CN110295179B - 芜菁抗病相关基因BrPGIP8及其应用 - Google Patents
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Abstract
本发明提供了芜菁抗病相关基因BrPGIP8及其应用,属于植物基因工程技术领域。所述芜菁BrPGIP8的DNA序列如SEQ ID No.1所示。通过农杆菌浸花法将该基因转化至哥伦比亚型拟南芥中,得到BrPGIP8异源表达拟南芥株系,结果发现,芜菁BrPGIP8的异源表达会导致拟南芥对菌核病的抗性出现提升,该表型是由于BrPGIP8的编码蛋白分别与菌核病的致病菌即核盘菌中SsPG3和SsPG5编码的多聚半乳糖醛酸酶的特异结合所致。这表明芜菁BrPGIP8与植物抗核盘菌病关系密切,可将该基因应用于白菜类及其他园艺植物育种,具有良好的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域,具体的说,涉及芜菁抗病相关基因BrPGIP8、其编码蛋白及其在植物抗病过程中的应用。
背景技术
芜菁(Brassica rapa L.syn.B.campestris L.)属十字花科芸薹属芸薹种作物,主要包含有结球白菜、不结球白菜、芜菁、菜薹、紫菜薹、薹菜、乌塌菜、日本水菜等8个变种。它们不仅是重要的蔬菜和油料作物,在生产上有较高的经济价值,而且与十字花科模式植物拟南芥有较近的亲缘关系,在植物基础科学中也有重要研究意义。核盘菌(Sclerotiniasclerotiorum(Lib.)de Bary)是一种兼性真菌,由其引起的菌核病(S.sclerotiorumstemrot,SSR)是一种在世界范围内广泛分布的植物病害,对十字花科作物的生长发育造成严重威胁。
植物细胞壁是植物抵御病原菌入侵的一道重要屏障,而果胶质是其重要组成成分。病原菌微生物对植物的定殖首先需要穿过细胞壁,为此,病原菌会在侵染植物的早期阶段时分泌多种细胞壁降解酶(Cell wall degrading emzymes,CWDEs)以破坏细胞壁结构,如果胶酶、木聚糖酶、纤维素酶、PGs等。PGs是糖苷水解酶家族28(GH28)的一员,它可以切断聚半乳糖醛酸(homogalacturonan,HG)中D-半乳糖醛酸残基之间的α-(1-4)糖苷键,从而破坏细胞壁,进一步导致寄主组织细胞分离和浸渍,为病原菌侵染创造有利条件。
而针对病原菌分泌的细胞壁降解酶类,植物也进化出了一系列相对应的抑制蛋白。多聚半乳糖醛酸酶抑制蛋白(PGIPs)即是其中的一种。它可以特异性结合病原菌分泌的PGs从而抑制其对细胞壁的降解。目前PGIPs-PGs互作已经成为经典的蛋白互作模式。同时,PGIPs与PGs的互作可以促进植物体内产生并累积OGs,进而激活下游特定的免疫响应,最终抑制病原菌的侵染。
发明内容
本发明的目的是针对现有技术的不足,提供芜菁抗病相关基因BrPGIP8及其应用。
本发明提供了一种多聚半乳糖醛酸酶抑制蛋白编码基因BrPGIP8,该基因为:从来源于品种为‘ECD-04’的芜菁中克隆得到的基因,其具有:
1)SEQ ID No.1所示的核苷酸序列;或
2)SEQ ID No.1所示的核苷酸序列经取代、缺失和/或增加一个或几个核苷酸;
本发明提供了含有上述芜菁抗病相关基因BrPGIP8的生物材料,所述生物材料为表达载体,表达盒,宿主细胞或工程菌。
本发明提供了上述芜菁抗病相关基因BrPGIP8在激发植物抗菌核病中的应用。
本发明提供了上述芜菁抗病相关基因BrPGIP8在制备转基因植物中的应用。
发明还提供了上述芜菁抗病基因BrPGIP8在植物种质资源改良中的应用。
本发明提供的芜菁抗病相关基因BrPGIP8序列如SEQ ID No.1所示。通过农杆菌介导法将该基因导入拟南芥中,获得芜菁抗病相关基因BrPGIP8异源表达的转基因拟南芥株系,结果发现,芜菁抗病相关基因BrPGIP8的表达变化可以抑制核盘菌在拟南芥叶片上形成病斑,从而明显提升植株对菌核病的抗性。这表明了芜菁抗病相关基因BrPGIP8与植物抗菌核病关系密切,将该基因应用于白菜或其他十字花科蔬菜育种,具有良好的应用前景。
附图说明
图1为芜菁抗病相关基因BrPGIP8克隆PCR电泳图。其中左泳道为markerFermentasSM0321,右泳道为目的片段。
图2为BrPGIP4表达特征分析结果。
图3为BrPGIP8与核盘菌SsPG基因在双分子荧光素酶互补(BiFC)实验中的互作荧光信号。A、B阳性对照。C、D阴性对照。E、F 35S:2YN-BrPGIP8与35s:2YC-SsPG1注射后48h的荧光信号。G、H 35S:2YN-BrPGIP8与35s:2YC-SsPG3注射后48h的荧光信号。I、J 35S:2YN-BrPGIP8与35s:2YC-SsPG5注射后48h的荧光信号。K、L 35S:2YN-BrPGIP8与35s:2YC-SsPG6注射后48h的荧光信号。
图4为BrPGIP8与核盘菌SsPG基因在分离单荧光素酶互补(Luc)实验中的互作荧光信号。A阳性对照。B阴性对照。C 35S:SsPG3-NLUC与35S:CLUC-BrPGIP8的互作荧光信号检测。D 35S:SsPG5-NLUC与35S:CLUC-BrPGIP8的互作荧光信号检测。
图5为BrPGIP8过表达及载体示意图。
图6为转基因菜心植株的PCR阳性检测,从左到右依次,泳道1~3为三条引物对OE-1的叶片DNA为模板的PCR产物。泳道5~7为三条引物对OE-2叶片DNA为模板的PCR产物。泳道9~11为三条引物对OE-3叶片DNA为模板的PCR产物。13~15为三条引物对空载体作为模板的PCR产物。泳道16为阴性对照。
图7为转基因拟南芥株系中BrPGIP8的相对表达量示意图。
图8为BrPGIP8异源表达植株与对照植株生长状况比较。A中左边为对照植株,右边为BrPGIP8异源表达植株。
图9为BrPGIP8异源表达植株与对照植株花的抗病性状比较。其中,A、D、G、J、M分别为CK于核盘菌侵染后0h、24h、48h、72h、96h的发病情况。B、E、H、K、N分别为OE-1于核盘菌侵染后0h、24h、48h、72h、96h的发病情况。C、F、I、L、O分别OE-3于核盘菌侵染后0h、24h、48h、72h、96h的发病情况。
具体实施方式
下面通过具体实施例对本发明进行说明,实施例中未作详细描述的技术手段属于本领域专业技术人员熟知的常规技术。实施例只用于说明本发明,但不限制本发明的范围,任何本领域的技术人员在不付出创造性劳动的情况下,以本发明的实施例为基础所获得的其他实施例均属于本发明的保护范围。
本发明实施例提供了一种芜菁抗病相关基因BrPGIP8,该基因为:从来源于品种为‘ECD-04’(B.rapa ssp.rapifera line AABBCC,由国家农作物基因库引进)的芜菁中克隆到的基因,其基因序列如SEQ ID No.1所示。
本发明实施例还提供了上述芜菁BrPGIP8在调控拟南芥抗菌核病中的应用,下面对其进行具体描述。
实施例1:芜菁抗病相关基因BrPGIP8异源表达及抑制表达载体的构建
1.1异源表达载体构建
(1)取芜菁‘ECD-04’的叶片组织样品用TRIzol试剂提取总RNA,cDNA的合成用TAKARA反转录试剂盒完成,具体方法如下:5×gDNA Eraser Buffer 2μL,gDNA Eraser 1μL,RNA 1μg,RNase Free H2O补足至10μL,42℃,2min去除基因组DNA,在上一步的反应液中加入5×Primer Script Buffer 4μL,RT Primer Mix 1μL,Primer Script RT EnzymeMix1μL,RNase Free H2O 4μL,吸打混匀后37℃20min,85℃5s即完成cDNA的合成,cDNA在-20℃冰箱保存。
(2)以芜菁叶片cDNA为模板,用表1中的引物通过高保真酶扩增获得BrPGIP8基因目的片段(图1),并对BrPGIP4在不同组织器官中的表达特征进行了分析,结果表明,该基因在茎中的表达丰度最高(图2)。同时,对BrPGIP8与核盘菌SsPG基因在双分子荧光素酶互补(BiFC)实验中观察到互作荧光信号(图3),并通过分离单荧光素酶互补(Luc)实验对蛋白的互作关系进行了验证(图4)。
随后,将图1中的目的片段回收后连接P-clone并测序确认获得目的序列(SEQ IDNo.1)后,以其为模板再次扩增回收后,用相应的限制性内切酶酶切。酶切体系如下:Buffer4μL,扩增回收的产物约2μg,Xba I和BamH I酶各2μL,双蒸水补足至40μL。37℃水浴1h后回收酶切产物。pBI121载体也用同样方法酶切并回收获得pBI121空载体于-20℃保存备用。将酶切并回收获得的pBI121空载体直接与基因的酶切产物反应连接,反应体系如下:10×Buffer 1μL,T4连接酶1μL,加入基因片段与载体片段的摩尔比约为3:1,总量约为0.5μg。双蒸水补足至10μL,4℃反应连接过夜后转化大肠杆菌。取阳性菌落PCR检测并测序证明连接正确后,菌液扩繁并抽提质粒获得pBI121-BrPGIP8载体(图5),于-20℃保存备用。
表1异源表达载体构建所用引物
实施例2拟南芥浸花法转化
2.1将实施例1中得到的载体pBI121-BrPGIP8载体及pBI121空载体分别转入农杆菌GV3101中,具体方法如下:取解冻的农杆菌感受态分别与5μL pBI121-BrPGIP8载体及pBI121空载体质粒混匀后,冰浴10min,液氮中反应5min,28℃水浴5min;在超净工作台上加入1mL不含任何抗生素的液体LB培养基,28℃摇床200rpm培养4~5h;离心10,000rpm,1min,弃大部分上清,剩余约100μL将菌体重悬后涂于含Rif(50mg·L-1)和Kan(50mg·L-1)的固体LB平板上;28℃正向放置30min后倒置培养1~2d。取阳性菌落PCR检测正确后,菌株用体积分数为25%甘油LB重悬后获得含有p×35S:BrPGIP8质粒及pBI121空载体质粒的农杆菌GV3101菌种,于-75℃保存备用。
2.2实施浸花法转化
根据需要种植一批野生型拟南芥,待拟南芥长出20~30个花序时,剪掉果夹。从超低温冰箱中取出含有p×35S:BrPGIP8质粒及pBI121空载体质粒的农杆菌GV3101菌种,分别在超净工作台上用接种环将菌种接种于含有利福平终浓度(Rif,50mg·L-1)、卡那霉素终浓度(50mg·L-1)的固体LB筛选平板上,活化菌种。接种后封口,将其倒置于28℃培养箱中,培养36h。挑取单菌落于15mL含有利福平终浓度(50mg·L-1)、卡那霉素(50mg·L-1)的液体培养基中。放到28℃摇床,200rpm培养12h以制成农杆菌母液,保存于4℃。侵染前一天,取1mL母液于50mL液体LB培养基(含抗生素)中28℃培养至OD值1.0。将菌液室温离心(4000rpm,10min),加入50mL的1mM MgCl2 5%(质量分数)蔗糖溶液,加入40μL表面活性剂Silwet77至终浓度为0.02%(体积分数),混匀后转移至50mL离心管。
侵染时,将拟南芥花序浸入装有菌液的离心管中,计时30s,移出花序,横放于铺有湿纸巾的穴盘中,避光放置24h,再正置放回人工气候室,正置培养约30天后停止浇水并套袋收种,将种子按照植株收集后编号。
2.3配制固体播种培养基
(1)播种培养基:4.43g MS粉(产品型号M519),20g蔗糖,8g琼脂粉,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,121℃高压蒸汽灭菌20min,待冷却至50℃左右时于超净工作台加入卡那霉素至终浓度为50mg/mL,分装至灭菌的培养皿中,每皿约10mL。
2.4转基因拟南芥阳性检测
(1)抗性平板筛选
于无菌环境下,用体积分数为75%的乙醇洗涤步骤2.2收集并编号的种子约30s,重复3次,在用双蒸水洗涤,重复2次,取适量种子均匀播种于步骤2.3获得的含有卡那霉素(Kan,50mg·L-1)的固体播种培养基上,封口后静置培养,待植株长出2片真叶时观察植株颜色,选取绿色植株移栽至普通栽培基质(泥炭土∶珍珠岩∶蛭石=5∶3∶2)中。
(2)PCR检测
取样:培养过程中,每个芽系的多个芽的叶片混合取样检测。
采用DNA简易提取法提取DNA。首先,配制DNA提取缓冲液,取20%(质量分数)的SDS水溶液0.5mL,0.5mol·L-1EDTA水溶液0.5mL,1mol·L-1Tris-HCl缓冲液(pH 9.0)2mL,2mol·L-1LiCl水溶液2mL,双蒸水补足至10mL,混合均匀即可使用。然后,取约0.1g样品放入2mL离心管中,加入200μL提取缓冲液及1个磁珠,放入磨样机中研磨(65Hz,120s),取出离心管中的磁珠后,13000rpm离心5min;转移100μL上清液至新的1.5mL离心管中,加入100μL异丙醇后,迅速温和颠倒混匀,室温静置5min后,13,000rpm离心10min;去掉上清液,用1mL体积分数为70%乙醇洗涤沉淀后,13000rpm离心3min,弃上清液;重复洗涤一次后,将离心管倒置于吸水纸上,吹干乙醇后,加入50μL双蒸水溶解DNA。
转基因阳性植株基因组中应有NPTII基因的插入,因此,所有转基因植株均用NPTII基因的引物(表2)检测,PCR体系如下:T5Mix 12.5μL,NPTII正反向引物各0.5μL,DNA模板2μL,双蒸水补足至25μL,98℃3min,35个循环(98℃10s,55℃10s,72℃15s),72℃3min,4℃保温。PCR产物通过1.2%(质量分数)的琼脂糖凝胶电泳鉴定扩增片段的长度,只有阳性植株用于进行后续实验(参见图6)。
表2转基因拟南芥PCR检测所用引物
(3)实时荧光定量PCR分析转基因拟南芥植株中BrPGIP8基因的相对表达量
取移栽后通过PCR检测的阳性株系中每个株系至少3株进行混合取材,为排除干扰,所有植株均取第3片叶子,做好标记后,迅速置入液氮中固定,全部取材完成后,提取总RNA并合成cDNA后,进行qRT-PCR分析。qRT-PCR分析所用引物通过Primer Premier 5设计,如表3所示。反应体系为15μL:7.5μL SYBR Green Master Mix,正反向引物各0.3μL,模板1μL,5.9μL双蒸水。qRT-PCR反应流程:95℃:30s,40个循环(95℃:5s,55℃:45s)。通过熔融曲线确定反应的特异性,内参基因为Atactin4,基因的相对表达量通过2-ΔΔCt方法计算。
表3转基因拟南芥植株qRT-PCR分析所用引物
引物名称 | 引物序列(5’-3’) |
BrPGIP8F | ATACCGAGTTCTCTGTCTTTGTTAC(SEQ ID No.10) |
BrPGIP8R | GATTGCCTAGTGTTTTTGGTATATA(SEQ ID No.11) |
Atactin7F | GGAACTGGAATGGTGAAGGCTG(SEQ ID No.12) |
Atactin7R | CGATTGGATACTTCAGAGTGAGGA(SEQ ID No.13) |
结果表明,在BrPGIP8异源表达株系(OE-1、OE-3)中BrPGIP8的均发生了异源表达(图7和图8)。
实施例3:转基因拟南芥植株抗病性鉴定
3.1将活化后的核盘菌用于侵染实验,用孔径为6mm的打孔器在培养核盘菌的PDA培养皿上打孔,将含有菌丝的部分朝下贴在拟南芥叶片背面上,将叶片背面朝上,在培养皿底部放入滤纸并加入1mL ddH2O保持滤纸湿润,再放入叶片封口。将培养皿放入28℃培养箱避光培养。侵染后每隔24h对叶片进行一次观察。
3.2从每个经过验证的转基因拟南芥株系的不同植株中取共3-4片叶片作为一组,取培养皿于底部垫入滤纸,加入1mL无菌水润湿,将同一组叶片置于同一培养皿中,用封口膜将培养皿封口。分别记录侵染后0h、24h、48h、72h、96h时叶片的发病情况,利用ImageJ软件对叶片上褐色病斑面积进行计算,再利用Excel的统计分析工具对各组病斑的差异显著性进行统计分析。
在转基因拟南芥植株的菌核病发病实验中发现,BrPGIP8异源表达的转基因拟南芥植株相比于对照组而言均出现了对菌核病抗性提升的表型,用Image J和Excel的统计分析结果表明,表达量较高的OE-1株系的叶片上,病斑面积最小,且相对于其他实验组有显著差异(图9)。
以上所述为本发明较佳的具体实施方式,但在其基础上可以进行一些改进或修改,这对任何熟悉本领域的技术人员而言是显而易见的。因此,在本发明基础上所作的这些修改和改进,均属于本发明要求保护的范围。
序列表
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无锡迪茉得生物种业科技有限公司
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Claims (1)
1.一种芜菁抗病基因BrPGIP8在激发植物抗菌核病功能中的应用,所述芜菁抗病基因BrPGIP8的核苷酸序列如SEQ ID No.1所示。
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Brassica Napus Polygalacturonase Inhibitor Proteins Inhibit Sclerotinia Sclerotiorum Polygalacturonase Enzymatic and Necrotizing Activities and Delay Symptoms in Transgenic Plants;Zafer Dallal Bashi等;《Can. J. Microbiol》;20130228;第59卷(第2期);第79-86页 * |
Brassica Napus Possesses an Expanded Set of Polygalacturonase Inhibitor Protein Genes That Are Differentially Regulated in Response to Sclerotinia Sclerotiorum Infection, Wounding and Defense Hormone Treatment;Dwayne D. Hegedus等;《Planta》;20080423;第228卷(第2期);第241-253页 * |
Overexpression of OsPGIP2 Confers Sclerotinia Sclerotiorum Resistance in Brassica Napus Through Increased Activation of Defense Mechanisms;Zhuanrong Wang等;《Journal of Experimental Botany》;20180410;第69卷(第12期);第3141-3155页 * |
PREDICTED: Brassica rapa polygalacturonase inhibitor 1-like (LOC103847026), mRNA;GenBank登录号; XM_009124055;《GenBank数据库》;20161013;参见序列部分 * |
Prokaryotic expression and protein function of Brassica napus PGIP2 and its genetic transformation;Haiyan HuangFu等;《Plant Biotechnology Reports》;20131211;第8卷;第171-181页 * |
内源BnPGIPs基因转化甘蓝型油菜的抗菌核病研究;王转茸等;《中国作物学会油料作物专业委员会第八次会员代表大会暨学术年会综述与摘要集》;20181119;第178页 * |
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