CN117535317A - Mapk基因及其在抗杨树真菌感染中的应用 - Google Patents
Mapk基因及其在抗杨树真菌感染中的应用 Download PDFInfo
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Abstract
本发明属于植物生物技术领域，尤其涉及一种MAPK基因及其在抗杨树真菌感染中的应用，本发明通过生物信息学分析，发现PtMAPK3‑1基因是杨树MAPK级联基因家族成员，在杨树的抗逆、抗病过程中扮演了重要的角色。本发明通过构建表达载体以及根癌农杆菌转基因过表达MAPK3‑1，过表达PagMAPK3‑1在S.musiva侵染后杨树体内ROS的积累，为病原真菌在植株体内的进一步侵染和繁殖提供了营养，发现K8杨中的PagMAPK3‑1在杨树抗病反应中起着负调控作用。本发明为筛选抗病的杨树品种提供了一种分子标记，同时也为提高杨树的抗病能力提供了一种调控手段。
Description
技术领域
本发明属于植物生物技术领域，尤其涉及一种MAPK基因及其在抗杨树真菌感染中的应用。
背景技术
随着经济和人口的不断增长，人类对木材产品资源和生物能源原料的需求不断增加，然而由于温度上升、可用水资源有限以及自然灾害发生的概率增加限制了林木的生产力。杨树具有生长速度快、适应性强、轮伐期短等优点，常作为板材和纸浆材在全球范围内广泛种植。然而，长期大面积的种植杨树，杨树也面临着大量的病虫害的侵扰。由Sphaerulinamusiva(亚球孢菌S.musiva)引起的溃疡病和叶斑病会导致杨树的过早落叶、光合作用面积下降和茎折断，不仅降低了木材的年产量，更严重会导致杨树的死亡和造林的失败。杨树叶斑病是一种由真菌引起的病害，主要侵染杨树及其亲缘植物。亚球孢属(Sphaerulinaspp.)真菌是叶斑病病原菌的一种，能够引起植物的落叶和腐烂，轻者导致产量下降，重者导致植物的死亡。Sphaerulinamusiva可引起杨树叶斑病和溃疡病。它原产于东部美洲黑杨(Populusdeltoides)，并且仅引起叶斑病症状。在容易被感染的杂交杨树上，S.musiva会导致叶片坏死，导致早期的落叶，并在茎和树枝上造成溃疡，从而降低生长速度，使树木容易被微生物定植。目前还没有办法阻止S.musiva在杨树种植地区的蔓延，一旦S.musiva传播进一个区域，就很难清除该真菌。目前，化学和生物防治措施可以很大限度的减少杨树的发病率，此外创制抗病转基因杨树是控制杨树病害的另一有效手段。中国专利CN202210837131.X提出在杨树中过表达因PtoCXE06基因，以增强杨树对杨树烂皮病菌抗性。中国专利CN202211588272.9发现了杨树在S.musiva感染中的效应蛋白SmCSEP3，并提出在杨树中瞬时表达或过表达SmCSEP3以增强杨树抗S.musiva感染的能力。因此，将基因工程技术和植物抗病性相结合，开展杨树的抗病研究、探索抗病的相关调控机制，对提高杨树人工林成活率、增加木材产量和保护景观生态都具有重要意义。
发明内容
目前针对S.musiva感染，探索杨树的相关染病、抗病基因、内部的信号通路，采用基因工程的手段提升杨树的抗真菌能力的研究还相对较少，需要进一步的深入。本发明的目的即在于探索相关机制机理，提出更多的手段以提升杨树的抗病能力。
基于此，本发明提出如下技术方案：
本发明的一个方面，本发明提供了一种杨树抗病的负调控基因，所述基因为PagMAPK3-1，其核酸序列如SEQ ID NO.3所示。
本发明的一个方面，本发明提供了一种表达载体，所述表达载体中含有PagMAPK3-1基因，所述PagMAPK3-1基因的序列如SEQ ID NO.3所示。所述表达载体可以是原核表达载体，也可以是真核表达载体。
本发明的一个方面，本发明提供了一种基因工程菌，所述工程菌中有含有本发明所述的表达载体。优选的，所述工程菌为大肠杆菌、根癌农杆菌中的一种。
本发明的一个方面，本发明提供了MAPK3-1基因在作为杨树抗S.musiva菌感染的筛选标记中的应用。优选的，所述杨树为毛果杨或84K杨。
当被试品种的PagMAPK3-1基因表达显著高于野生型杨树K8的表达水平时，则该品种为S.musiva菌感染抗病性较差的品种；当被试品种的PagMAPK3-1基因表达显著低于野生型杨树K8的表达水平时，则该品种为S.musiva菌感染抗病性较好的品种。
本发明的一个方面，本发明提供了一种提高杨树抗S.musiva菌感染能力的方法，通过瞬时转染或稳定转染降低杨树体内的MAPK3-1基因表达。
有益效果
通过基因组学分析发现了在盐、干旱和M.brunnea真菌胁迫下，PtMAPK3-1、PtMKK7、PtMKK9和PtRaf23-1在各种生物和非生物胁迫下的转录水平有明显变化，尤其是PtMAPK3-1能够响应多种逆境影响是一种新的MAPK家族成员。
对过表达PagMAPK3-1转基因杨树进行了S.musiva接种后，转基因杨树均表现出更多的坏死病斑，DAB染色、PAL活性、CAT活性、POD活性和MDA含量等生理生化指标进一步表明PagMAPK3-1在杨树抗病反应中起着负调控作用，为筛选抗病的杨树品种提供了一种分子标记，也为提高杨树的抗病能力提供了一个调控手段。
附图说明
图1：PagMAPK3-1基因PCR扩增后的凝胶电泳分析。
图2：PagMAPK3-1基因序列及保守域预测。
图3：转基因杨树基因组PCR电泳检测。
图4：PagMAPK3-1过表达转基因杨树基因表达量检测。
图5：PagMAPK3-1转基因杨树接种S.musiva后的表型分析。
图6：PagMAPK3-1转基因杨树接种S.musiva两天后的生理生化指标检测。
具体实施方式
本发明中植物材料杂交杨84K(Populusalba×Populusglandulosa)，由中国林科院的王留强老师赠送；其他的生物学试剂除特殊说明外均为常规试剂来自的市场上的生物试剂公司，相关分子生物学操作方法除特殊说明外均参照《分子克隆实验指南》(J.萨姆布鲁克、D.W.拉塞尔编著，科学出版社)。
实施例1：杨树MAPK级联基因家族成员的鉴定
从PhycoCosm数据库(http://genome.jgi-psf.org/Poptr1/Poptr1.home.html)和Phytozome数据库(https://phytozome.jgi.doe.gov/pz/portal.html)获得了毛果杨基因组组装版本v1.0和v3.0的基因组数据。根据先前的研究，获得了杨树基因组v1.0版中21个PtMAPKs和11个PtMAPKKs的蛋白质序列(Hamel L P,Nicole M C,Sritubtim S,etal.Ancient signals:comparative geno mics of plant MAPK and MAPKK genefamilies[J].Trends in plant science,2006,11(4):192-198)；以这32条蛋白序列作为查询序列，通过TBtools中的BLAST工具在毛果杨的v3.0版本的蛋白质库中得到了PtMAPKs(即毛果杨的MAPKs)和PtMAPKKs基因ID。为了确定PtMA PKKK基因家族的潜在成员，从植物基因组数据库(https://phytozome.jgi.doe.gov/pz/portal.html)下载了毛果杨的基因组序列和蛋白质序列。用查询到的拟南芥，枣，麻风树，猕猴桃的MAPKKK的蛋白序列作为查询序列对毛果杨蛋白质数据库进行BLAST搜索。随后，使用隐马尔可夫模型(HMM)搜索毛果杨中含有丝氨酸/苏氨酸蛋白激酶结构域(PF00069)的基因。比对BLAST搜索和HMM搜索得到的基因，去除不能同时满足二者条件的基因，并且去除多余转录本。最终获得了新版本中的21个PtMAPKs和11个PtMAP KKs的基因号。为了更好地了解MAPK级联基因响应逆境的基因表达水平，本发明从NCBI的GEO数据库中获得了杨树与非生物胁迫(干旱和盐)和生物影响(Marssoninabrunnea感染)相关的转录组数据，结果发现感染病原菌后，PtMPK3-1、PtMKK9、PtMKK7和PtRaf23-1显著上调，PtMAPK3-1对多种胁迫有响应。基于此，将MAPK3-1基因对于杨树的抗逆、抗病的重要候选基因。
实施例2杨树MAPK级联基因家族成员PagMAPK3-1基因克隆和分析
毛果杨主要是国外的杨树品种，基于实施例1中毛果杨基因组分析的结果，发明人研究了MAPK3-1基因在中国的杨树品种84K杨中的相关情况。
以84K杨的全长cDNA为模板克隆PagMAPK3-1(即84K杨MAPK3-1基因)，PCR实验所用的相关引物如下：
PagMAPK3-1-F：ATGGCGAATTATGCACAGGGAAATG(SEQ ID NO.1)
PagMAPK3-1-R：CTAGCATGCATATTCTGGATTAAGTGC(SEQ ID NO.2)
将PCR产物胶回收，将回收的PCR产物连接到克隆载体pEasy-BluntSimple上，转化大肠杆菌。
结果分析：将PCR产物通过1％琼脂糖凝胶电泳凝纯化分离，结果显示在2000bp到1000bp之间有一条明亮且单一的条带(图1)。基因组序列注释中显示PagMAPK3-1基因大小1116bp，初步说明PagMAPK3-1基因扩增成功。将明亮的胶块切下进行胶回收后连接克隆载体，送至测序公司进行测序。测序结果通过DNAMAN软件进行比对，结果表明序列一致，证明目的基因克隆成功。将PagMAPK3-1和PtMAPK3-1序列进行比对后发现，毛果杨和84K杨中MAPK3-1的CDS序列相差8个碱基，翻译成蛋白质后有3个氨基酸的差别。
经测序84K杨PagMAPK3-1基因序列如SEQ ID NO.3所示，如图2中(a)所示，PagMAPK3-1基因大小1116bp，PagMAPK3-1基因的结构保守域预测分析如参见图2中(b)。
实施例3过表达转基因杨树抗病性功能鉴定
3.1过表达转基因株系的获得
(1)载体构建
采用Gateway技术构建PagMAPK3-1过表达载体：
a.在PagMAPK3-1基因克隆引物的F、R端分别加上gateway接头，实施例2中构建的PagMAPK3-1克隆载体为模板进行克隆，加上接头引物序列如下：
然后进行PCR产物胶回收；
b.通过BP反应将上述PCR产物克隆到中间载体pDONR207上，反应体系如下：
| 试剂 | 用量 |
| PCR产物 | 150ng |
| 载体 | 75ng |
| BP酶 | 0.8μl |
25℃,连接6h，然后进行PCR产物胶回收，转化，测序。
c.上一步中间载体的构建测序正确后，利用LR反应将两个基因构建到过表达载体pMDC32上，反应体系如下：
25℃,连接6h，然后进行PCR产物胶回收，转化，测序，提质粒，最后导入农杆菌感受态细胞GV3101中。
(2)杨树遗传转化：
a.将上述(1)中获得的农杆菌加入含有相应Kan、Rif的LB培养基中进行扩增，使其OD600值达到0.6左右；
b.在超净工作台中，将切好的带有伤口的84K杨叶片放入上一步的菌液中，低速水平摇床20min左右；
c.将侵染完的叶片吸干多余菌液后放入分化培养基中，在24℃暗培2d；
d.暗培养2d后，将叶片转入含有潮霉素和特美汀的筛选培养基中进行筛选，一般20d左右会出现抗性芽；
e.将抗性芽苗切下转至含相同抗性的生根培养基中，待生根后扩繁并进行表达量的检测。
分化培养基体系1L：
筛选培养基体系1L：分化培养基1L，待灭菌后冷却至50℃在超净台下加入潮霉素(0.0003g/L)和特美汀(0.2g/L)。
生根培养基体系1L：
待灭菌后冷却至50℃在超净台下加入潮霉素(0.0003g/L)和特美汀(0.2g/L)。
(3)基因组PCR验证：
CTAB法提取杨树基因组，然后以提取的基因组为模板进行PCR实验检测PagMAPK3-1基因。以过表达载体质粒为阳性对照组。PCR反应结束后进行凝胶电泳检测，在阳性对照对应的位置有PCR条带的植株即可能为阳性植株(参见图3)。
(4)阳性株系的相对表达水平分析：
分别提取阳性植株的RNA并进行反转录，将获得的cDNA作为qRT-PCR实验的模板。通过qRT-PCR实验检测每个株系中目的基因的相对表达量。
结果显示：PagMAPK3-1过表达转基因株系中，OE-1和OE-3的PagMAPK3-1基因表达量达到20倍以上(参见图4)，因此选择这两个株系进行后续的分析。
实施例4PagMAPK3-1转基因杨树的抗病性研究
4.1S.musiva侵染
S.musiva菌分离自有叶斑病的毛白杨叶片，并使用PDA培养基培养，将菌丝体用枪头或接种针刮下，并使用无菌的0.05％吐温80水溶液溶解，调整孢子悬浮液的中孢子数量，采取杨树移栽苗的健康叶片，将其放置在铺有湿润纱布的培养皿中，叶片背面朝上，在叶片两侧避开叶脉的地方滴加25μl制备好的孢子悬浮液。培养皿置于25℃培养箱黑暗培养，每日观察其发病情况。
结果：在84K杨(野生型对照WT)、PagMAPK3-1转基因杨树叶片分别接种S.musiva孢子悬浮液4天后对病斑情况的观察，如图5所示，84K杨叶片在接种S.musiva后的第六天出现轻微病斑，而PagMAPK3-1转基因杨树叶片的坏死区域多于WT。对接种病菌第六天后的病斑大小进行统计分析后发现，OE-1和OE-3的病斑大小显著高于WT。以上结果表明，过表达PagMAPK3-1降低了杨树对S.musiva的抗性。
4.2生理生化指标测定
植物在受到病原菌胁迫后会迅速积累活性氧(ROS)，活性氧的大量积累会导致植物细胞的死亡。因此，植物为了清除过多的ROS会激活体内的抗氧化酶如过氧化氢酶(CAT)、过氧化物酶(POD)和PAL(苯丙氨酸酶)。丙二醛(MDA)是衡量植物受到氧化胁迫程度的常用指标之一，能反映植物膜脂过氧化的程度。对接种S.musiva后第二天的WT、OE-1和OE-3植株叶片进行DAB染色可以检测植物中ROS即H2O2积累情况。
(1)CAT(过氧化氢酶)、POD(过氧化物酶)活性测定、PAL(苯丙氨酸酶)，其中POD、CAT检测方法参考：Maehly A C,Chance B.The assay of catalases and peroxidases[J].Methods of Biochemical Analysis,1954,1:357-424.PAL检测方法参考：Beaudoin-Eaga n LD,Thorpe TA.Tyrosine and phenylalanine Ammonia Lyase activitiesduring shoot initia tion in tobacco callus cultures.Plant Physiol.1985；78:438–41。
(2)MDA含量测定：使用硫代巴比妥酸方法测定丙二醛含量，具体实验方法参考2017年科学出版社出版的由王三根主编的《植物生理学实验教程》。
(3)DAB染色：取发病区域杨树叶片放于50ml的离心管中，加入DAB染液使其浸没叶片。避光条件下于摇床(25～28℃，80r/min)震荡4-7h，弃染液后加入脱色液(脱色液配制：乙醇:乙酸:甘油＝3:1:1)，并将离心管移至恒温水槽(95℃)中脱色15min，期间可更换1-2次脱色液。
以上各项指标均取发病区域杨树叶片，每个样品3个重复，并用GraphPadPrism5软件呈现直方图和ANOVA差异显著性分析(*p<0.05；**p<0.01)。
结果：
参见图6中(b)所示，对PAL、CAT和POD活性测定表明，野生型杨树(WT)的PAL和CAT活性升高，而过表达PagMAPK3-1转基因杨树的PAL和CAT活性下降并且显著低于WT。WT的POD活性在接种后第二天有所上升，但过表达PagMAPK3-1转基因杨树的POD活性却有所下降，并且显著低于WT。
参见图6中(b)所示，在病原菌处理后，转基因株系的MDA含量明显高于WT植株。
参见图6中(a)所示，在接种S.musiva后的第二天，WT、OE-1和OE-3植株叶片中均产生黄褐色的沉淀，表明S.musiva侵染杨树后，可诱导植株的活性氧积累。WT叶片中的沉淀物比两个转基因植株更少，表明过表达PagMAPK3-1转基因杨树在S.musiva入侵后引起更多的细胞死亡与H2O2积累，为病原真菌在植株体内的进一步侵染和繁殖提供了营养。
以上内容是结合具体实施方式对本发明作进一步详细说明，不能认定本发明具体实施只局限于这些说明，对于本发明所属技术领域的普通技术人员来说，在不脱离本发明的构思的前提下，还可以做出若干简单的推演或替换，都应当视为属于本发明所提交的权利要求书确定的保护范围。
Claims (8)
1.一种杨树抗病的负调控基因，其特征在于，所述基因为PagMAPK3-1，其核酸序列如SEQ ID NO.3所示。
2.一种表达载体，其特征在于，所述表达载体中含有PagMAPK3-1基因，所述PagMAPK3-1基因的序列如SEQ ID NO.3所示。
3.一种基因工程菌，其特征在于，所述工程菌中有含有权利要求2所述的表达载体。
4.根据权利要求3所述的工程菌，其特征在于，所述工程菌为根癌农杆菌。
5.一种如权利要求1所述的基因在作为杨树抗S.musiva菌感染的筛选标记中的应用。
6.根据权利要求5所述的应用，其特征在于所述杨树为毛果杨或84K杨。
7.根据权利要求5所述的应用，其特征在于，当被试品种的PagMAPK3-1基因表达显著高于野生型杨树K8的表达水平时，则该品种为S.musiva菌感染抗病性较差的品种；当被试品种的PagMAPK3-1基因表达显著低于野生型杨树K8的表达水平时，则该品种为S.musiva菌感染抗病性较好的品种。
8.一种提高杨树抗S.musiva菌感染能力的方法，其特征在于，通过瞬时转染或稳定转染降低杨树体内的MAPK3-1基因表达。
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| US20210371874A1 (en) * | 2020-07-30 | 2021-12-02 | Nanjing Agricultural University | Gene PpHSP21 with black spot disease resistance in Pyrus pyrifolia and its applications in improving black spot disease resistance in plants |
| CN116064566A (zh) * | 2022-07-15 | 2023-05-05 | 中国林业科学研究院 | 一种杨树抗烂皮病菌的基因PtoCXE06及其应用 |
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| WO2001041556A1 (en) * | 1999-12-06 | 2001-06-14 | Københavns Universitet | Method of using mapk4 and orthologues thereof to control plant disease resistance and plant growth |
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| US20210371874A1 (en) * | 2020-07-30 | 2021-12-02 | Nanjing Agricultural University | Gene PpHSP21 with black spot disease resistance in Pyrus pyrifolia and its applications in improving black spot disease resistance in plants |
| CN116064566A (zh) * | 2022-07-15 | 2023-05-05 | 中国林业科学研究院 | 一种杨树抗烂皮病菌的基因PtoCXE06及其应用 |
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