(含有相应的抗生素)中,28℃、220rpm摇菌过夜。将小摇的菌液按照1:100比例加入到250mL的LB液体培养基(含有相应抗生素)中进行扩大培养,28℃、220rpm摇菌过夜,6000×g、10min离心收集菌体,舍弃上清,用表2中的浸染液重悬菌体。把菌液转移到烧杯中,使植株的花序倒置放在浸染液中,放置2min,将植株平放于黑盘中(保湿处理),黑暗处理24h后,重新放置于光下。筛选阳性的转基因植株,进性RNA水平和蛋白水平的检测,结果如图6所示。
2、CCP基因的突变植株的构建
利用CRISPR-Cas9技术对CCP的基因进行编辑(Xing et al.,2014A CRISPR/Cas9toolkit for multiplex genome editing in plants.BMC Plant Biol.14,327.)。在https://www.genome.arizona.edu/crispr/CRISPR search.htmL网站上查找基因CCP的靶点TCTTGATGTCGGTAAGTATGAGG(图7的A),针对该靶点序列对CCP的基因进行编辑。通过提取DNA筛选(如图7的B)以及测序验证(如图7的C和D)筛选CCP基因编辑的拟南芥CCP基因敲除突变株系(ccp)。
实施例3 CCP基因的过表达以及敲除突变体植株的抗性分析
1、对丁香假单胞杆菌的抗性分析
分析实施例2构建的CCP基因的过表达植株和敲除突变体植株对丁香假单胞杆菌的抗性,具体方法如下:
将保存在-80℃冰箱的丁香假单胞杆菌Pst.DC3000菌种在含有利福平(25μg/mL)抗生素的KB培养基上活化,在30℃温箱培养2-3d,挑取单菌落,在含有利福平(25μg/mL)抗生素液体KB培养基中进行培养过夜。800rpm离心10min,收集菌体,弃上清,用10mM的MgCl2重悬菌体。将菌液稀释到OD600=0.0002,用注射器将菌液注射到叶片中,接种量为每平方厘米叶片上大约接种1000cfu。盖上透明的塑料盖子保湿以有利于细菌的侵染。以接种10mM的MgCl2缓冲液作为对照。
结果显示,在野生型拟南芥Col-0、突变体npr1-1、CCP的超表达株系和突变体ccp上接种缓冲液的植株长势良好(图8的A和B);在接种Pst DC3000的野生型拟南芥Col-0、突变体npr1-1、CCP的超表达株系和突变体ccp中,与Col-0相比,CCPOE-3和CCPOE-4株系的接种叶片中Pst DC3000细菌的积累量明显降低,而突变体株系ccp-1和ccp-2接种叶片中的PstDC3000细菌的积累量则显著提高(图8的C和D)。结果表明,CCP能够增强拟南芥对丁香假单胞菌的抗病性。
2、对CMV和TuMV的抗性分析
分析实施例2构建的CCP基因的过表达植株和敲除突变体植株对CMV和TuMV的抗性,具体方法如下:
将储存提取CMV的病毒粒子用缓冲液稀释到要使用的浓度,放置到冰上,将病毒粒子滴到喷洒了硅藻土的叶子上,进行摩擦接种。10min后,在接种叶片上喷洒水,盖上透明罩子进行保湿。
将含有TuMV-GFP的侵染性克隆通过农杆菌介导的方法注射到生长5周的本生烟叶片中,注射7-8d之后,用紫外灯观察GFP荧光。采取注射本生烟的叶片0.1g,加入200μL的磷酸缓冲液(10mM NaH2PO4/Na2HPO4,pH=7.4,0.2%PVP-40),取汁液摩擦接种拟南芥。
拟南芥的幼苗上接种CMV-2blm(25μg/mL),12天后观察拟南芥接种后的表型(图9的A);RT-PCT检测mRNA积累水平(图9的B);采取接种后拟南芥的系统叶,提取植物的总蛋白进行Western blotting检测CMV-2blm病毒粒子在叶片内的积累量(图9的C)。结果显示,CCP超表达植株相较于野生型拟南芥系统叶片皱缩卷曲较轻,症状较弱。突变体ccp植株接种病毒后发病相较于野生型的系统叶片皱缩卷曲症状更加严重。RT-PCR检测结果表明CCP的超表达转基因植株中CCP的积累量较高。检测CMV-2blm病毒粒子在接种拟南芥叶片中的积累量,结果表明,与野生型拟南芥相比,在CCP的超表达转基因植株中CMV-2blm病毒粒子的积累量显著降低,在突变体ccp中的积累量显著提高,与表型相一致。
对生长4周的拟南芥叶片接种TuMV-GFP,注射接种的OD600是0.5,大约是2000000个菌落,接种10d和13d后紫外灯下观察GFP蛋白的表达情况(图9的D);RT-PCT检测mRNA积累水平(图9的E);提取植物的总蛋白进行Western blotting检测GFP在叶片内的积累量(图9的F)。结果显示,在接种TuMV-GFP 10d和13d后GFP在CCP转基因拟南芥上的面积相较于野生型拟南芥显著缩小,GFP在突变体ccp植株上的积累量相较于野生型拟南芥的面积显著增大。通过RT-PCR检测发现CCP的超表达转基因植株中CCP基因的表达量较高。GFP在侵染叶片上的积累量检测结果表明,与野生型植株相比,在CCP超表达转基因植株样品中GFP的积累量显著降低,与表型相一致。
以上结果表明,CCP能够增强植物对CMV和TuMV的抗性。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
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<120> 一类植物抗病相关蛋白及其应用
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Claims (7)
1.植物抗病相关蛋白或编码所述植物抗病相关蛋白的核酸或含有所述核酸的生物材料在调控植物免疫力或抗病性中的应用,所述植物抗病相关蛋白的氨基酸序列如SEQ IDNO.1所示;
所述生物材料为表达盒、载体、宿主细胞或转基因细胞系。
2.植物抗病相关蛋白或编码所述植物抗病相关蛋白的核酸或含有所述核酸的生物材料在抗病植物遗传育种中的应用,所述植物抗病相关蛋白的氨基酸序列如SEQ ID NO.1所示;
所述生物材料为表达盒、载体、宿主细胞或转基因细胞系。
3.植物抗病相关蛋白或编码所述植物抗病相关蛋白的核酸或含有所述核酸的生物材料在植物病害防治中的应用,所述植物抗病相关蛋白的氨基酸序列如SEQ ID NO.1所示;
所述生物材料为表达盒、载体、宿主细胞或转基因细胞系。
4.一种抗病转基因植物的构建方法,其特征在于,提高植物中序列如SEQ ID NO.1所示的植物抗病相关蛋白的表达量。
5.一种防治植物病害的方法,其特征在于,提高植物中序列如SEQ ID NO.1所示的植物抗病相关蛋白的表达量。
6.根据权利要求5所述的方法,其特征在于,同时给植物施用含铜离子的抗病药剂。
7.根据权利要求6所述的方法,其特征在于,所述含铜离子的抗病药剂为波尔多液。
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