CN110699360B - 白菜抗病相关基因BrPGIP4及其应用 - Google Patents
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Abstract
本发明提供了白菜抗病相关基因BrPGIP4及其应用,属于植物基因工程技术领域。所述白菜抗病相关基因BrPGIP4的DNA序列如SEQ ID No.1所示。通过农杆菌浸花转化法将该基因转化至哥伦比亚型拟南芥中,得到BrPGIP4异源表达拟南芥株系,结果发现,白菜抗病相关基因BrPGIP4的异源表达会导致拟南芥对菌核病的抗性出现显著提升。这表明白菜抗病相关基因BrPGIP4与植物抗核盘菌病关系密切,可将该基因应用于白菜类蔬菜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,涉及白菜抗病相关基因BrPGIP4、其编码蛋白及其在植物抗病过程中的应用。
背景技术
白菜(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、白菜、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,提供白菜抗病相关基因BrPGIP4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BrPGIP4,该基因为:从来源于品种为‘矮脚黄’(由本实验室保存多年的高代自交系)的白菜中克隆到的基因,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白菜抗病相关基因BrPGIP4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。
本发明提供了上述白菜抗病相关基因BrPGIP在激发植物对菌核病的抗性中的应用。。
本发明提供了上述白菜抗病相关基因BrPGIP4在制备转基因植物中的应用。
本发明还提供了上述白菜抗病相关基因BrPGIP4在植物种质资源改良中的应用。
本发明提供的白菜BrPGIP4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,获得白菜BrPGIP4异源表达的转基因拟南芥株系,结果发现,白菜抗病相关基因BrPGIP4的表达变化可以抑制核盘菌在拟南芥叶片上形成病斑,从而显著提升植株对菌核病的抗性。这表明了白菜抗病相关基因BrPGIP4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为白菜抗病相关基因BrPGIP4克隆PCR电泳图。其中M泳道为DNA marker,1,2泳道为目的片段。
图2为BrPGIP4表达特征分析结果。
图3为BrPGIP4过表达及载体示意图。
图4为BrPGIP4异源表达植株与对照植株生长状况比较。A为对照植株,B,C为BrPGIP4异源表达植株。
图5为转基因拟南芥株系中BrPGIP4的相对表达量示意图。
图6为BrPGIP4异源表达植株与对照植株花的抗病性状比较。A、E、I、M分别为空白对照组叶片于0、24、48、72h时的情况。B、F、J、N分别为CK于核盘菌侵染后0、24、48、72h的发病情况。C、G、K、O分别OE-1于核盘菌侵染后0、24、48、72h的发病情况。D、H、L、P分为为OE-3于核盘菌侵染后0、24、48、72h的发情情况。
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白菜抗病相关基因BrPGIP4,该基因为:从来源于品种为‘Chiffu-1-4’的白菜中克隆到的基因,其基因序列如SEQ ID No.1所示。
本发明实施例还提供了上述白菜抗病相关基因BrPGIP4在激发拟南芥对菌核病的抗性中的应用,下面对其进行具体描述。
实施例1:白菜BrPGIP4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)取白菜‘Chiffu-1-4’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 Eraser1μL,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 Enzyme Mix 1μL,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)以白菜叶片cDNA为模板,用引物(序列如SEQ ID No.2和SEQ ID No.3所示)通过高保真酶扩增获得BrPGIP4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,该基因在叶中的表达丰度最高(图2)。
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,回收产物约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后,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,加入酶切后回收的基因片段与酶切后pBI121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℃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(图3),-20℃保存备用。
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-BrPGIP4及pBI121空载体转入农杆菌GV3101中,具体方法如下:取解冻的农杆菌感受态与5μL实施例中获得的质粒混匀后,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,弃大部分上清液,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25%甘油LB重悬后,含有p×35S:BrPGIP4质粒及pBI121空载体质粒的农杆菌GV3101菌种并于-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:BrPGIP4质粒及pBI121空载体质粒的农杆菌GV3101菌种,在超净工作台上用接种环将菌种接种于含有终浓度的利福平(Rif,50mg·L-1)、卡那霉素(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含有终浓度的利福平(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)的蔗糖溶液,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上述菌液的离心管中,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/L,分装至灭菌的培养皿中,每皿约10mL。
2.4转基因拟南芥的阳性检测
(1)抗性平板筛选
于无菌环境下,用体积浓度75%的乙醇洗涤步骤2.2中收集、编号的种子约30s,重复3次,再用双蒸水洗涤,重复2次,取适量种子均匀播种于步骤2.3配置的含有卡那霉素(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1)检测,PCR体系如下:T5 Mix 12.5μL,NPTII正反向引物(见表1)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)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。
表1转基因拟南芥PCR检测所用引物
引物名称 | 引物序列(5’-3’) |
NPTII-F | GTCACTGAAGCGGGAAGGG(SEQ ID No.4) |
NPTII-R | CGGCGATACCGTAAAGCAC(SEQ ID No.5) |
BrPGIP4 | ATGGATAAGACAACGACACTGCTC(SEQ ID No.6) |
BrPGIP4 | TCACTTGCAACTCTGAAGAGGTG(SEQ ID No.7) |
pBI121-F | CCGACAGTGGTCCCAAAGAT(SEQ ID No.8) |
pBI121-R | CGGCTTCAAATGGCGTATAG(SEQ ID No.9) |
(3)实时荧光定量PCR分析转基因拟南芥植株中BrPGIP4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(图4),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PrimerPremier 5设计,如表2所示。反应体系为15μL:7.5μL SYBR Green Master Mix,正向和反向引物各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2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’) |
BrPGIP4F | CATTCTCTAGACATATCAAACGAC(SEQ ID No.10) |
BrPGIP4R | TAGATTCTTGAGCTCAGTAAT(SEQ ID No.11) |
Atactin7F | GGAACTGGAATGGTGAAGGCTG(SEQ ID No.12) |
Atactin7R | CGATTGGATACTTCAGAGTGAGGA(SEQ ID No.13) |
结果表明,在BrPGIP4异源表达株系(OE-1、OE-3)中BrPGIP4的均发生了异源表达(图5)。
实施例3:转基因拟南芥植株的抗病性鉴定
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,BrPGIP4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-3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6)。
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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ttacctaaac tcgagattct tgacctcagc aggaacaagc ttacaggttc gataccagag 180
tcatttggat cctttaaagg agtgatgtat gctcttttcc tatctcacaa ccagctgtcc 240
ggttctattc cgaaatcatt agaaaacctg gacattaacc agattgatct ttcccggaac 300
aagcttgaag gtgacgcgtc gatgttgttt ggagcccaaa agactacaca taacattgac 360
ctatcaagaa acatgttcca gttcaatatc tccatggtta aagtctctaa aacagttaat 420
ttcttgcact tgaatcacaa cgggctcaca gggactatcc cgattcaatg gacccaactt 480
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Claims (2)
1.一种白菜抗病相关基因BrPGIP4在激发植物对菌核病的抗性中的应用,其特征在于,所述白菜抗病相关基因BrPGIP4的核苷酸序列如SEQ ID No.1所示。
2.一种白菜抗病相关基因BrPGIP4在植物种质资源改良中的应用,其特征在于,具体为:在拟南芥中过表达如SEQ ID No.1所示的白菜抗病相关基因BrPGIP4,提高拟南芥对菌核病的抗性。
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