CN116218877A - 三七WRKY转录因子PnWRKY12的应用 - Google Patents
三七WRKY转录因子PnWRKY12的应用 Download PDFInfo
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Abstract
本发明公开了一种三七WRKY转录因子基因PnWRKY12的新用途，即其在提高烟草对棕黑腐质霉（Humicola fuscoatra）抗性中的应用，三七WRKY转录因子基因PnWRKY12的核苷酸序列如SEQ ID NO：1所述，本发明通过功能基因组学相关技术研究证实PnWRKY12基因具有提高植物抗真菌的功能，将本发明抗真菌的PnWRKY12基因构建到植物表达载体上并转入烟草中过量表达，转基因烟草植株具有很强的抗真菌侵染能力，实验结果显示超表达PnWRKY12的转基因烟草对棕黑腐质霉的侵染具有高水平的抗性。
Description
技术领域
本发明属于分子生物学以及基因工程相关技术领域，特别是一种具有抗真菌侵染能力的三七WRKY转录因子PnWRKY12的应用。
背景技术
植物生长过程中难免遭受到各种各样病虫害的侵染，这一直是农业生产中一个不容忽视的问题。在与生物胁迫互作的过程中，植物也进化出了独特的免疫系统，主要包括由植物细胞表面模式识别受体（PRRs）识别病原体从而激活的模式触发免疫（PTI）和富含亮氨酸的重复受体（NLR）蛋白识别病原体分泌的效应因子激活的效应触发免疫（ETI）（NgouBPM, Ding P, Jones JDG. Thirty years of resistance: Zig-zag through the plantimmune system. The Plant Cell, 2022, 34 (5): 1447-1478）。在植物对病原体的防卫反应过程中，需要转录因子调节各种防御相关基因的表达（Prom LK, Egilla J. Effectof chitinase and thaumatin on mycelial growth of five sorghum [Sorghum bicolor (L.) Moench] grain molding fungi under in vitro conditions. Journalof Tropical Agriculture, 2011, 49: 88-90）。其中，WRKY转录因子是植物中最大的转录因子家族之一，其N端一般是由WRKYGQK 七肽序列组成的WRKY结构域，C端则是CX4-5CX22-23HXH (C2H2)或CX7CX23-HXC (C2HC)组成的锌指结构（Zou SH, Tang YS, Xu Y, Ji JH,Lu YY, Wang HM, Li QQ, Tang DZ. TuRLK1, a leucine-rich repeat receptor-likekinase, is indispensable for stripe rust resistance of YrU1 and confers broadresistance to multiple pathogens. BMC Plant Biology, 2022, 22(1): 280）。
WRKY转录因子能与靶基因启动子中的W-box元件结合，调控目标基因表达，在植物的生长发育过程中发挥重要作用（Xin YC, Wang DH, Han SM, Li SX, Gong N, Fan YT,Ji XL. Characterization of the chitinase gene family in mulberry (Morus notabilis) and MnChi18 involved in resistance to Botrytis cinerea. Gene,2021, 13(1): 98）。水稻（Oryza sativa）中的转录因子OsWRKY50在脱落酸（ABA）处理后被激活，过表达OsWRKY50的水稻株系对ABA介导的种子萌发和幼苗生长起负调控作用（HuangSZ, Hu LJ, Zhang SH, Zhang MX, Jiang WZ, Wu T, Du XL. Rice OsWRKY50 mediatesABA-dependent seed germination and seedling growth, and ABA-independent saltstress tolerance. International Journal of Molecular Sciences, 2021, 22(16):8625）。相反，转录因子OsWRKY36与水稻SLR1基因的启动子结合后，促进了GA信号途径负调控因子DELLA蛋白的表达，从而抑制水稻的植株高度和种子大小（Lan J, Lin QB, ZhouCL, Ren YK, Liu X, Miao R, Jing RN, Mou CL, Nguyen T, Zhu XJ, Wang Q, ZhangX, Guo XP, Liu SJ, Jiang L, Wan JM. Small grain and semi-dwarf 3, a WRKYtranscription factor, negatively regulates plant height and grain size bystabilizing SLR1 expression in rice. Plant Molecular Biology, 2020, 104(4-5):429–450）。
WRKY转录因子通过调控植物转录组重新编程以应对不同病原物的入侵。WRKY转录因子上调或抑制靶基因的表达水平，同时参与激素信号通路，还与WRKY转录因子家族的其他成员相互作用。一些WRKY转录因子在植物对病原体的防卫反应中起正调控作用，例如过表达AtWRKY30的拟南芥（Arabidopsis thaliana）株系对黄瓜花叶病毒（Cucumber mosaic virus, CMV）的抗性增强（Zou LJ, Yang F, Ma YH, Wu QG, Yi KX, Zhang DW.Transcription factor WRKY30 mediates resistance to Cucumber mosaic virus inArabidopsis. Biochemical and Biophysical Research Communications, 2019, 517(1): 118-124）。FvWRKY42的过表达提高了草莓（Fragaria vesca）对白粉病（Powdery mildew）的抗性，并且增强了对盐胁迫和干旱胁迫的耐受程度，也能诱导根的生长和种子萌发，同时上调了转基因草莓株系中PR1的转录水平（Wei W, Cui MY, Hu Y, Gao K, XieYG, Jiang Y, Feng JY. Ectopic expression of FvWRKY42, a WRKY transcriptionfactor from the diploid woodland strawberry (Fragaria vesca), enhancesresistance to powdery mildew, improves osmotic stress resistance, andincreases abscisic acid sensitivity in Arabidopsis. Plant Science, 2018, 275:60-74）。也有一部分WRKY转录因子在植株对病原菌的防卫反应中起抑制作用，水稻的OsWRKY62在丝裂原激活蛋白激酶基因OsMAPK17过表达的水稻中大量表达，协同OsMAPK17参与水稻对水稻白叶枯病（Xanthomonas oryzae pv. oryzae）的免疫反应，并且呈负调控（Zhu Z, Wang TXZ, Lan JP, Ma JJ, Xu HQ, Yang ZX, Guo YL, Chen Y, Zhang JS,Dou SJ, Yang M, Li LY, Liu GZ. Rice MPK17 plays a negative role in the Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae. Rice, 2022, 15(1):41）。
三七[Panax notoginseng (Burk) F.h. Chen]属于伞形目五加科人参属，是我国名贵中药材，主要生长于云南省文山州。由于三七要求生长环境温暖湿阴，导致三七在种植过程中极易受到病虫害的威胁，其中由茄腐镰刀菌、尖孢镰刀菌等真菌引起的根腐病最为严重。三七感染根腐病后，会导致根部腐烂和坏死，并伴随着植株发育不良、叶片变黄、叶片边缘枯萎等症状，最终导致植株死亡。WRKY转录因子是调节植物非生物或生物胁迫反应的关键调节因子，是植物防御反应的重要组成部分，因此对三七中WRKY转录因子基因的发掘以及功能分析具有重要的研究及其应用价值。
发明内容
本发明提供了一种三七WRKY转录因子基因PnWRKY12的新用途，即其在提高烟草对棕黑腐质霉（Humicola fuscoatra）抗性中的应用。
本发明从三七中克隆获得WRKY转录因子12基因，WRKY转录因子基因PnWRKY12核苷酸序列如SEQ ID NO:1所示，该基因cDNA全长序列为1118 bp，包含一个849 bp的开放阅读框、143 bp的5’非翻译区、126 bp的3’非翻译区，编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明中PnWRKY12基因的编码区是序列表SEQ ID NO:1中第144-992位所示的核苷酸序列。本发明分离克隆三七的一个抗真菌相关基因的完整cDNA片段，利用根癌农杆菌（Agrobacterium tumefaciens）介导将目的基因转入受体植物烟草（Nicotiana tabacum）中过量表达，通过进一步实验验证该基因是否具有抗真菌的活性，为后期利用该基因改良烟草及其他植物抵御真菌病害的能力奠定基础。
上述PnWRKY12基因应用在提高烟草的抗真菌特性的具体操作如下：
（1）采用扩增PnWRKY12的特异引物，从接种茄腐镰刀菌的三七根中提取总RNA，通过逆转录-聚合酶链式反应（reverse transcription-polymerase chain reaction, RT-PCR）扩增出PnWRKY12的全长编码区，然后将其连接到pGEM-T easy载体上，经测序获得具有目的基因的克隆；
（2）用限制性内切酶PstI和SmaI酶切pGEM-T easy-PnWRKY12载体，通过胶回收得到目的基因片段，用同样的内切酶酶切植物表达载体pCAMBIA2300s，胶回收获得所需载体大片段，再将所获得的PnWRKY12基因片段与pCAMBIA2300s片段连接，构建植物超表达载体，之后将所构建的重组载体通过根癌农杆菌介导转入烟草中表达；
（3）以重组载体T-DNA上具有的抗性标记筛选转化子，并通过PCR以及RT-PCR检测得到阳性转基因植株，分析转基因植株对于病原真菌的抗性，最后筛选出抗病性明显增强的转基因植株。
本发明为提高植物对真菌病害的抗性提供了一种新的方法，通过基因工程手段培育抗病植物可以克服传统育种的不足，不仅缩短了育种周期，而且操作简单，容易获得高抗材料。本发明中来自三七的PnWRKY12基因能增强植物对棕黑腐质霉的抗性，将该基因导入烟草中，可以产生具有真菌抗性的新品种和新材料。利用基因工程技术培育抗性植物品种和材料具有明显的优势和不可取代的重要性；它不仅可以为大规模生产作物、花卉、药材等提供方便，减少化学农药的使用，还可以为农业生产节约成本、减少环境污染，因此本发明具有广阔的市场应用前景。
附图说明
图1是本发明PnWRKY12转基因烟草基因组DNA的PCR检测结果图，图中：Marker为DL2501 DNA Marker；阳性对照为质粒pGEM-T easy-PnWRKY12为模板的PCR结产物；WT为非转基因烟草（野生型）总DNA为模板PCR的产物；
图2是本发明阳性PnWRKY12转基因烟草中PnWRKY12转录水平的表达分析结果图；图中：Marker是DL2501 DNA Marker；WT是非转基因烟草总RNA逆转录cDNA为模板的PCR产物；阳性对照是质粒pGEM-T easy-PnWRKY12为模板的PCR产物；
图3是本发明PnWRKY12转基因烟草抗病性分析的结果图；图中是接种棕黑腐质霉的PnWRKY12转基因烟草叶片；WT为野生型烟草的叶片，6、14、15、19为PnWRKY12转基因烟草的叶片。
具体实施方式
下面通过附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：转录因子PnWRKY12的克隆
用茄腐镰刀菌接种三七，用接种24 h后的三七根提取总RNA。以逆转录得到的cDNA为模板，克隆目的基因PnWRKY12。所用上下游引物序列分别为：5’GCTGCAGCATGGACGGCAGAATCAAT 3’及5’ CCCGGGCTACAAGGTTGTCAAGCT 3’。通过PCR扩增出目的基因；PCR反应条件：94℃、5 min；94℃、30 s，60℃、30 s，72℃、1 min，32个循环；72℃、7min；PCR反应体系（50 μL）为：2.5 μL cDNA、5 μL 10× Ex Taq Buffer、4.5 μL dNTP、0.5μL 正向引物、0.5 μL 反向引物、0.5 μL E× Taq、36.5 μL ddH2O。PCR结束后，将全部PCR产物用于琼脂糖凝胶电泳，检测扩增产物的特异性以及大小。
反应结束后，用SanPrep柱式PCR产物纯化试剂盒（上海生工）回收目的片段，进行T-A克隆，反应体系和操作过程为：取3.4 μL 胶回收产物，依次加入0.7 μL pGEM-T Easyvector，0.9 μL T4 DNA Ligase，5 μL 2×Rapid Ligation Buffer混匀后置于4℃过夜反应。采用热激转化法将连接产物转入大肠杆菌DH5α中。使用含有氨苄青霉素（ampicillin，Amp）的LB固体培养基筛选阳性克隆，挑选若干个单菌落，过夜培养后用扩增PnWRKY12的特异引物鉴定出多克隆位点插入PnWRKY12的克隆，将所鉴定的克隆进行测序，最终获得的PnWRKY12全长cDNA为1118 bp，通过NCBI ORF finder （http://www.ncbi.nlm.nih.gov/gorf/gorf.html）分析发现其包含一个849 bp的开放读码框（见序列表），PnWRKY12编码一个含282个氨基酸的蛋白质PnWRKY12，其分子量约为31.38 KDa，等电点约为5.731。
实施例2：植物超表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒（上海生工）提取插入PnWRKY12的大肠杆菌质粒pGEM-T Easy-PnWRKY12以及植物表达载体pCAMBIA2300s的质粒，取1 μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性及浓度高低；用限制性内切酶PstI（TaKaRa）和SmaI（TaKaRa）分别对质粒pGEM-T Easy-PnWRKY12和pCAMBIA2300s进行双酶切（50 μL体系），反应体系和操作过程为：取20 μL pGEM-T Easy-PnWRKY12和 20 μL pCAMBIA2300s质粒、分别依次加入7.5 μL 10×T+BSA Buffer、2.5 μL PstI、2.5 μL SmaI、17.5 μL ddH2O，混匀后短时离心，置于37℃水浴1.5 h；将所有酶切产物点于琼脂糖凝胶中进行电泳，然后对PnWRKY12片段和pCAMBIA2300s载体大片段分别进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒（上海生工）；取1 μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度，置于-20℃保存备用。
利用T4 DNA Ligase（TaKaRa），将回收的PnWRKY12 DNA片段和pCAMBIA2300s载体片段连接起来，反应体系（10 μL）和操作过程为：取6 μL PnWRKY12 DNA片段依次加入2 μLpCAMBIA2300s载体DNA、1 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase，混匀后短时离心，然后16℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌DH5α中，用含有50 mg/L卡那霉素（kanamycin，Kan）的固体培养基筛选阳性克隆。挑选单菌落摇菌，以菌液为模板用扩增PnWRKY12的特异引物进行PCR，挑选出PnWRKY12与pCAMBIA2300s成功连接的克隆，所检测的菌株若为阳性，加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌中的pCAMBIA2300s-PnWRKY12质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-PnWRKY12转入根癌农杆菌LBA4404感受态细胞中。操作步骤为：取5 μL pCAMBIA2300s-PnWRKY12质粒加入含有50 μL感受态细胞的离心管中，轻轻混匀后冰浴30 min，随后转入液氮中冷冻5 min，然后迅速置于37℃水浴5 min，之后立即冰浴2 min，加入600 μL LB液体培养基于28℃振荡培养4 h。将活化后的农杆菌涂于含有50 mg/L Kan和25 mg/L Rif的LB固体培养基上，28℃静止培养两天。挑选单菌落摇菌，再用扩增PnWRKY12的特异性引物进行PCR，检测pCAMBIA2300s-PnWRKY12是否转入农杆菌中，对于阳性克隆，加入甘油后置于-80℃保存备用。
实施例3：农杆菌介导的植物遗传转化以及转基因植物筛选
将烟草种子用75%的酒精浸泡30 s，用无菌水洗涤后用0.1%的HgCl2浸泡8 min，然后再用无菌水洗涤若干次，播种于1/2 MS培养基上，28℃暗培养6 d，发芽后转至光照培养箱（25℃，16 h/d光照），以后每月用1/2 MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300s-PnWRKY12质粒的农杆菌LBA4404菌种，接种于5 mL含有50 mg/L Kan和25 mg/L Rif的LB液体培养基中，28℃培养至培养基浑浊。吸取1 mL浑浊的菌液至含有50 mg/L Kan的LB固体培养基上，28℃培养48 h；随后将LB固体培养基上的农杆菌刮下适量接种于附加有50 mg/L的乙酰丁香酮的MGL液体培养基中，28℃振荡培养2-3 h以活化农杆菌。
取烟草无菌苗叶片切成1 cm2左右的叶盘，完全浸泡于上述含有活化农杆菌的MGL液体培养基中，浸染时间为15 min，用无菌滤纸吸干叶片表面的菌液，将叶盘置于共培养基上进行室温培养，烟草转化的共培养基为MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/L葡萄糖+6 g/L琼脂，22℃无光条件下共培养2 d。
将共培养后的叶盘转到加有抗生素的MS筛选培养基中分化成苗，同时筛选转基因植株。烟草筛选培养基为MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L葡萄糖+6 g/L琼脂+50mg/L Kan+300 mg/L头孢霉素（cefotaxime sodium salt，Cef）；筛选培养时将培养瓶转移至光照培养箱培养（25℃，16 h/d光照，8 h/d黑暗），待烟草长出芽后用含有50 mg/L Kan和200 mg/L Cef的MS培养基继代培养，最后选择生根较好的再生苗通过PCR筛选阳性转基因烟草。
采用CTAB法提取转基因烟草植株叶片的基因组DNA，取1 μL基因组DNA通过琼脂糖凝胶电泳检测其完整性和浓度。以转基因植株的基因组DNA为模板用扩增PnWRKY12的特异引物进行PCR，PCR结束后取8 μL产物用于琼脂糖凝胶电泳以检测阳性转基因植株，部分烟草转基因植株的扩增结果如图1所示，PnWRKY12转基因烟草共筛选到31株阳性转基因植株。
实施例4：转基因烟草中PnWRKY12的表达分析以及转基因植株抗性分析
取阳性转基因单株以及非转基因烟草（野生型）的嫩叶提取总RNA，以逆转录得到的cDNA为模板，用扩增PnWRKY12的特异引物进行PCR，根据PCR结果分析各转基因单株中PnWRKY12转录水平的表达，总RNA提取以及RT-PCR的方法与实施例1中相同，PCR结束之后，取8 μL用于琼脂糖凝胶电泳，部分单株的检测结果如图2所示，共检测到21个转基因单株中PnWRKY12在转录水平大量表达，这些单株的编号为1~21。
将实验室保存的棕黑腐质霉接种于PDA固体培养基（200 g/L马铃薯，20 g/L琼脂，20 g/L葡萄糖）上，28℃暗培养7 d。选取温室中生长良好、大小均一且完全伸展的野生型烟草和PnWRKY12转基因烟草叶片，用手术剪从叶柄除剪下。用无菌塑料枪头在叶片约相同位置形成大小一致的伤口，分别接种大小相等的棕黑腐质霉菌丝块。将接种后的叶片置于铺有无菌水浸湿滤纸的平板中，于28℃光照培养箱中培养，每天加水保湿。培养7 d后收集叶片并观察各株系叶片的发病情况。结果如图3所示，接种棕黑腐质霉后，野生型烟草的叶片形成较大的病斑，叶片出现黄化和腐烂的现象，而转基因烟草叶片的症状很轻微，形成的病斑面积也远小于野生型烟草。显然，PnWRKY12转基因烟草对棕黑腐质霉具有很强的抗性。
Claims (1)
1.一种三七WRKY转录因子基因PnWRKY12在提高烟草对棕黑腐质霉（Humicola fuscoatra）抗性中的应用。
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