CN117305267B - 葡萄类受体蛋白激酶herk1的新用途 - Google Patents
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Abstract
本发明涉及一种葡萄类受体蛋白激酶的新用途。本发明所述的葡萄类受体蛋白激酶HERK1(VvHERK1)、编码该VvHERK1的编码基因、含有前述编码基因的重组表达载体可以抑制葡萄卷叶伴随病毒2号(GLRaV‑2)基因组的复制。本发明分别通过在葡萄愈伤组织和组培苗中用农杆菌介导法共转化能表达葡萄类受体蛋白激酶HERK1(VvHERK1)的质粒和GLRaV‑2侵染性cDNA克隆，发现过表达VvHERK1可将GLRaV‑2的RNA和蛋白水平均降低约50％，这表明过表达VvHERK1可极有效抑制GLRaV‑2基因组复制，可以有效防治葡萄卷叶病，同时有利于葡萄的分子育种。
Description
技术领域
本发明涉及植物基因工程技术领域，具体说是一种葡萄类受体蛋白激酶HERK1的新用途。
背景技术
葡萄是我国重要的栽培物种，据统计，2019年我国葡萄种植面积高达1062万亩左右。而葡萄也是感染病毒最多的果树树种之一，在生产中发生严重的葡萄病毒病主要有葡萄卷叶病、扇叶病和栓皮综合症，其中葡萄卷叶病是世界范围内发生最普遍、危害最严重的葡萄病毒病害（Naidu R A, Maree H J, Burger J T. Grapevine leafroll disease andassociated viruses: a unique pathosystem. Annual Review of Phytopathology,2015, 53: 613–634），在我国的发生率更是高达80–90%（Liu M H, Li M J, Qi H H, etal. Occurrence of grapevine leafroll-associated viruses in China. PlantDisease, 2013, 97: 1339–1345）。该病可造成葡萄产量减少30–40%左右，葡萄果粒大小不等、着色差、且可溶性固形物含量显著降低，严重影响葡萄产量和品质（Song Y, Hanner RH, Meng B. Probing into the effects of grapevine leafroll-associated viruseson the physiology, fruit quality and gene expression of grapes. Viruses,2021, 13: 593）。目前，葡萄卷叶病已成为制约我国葡萄产业发展的关键因素。已知有6种葡萄卷叶伴随病毒（GLRaVs）与该病相关，而葡萄卷叶伴随病毒2号（GLRaV-2）是与葡萄卷叶病相关最重要的病原物之一（Song Y, Hanner R H, Meng B. Probing into the effectsof grapevine leafroll-associated viruses on the physiology, fruit quality andgene expression of grapes. Viruses, 2021, 13: 593）。此外，GLRaV-2侵染也与葡萄嫁接不亲和综合症的发生有关（Meng B , Martelli G P , Golino D A , et al.Grapevine Viruses: Molecular Biology, Diagnostics and Management || Grapevineleafroll-associated virus 2. 2017, 10.1007/978-3-319-57706-7: 141-165）。与其它植物病毒病害一样，葡萄植株一旦被GLRaVs等病毒入侵，则难以用化学药剂防治。因此鉴定葡萄植物在抗GLRaVs防卫反应中起重要作用的蛋白，可拓宽对葡萄卷叶病的防控途径，并为最终通过转基因技术获得抗GLRaVs葡萄新种质提供重要的理论依据和试验材料。
在病毒入侵时，植物会采取多种防御途径如促分裂原活化激酶（mitogen-activated protein kinase，MAPK）级联信号途径（Hu T, Huang C, He Y, et al. βC1protein encoded in geminivirus satellite concertedly targets MKK2 and MPK4 tocounter host defense. PLoS Pathogen, 2019, 15: e1007728）及SA、JA等介导的信号传导途径（Alazem M, Lin N S. Roles of plant hormones in the regulation of hostvirus interactions. Molecular Plant Pathology, 2015, 16: 529–540）来抵抗病毒侵染。目前已知植物类受体蛋白激酶（receptor-like kinase, RLK）在其中起着极其关键的作用（Antolin-overa M, Ried M, Binder A, et al. Receptor kinase signalingpathways in plant-microbe interactions. Annual Review of Phytopathology,2012, 50: 451–473）。
RLK是一类跨膜蛋白激酶家族，是蛋白激酶中的重要一类，也是信号分子的重要受体，能够感受外界刺激，从而在信号转导过程中起着重要作用。植物遭受病原物入侵时，RLK的胞外结构域识别细胞外的病原信号分子后将信号传递到胞内，其胞内激酶结构域随后通过磷酸化将信号传导到下游蛋白，从而启动一系列下游信号转导途径，如MAPK、SA、JA等信号通路，最终诱导植物抗病防卫基因如病程相关蛋白基因、防御素基因等的表达。目前已发现有几种RLKs如BAK1、BAM1和NIK1在植物抗病毒防卫反应起十分重要的作用。如植物在遭受病毒入侵时可以通过BAK1激发植物病原相关分子模式（PTI）防御机制（Kørner C J,Klauser D, Niehl A, et al. The immunity regulator BAK1 contributes toresistance against diverse RNA viruses. Molecular Plant-Microbe Interaction,2013, 26: 1271–1280）。BAM1通过促进RNA沉默信号在细胞间移动而在植物抗病毒防卫反应中起重要作用（Rosas-Diaz T, Zhang D, Fan P, et al. A virus-targeted plantreceptor-like kinase promotes cell-to-cell spread of RNAi. PNAS. 2018, 115:1388-1393)。此外，NKI1也介导植物抗病毒信号途径（Carvalho C , Santos A , Pires S, et al. Regulated nuclear trafficking of rpL10A mediated by NIK1 representsa defense strategy of plant cells against viruses. PLoS Pathogen, 2008, 4:e1000247）。但目前尚不清楚其它众多RLK家族成员在病毒侵染时的功能。
发明内容
针对现有技术中存在的缺陷，本发明的目的在于提供一种葡萄类受体蛋白激酶HERK1的新用途。本发明分别通过在葡萄愈伤组织和组培苗中用农杆菌介导法共转化能表达葡萄类受体蛋白激酶HERK1（VvHERK1）的质粒和GLRaV-2侵染性cDNA克隆，发现过表达VvHERK1可极有效抑制GLRaV-2 基因组复制，有利于防治葡萄卷叶病和葡萄的分子育种。
为达到以上目的，本发明采取的技术方案是：
葡萄类受体蛋白激酶HERK1（VvHERK1）的新用途，其特征在于，所述葡萄类受体蛋白激酶HERK1可以抑制GLRaV-2基因组的复制，上述葡萄类受体蛋白激酶HERK1的序列如SEQID NO:1所示。
编码上述葡萄类受体蛋白激酶HERK1的编码基因的应用，其特征在于：所述编码基因通过其编码的葡萄类受体蛋白激酶HERK1可以抑制GLRaV-2基因组的复制，上述编码基因的序列如SEQ ID NO:2所示。
含有上述编码基因的重组表达载体的应用，其特征在于：所述重组表达载体上包含的编码基因通过其编码的葡萄类受体蛋白激酶HERK1可以抑制GLRaV-2基因组的复制；
所述重组表达载体为在pK2GW7载体将ccdB序列替换为SEQ ID NO:2所示序列得到的重组表达载体。
本发明所述的葡萄类受体蛋白激酶HERK1的新用途，其有益效果为：
通过在葡萄愈伤组织和组培苗中用农杆菌介导法共转化能表达葡萄类受体蛋白激酶HERK1（VvHERK1）的质粒和GLRaV-2侵染性cDNA克隆，发现过表达VvHERK1可将GLRaV-2的RNA和蛋白水平均降低约50%，表明过表达VvHERK1可极有效抑制GLRaV-2 基因组复制。这将在防治葡萄卷叶病中发挥重要作用，同时为葡萄的分子育种打下良好的基础。
附图说明
本发明有如下附图：
图1 为pK2GW7- VvHERK1重组载体示意图；
图2为菌落PCR筛选pK2GW7- VvHERK1重组载体；
图3 为在葡萄愈伤组织中过表达VvHERK1对GLRaV-2基因组复制的影响；
图4 为在葡萄组培苗中过表达VvHERK1对GLRaV-2基因组复制的影响。
具体实施方式
以下结合附图对本发明作进一步详细说明。
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。
实施案例1、过表达葡萄类受体蛋白激酶VvHERK1抑制葡萄卷叶伴随病毒2号基因组复制。
一、构建VvHERK1的植物表达载体（pK2GW7- VvHERK1）
CTAB法提取葡萄组培苗茎叶RNA，将500 ng RNA用NovoScript® Plus All-in-one 1st Strand cDNA Synthesis SuperMix （gDNA Purge）反转录试剂盒（购自诺唯赞生物科技股份有限公司）所提供的随机引物和反转录试剂进行反转录得到cDNA。然后通过Gateway技术构建pK2GW7- VvHERK1。操作按Gateway™ BP Clonase™ II Enzyme Mix和Gateway™ LR Clonase™ II Enzyme Mix试剂盒说明书进行。以上述cDNA为模板，用上下游引物：attB1：5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGATGGGTTGTGGGAAAAT-3′、attB2：5′-GGGGACCACTTTGTACAAGAAA GCTGGGTGCGTAATCTGGAACATCGTAT-3′进行PCR扩增得到VvHERK1蛋白的编码基因序列，该编码基因的核苷酸序列如序列表中序列2所示，该序列所编码的VvHERK1氨基酸序列如序列表中序列1所示。将经过两轮PCR扩增到带有attB接头的产物用DNA胶回收试剂盒（购自诺唯赞生物科技股份有限公司）进行目的片段胶回收，然后按照说明书进行BP反应，将BP反应后的连接产物转入大肠杆菌DH5α感受态细胞，后将菌液涂布于含有卡那霉素的LB平板上，用菌落PCR筛选阳性克隆，将其中1个阳性克隆送擎科公司测序。将测序正确的样品提质粒按照说明书方法进行LR反应，将LR反应后的连接产物转入大肠杆菌DH5α感受态细胞，后将菌液涂布于含有壮观霉素的LB平板上，用菌落PCR筛选阳性克隆, 检测结果见图2（图2中泳道M为DNA分子量标准；泳道1、2、5、8、10为阳性克隆）。从图2可见，泳道1、2、5、8、10均有2490 bp大小的片段，与预期结果一致。将其中1个阳性克隆进行测序和比对后证明该阳性克隆确实插入了VvHERK1基因序列，且阅读框正确，表明重组质粒构建正确。将该重组质粒命名为pK2GW7- VvHERK1（图1）。
二、用冻融法分别将pK2GW7-VvHERK1、pK2GW7和GLRaV-2侵染性cDNA克隆质粒转化农杆菌GV3101。
将各装有30 ul农杆菌GV3101感受态（唯地公司产品）的3个离心管（将其分别编号为1、2和3号）放在冰上，依次在1、2、3号离心管中加入量为1ug 的pK2GW7、pK2GW7-VvHERK1和GLRaV-2侵染性克隆质粒（公众可从中国农业大学获得，记载过该载体的非专利文献是：（Zhang C, Wang X, Li H, et al. GLRaV-2 p24 protein suppresses host defensesby interaction with a RAV transcription factor from grapevine. PlantPathology, 2022, 189: 1848–1865），充分混匀。冰上放置5 min、液氮快速冷却5 min后迅速转入37 ℃水浴5 min、放置冰上5 min。然后在离心管中各加500 ul无抗生素的YEP液体培养基后在28 ℃条件下用200 r/min 摇培养2–4 h，吸取150 ul菌液涂布到含相关抗生素的YEP固体培养基上，28 ˚C恒温倒置培养48-60 h。挑取单个、饱满菌落接种至800 ul含相关抗生素的YEP液体培养基中，28 ℃ 200 rpm摇床中摇菌12-16 h。用菌落PCR筛选阳性克隆。取2 ml 阳性单克隆菌液，28 ℃ 180 rpm摇培24 h后，按照1:100加菌液于三角瓶大摇过夜。用6000 rpm转速离心5 min收集菌液，将分别携带pK2GW7–VvHERK1和GLRaV-2侵染性克隆质粒的菌液沉淀用30 ml 农杆菌悬浮液（10 mM MES, 100 mM MgCl2和10 uMacetosyringone，MMA）重悬，使OD600达到1.0。用同样的方法得到含有pK2GW7和GLRaV-2侵染性克隆质粒的农杆菌悬浮液作为后面试验的对照。上述摇菌及集菌等过程均需无菌操作。
三、在无病毒葡萄愈伤组织中分别共转化pK2GW7–VvHERK1/GLRaV-2、pK2GW7/GLRaV-2。
将每份葡萄愈伤组织（10 g）（公众可从中国农业大学获得，记载过该试验材料的非专利文献是：（Zhang C, Wang X, Li H, et al. GLRaV-2 p24 protein suppresseshost defenses by interaction with a RAV transcription factor from grapevine.Plant Pathology, 2022, 189: 1848–1865）放入30 ml上述携带有两种质粒的农杆菌悬浮液中，常温条件下用100 rpm摇30 min 。然后将液体愈伤组织用两层无菌纱布过滤农杆菌悬浮液后将其置于无菌滤纸上进一步吸干液体。最后将愈伤组织置于B5培养基上，在25 °C暗培养3天。以上实验过程均需无菌操作。3天后分别提取愈伤组织的总RNA和总蛋白，-80 °C保存备用。
四、在无病毒夏黑葡萄组培苗中用真空渗透法分别共转化pK2GW7–VvHERK1/GLRaV-2、pK2GW7/GLRaV-2。
在无菌广口瓶加入40 ml上述携带有两种质粒的农杆菌悬浮液，将继代培养4周的夏黑葡萄组培苗浸没其中。将广口瓶放在玻璃真空干燥器中用-83 kPa抽滤，20 min后取出组培苗移入WPM固体培养基中培养。3天后提取组培苗细嫩茎叶的总RNA和总蛋白，-80°C保存备用。
五、Real-time qPCR检测GRLaV-2 RNA积累水平。
分别将上述步骤三和四得到的总RNA用DNase I消解后，在紫外分光光度计上分别测定其在230 nm、260 nm和280 nm的吸光值以确定其纯度和浓度。用试剂盒ChamQ SYBRqPCR Master Mix（High ROX Premixed）（诺唯赞生物科技股份有限公司产品）进行Real-time qPCR，检测葡萄Actin基因（VvActin, XM_002282480.4）的表达作为内参。以500 ng总RNA作为模板，以试剂盒自带随机引物作为反向引物，进行RT反应。每个反应管依次加入0.5ul gDNA Purge和 500 ng总RNA, 用RNase Free Water补足至5 ul。42 ℃孵育5 min，反应结束后冰上放置。向反应物中加入5 ul 2×NovoScript® Plus 1st Strand cDNASynthesis SuperMix。50 ℃孵育 15 min，75 ℃孵育 5 min，终止反应。反应产物-20°C 保存。
每个PCR反应管中加入1.0 ul用RNase Free Water稀释10倍的RT产物、5 ul 2 ×ChamQ SYBR qPCR Master Mix、正反向引物各0.2 ul、最后加3.6 ul dd H2O补充至10 ul。在ABI公司生产的荧光定量PCR-ABI7500扩增仪进行反应。反应条件是95 oC，30 s；95 oC，10s；59 oC，30 s；40个循环。Real time PCR结束以后，根据扩增仪自带的软件对数据进行分析。
检测VvHERK1 mRNA的引物如下：
正向引物：5′-TGTTCAGTTCTTTCGATGGGTT-3′；
反向引物：5′-AGGATGAGTCAGATTTCGTGGC-3′。
检测 GLRaV-2 基因组RNA的引物如下：
正向引物：5′-GCTCAACCTCTCGTGCG-3′；
反向引物：5′-CGCTTCTCTCACTCCCAC-3′。
检测VvActin的mRNA的引物如下：
正向引物：5′-CCCCATGCTATCCTTCG-3′；
反向引物：5′-AGGCAGCTCATAGTTCTTCTC-3′。
实验结果见图3A和图4A，图中标注的VvHERK1和pK2GW7分别表示共转化pK2GW7–VvHERK1/GLRaV-2和pK2GW7/GLRaV-2。从图3A和图4A可见，与对照（共转化pK2GW7/GLRaV-2）相比，转入pK2GW7–VvHERK1/GLRaV-2的葡萄愈伤组织和组培苗中的VvHERK1 mRNA水平显著增加，而GLRaV-2基因组RNA水平显著下降，降幅为50%以上。这说明过表达VvHERK1能有效抑制GLRaV-2基因组复制。
六、Western blotting检测GRLaV-2 蛋白积累水平。
将上述步骤三和步骤四得到的植物总蛋白进行Western blot检测GLRaV-2蛋白积累水平。GLRaV-2基因组第10个阅读框所编码的蛋白为24kDa蛋白（p24），本实验用p24的抗血清检测p24来代表GLRaV-2蛋白积累水平。
将样品放入1.5 ml离心管中加入200 ul蛋白提取液，1 ul蛋白酶抑制剂，混匀。4℃下12000 g离心5 min。取上清液并加入SDS-蛋白上样缓冲液（含DTT），100 ℃煮沸5 min。将变性后的样品放于冰上，分别制备分离胶和浓缩胶，凝30 min后可用。 缓慢上样，先用80V电泳20 min、后用120 V电泳1 h。在滤纸上依次放置胶、PVDF膜和滤纸，然后用200 mA电流低温转膜90 min。转膜后用TBST漂洗膜3次，每次5 min。用封闭液（5%脱脂奶粉）封闭60min。用TBST漂洗膜3次，每次5 min。随后进行Western blot检测。先用一抗（p24抗血清1：2000）孵育PVDF膜60 min，用TBST漂洗膜3次。然后用羊抗兔二抗（康为公司产品）孵育PVDF膜60 min，用TBST漂洗膜3次。将发光剂 eECL-A和eECL-B（1：1的混合物）均匀加到膜上，最后将膜放置到化学发光仪中曝光并拍照。
实验结果见图3B和图4B。图上方显示GLRaV-2的蛋白积累水平，下方为各泳道的上样量，用考马斯亮蓝染色。从图3B和图4B可见，与对照（共转化pK2GW7/GLRaV-2）相比，转入pK2GW7–VvHERK1/GLRaV-2的葡萄愈伤组织和组培苗中GLRaV-2蛋白积累水平显著下降，降幅约为50%。这再次说明过表达VvHERK1能有效抑制GLRaV-2基因组的复制。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
Claims (3)
1.葡萄类受体蛋白激酶HERK1的用途，其特征在于，所述用途为葡萄类受体蛋白激酶HERK1抑制GLRaV-2基因组的复制，上述葡萄类受体蛋白激酶HERK1的序列如SEQ ID NO:1所示。
2.如权利要求1所述的葡萄类受体蛋白激酶HERK1的编码基因的应用，其特征在于：所述编码基因通过其编码的葡萄类受体蛋白激酶HERK1抑制GLRaV-2基因组的复制，上述编码基因的序列如SEQ ID NO:2所示。
3.含有如权利要求2所述的编码基因的重组表达载体的应用，其特征在于：所述重组表达载体上包含的编码基因通过其编码的葡萄类受体蛋白激酶HERK1抑制GLRaV-2基因组的复制；
所述重组表达载体为在pK2GW7载体将ccdB序列替换为SEQ ID NO:2所示序列得到的重组表达载体。
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