CN116286752A - 苦荞天冬氨酸蛋白酶及其编码基因和应用 - Google Patents
苦荞天冬氨酸蛋白酶及其编码基因和应用 Download PDFInfo
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Abstract
本发明提供了一种核苷酸序列及其编码的氨基酸序列，所述的氨基酸序列具有抗立枯丝核菌的效果，其编码序列通过构建转基因植株，能够提高植株的立枯病抗性，特别是针对苦荞植株的立枯病抗性。本发明的方法简单，效果可靠、成本低廉，生产过程高效、绿色、安全、无环境污染，对农业发展具有一定推动作用。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种天冬氨酸肽酶基因FtASP及其编码蛋白，及其在抗立枯丝核菌中的应用。
背景技术
苦荞(Fagopyrum tataricum)为蓼科(Polygonaceae)荞麦属(Fagopyrum)的双子叶植物，富含蛋白质、维生素及各种药用类黄酮物质。近年来随着种植面积的不断增加，立枯病也呈逐年加重的趋势，现已成为荞麦第一大病害。
荞麦立枯病病原体是一种多核的立枯丝核菌，具3个或3个以上的细胞核，菌丝较大。在土壤中能形成薄层蜡质状或白粉色网状至网膜状子实层，产生的担子桶形至亚圆筒形，担子具3-5个小梗，其上着生担孢子；担孢子椭圆形至宽棒状，担孢子能重复萌发，在担子上形成2次担子。该菌由单一菌丝尖端的分枝密集而形成或是由尖端紧密地和菌丝密集而形成菌丝结。由致病菌菌侵染引起的荞麦立枯病主要在幼苗期危害地下种子或幼苗茎基部，严重时可导致种子腐烂，幼苗萎蔫枯死，导致产量和品质显著下降，荞麦立枯病抗性问题亟待解决。
天冬氨酸蛋白酶(aspartic protease)是一类蛋白水解酶，其可以在酸性pH下以天冬氨酸残基作为催化活性位点，在植物生长发育和防御反应过程中起着重要作用。植物天冬氨酸蛋白酶均含有保守结构，即天冬氨酸-苏氨酸-甘氨酸(DTG)和天冬氨酸-丝氨酸-甘氨酸(DSG)保守活性中心，其次还有N端的信号肽、前导片段、PSI和C端结构。天冬氨酸蛋白酶已被证明在胡萝卜、番茄和小麦中能够抑制病原菌分泌的糖基水解酶和木聚糖酶等。然而荞麦中天冬氨酸生物代谢过程及其主要功能研究领域几乎仍是空白。在本研究中通过基因克隆和筛选获得了一个重要的FtASP基因，其对立枯病的抗性功能进行了较系统的探究，填补了苦荞中天冬氨酸蛋白酶功能研究的空白。
发明内容
本发明提供了一种基于苦荞天冬氨酸蛋白酶研究的核苷酸序列和氨基酸序列，旨在提高植株的立枯病抗性，为农业生产提供新的方法。
一方面，本发明提供了一种氨基酸序列。
所述的氨基酸序列为包括SEQ ID NO.1所示的序列。
所述的氨基酸序列所表示的蛋白的分子量约24.1KDa，所编码蛋白的等电点(pI)为9.10，为疏水蛋白。预测该蛋白的三级结构如图4。
另一方面，本发明提供了编码前述的氨基酸序列的核苷酸序列。
所述的核苷酸序列可以用于表达前述的氨基酸序列，包括基于密码子简并性具有表达前述氨基酸序列功能的任意核苷酸序列。
作为优选地，所述的核苷酸序列为SEQ ID NO.2，或与SEQ ID NO.2具有93.5％以上相似性的核苷酸序列。
本发明同时提供了前述的核苷酸序列的表达载体以及包含该表达载体的基因工程细胞。
所述的表达载体上还可以包括编码His标签的序列；所述的His标签上包括4-10个His，优选为6个。
再一方面，本发明提供了前述的氨基酸序列(蛋白)的制备方法。
所述的制备方法通过基因工程表达前述的核苷酸序列实现。
具体地，所述的制备方法中包括：将前述的核苷酸序列插入表达载体，转染细胞后培养细胞进行表达。
优选地，所述的制备方法中包括将前述的核苷酸序列插入His标签载体后获得His-FtASP重组质粒进行表达。
优选地，所述的细胞包括但不限于大肠杆菌BL21(DE3)。
所述的制备方法中还可以包括对前述的核苷酸进行扩增。所述的扩增可以是PCR扩增。所述的PCR扩增的引物可以选自SEQ ID NO.3-6。
再一方面，本发明提供了前述的氨基酸序列或核苷酸序列或表达载体或基因工程细胞在抑制立枯丝核菌中的应用。
本发明同时提供了包含前述的氨基酸序列或表达载体或基因工程细胞的抑制立枯丝核菌的制剂，具体为抗菌剂。
又一方面，本发明提供了前述的核苷酸序列或表达载体或基因工程细胞在构建抗立枯病的植株中的应用。
具体地，所述的立枯病的病原体为立枯丝核菌。
又一方面，本发明提供了一种过表达FtASP基因的植株的构建方法。
所述的方法中包括使用前述的核苷酸序列插入载体构建重组载体，重组载体转染菌株获得重组菌，重组菌株侵染目标植株获得过表达FtASP基因的植株。
优选地，所述的重组载体为过表达载体pCAMBIA 1307-FtASP。
所述的过表达载体pCAMBIA 1307-FtASP上包括FtASP的全长序列。
所述的全长序列的扩增引物为SEQ ID NO.5和SEQ ID NO.6。
所述的全长序列的扩增模板为FtASP-T载体。
所述的FtASP-T载体的制备方法为：引物扩增来源于苦荞的cDNA模板。
所述的引物可以是SEQ ID NO.3-4。
所述的苦荞的品种可以是苦荞1号。
优选地，所述的转染菌株为转染农杆菌。
优选地，所述的农杆菌采用蘸花法侵染目标植株。
优选地，所述的植株为拟南芥或苦荞。
本发明的有益效果：
通过基因克隆获得了一个重要的FtASP基因，利用基因工程手段，将天冬氨酸蛋白酶FtASP基因进行遗传转化获得过表达的转基因拟南芥，并进行抗立枯丝核菌鉴定发现FtASP转基因拟南芥具有更高的病菌抗性；创建了His-FtASP、His-FtASP-N及His-FtASP-C融合载体，将其转化至宿主菌进行原核表达以快速获得重组His-FtASP、His-FtASP-N及His-FtASP-C蛋白，并完成了重组蛋白对立枯丝核菌生长的影响鉴定；同时，为后续深入研究天冬氨酸的代谢机制奠定了理论基础。本发明效果可靠、成本低廉；生产过程高效、绿色、安全、无环境污染。
附图说明
图1为pCAMBIA 1307载体图谱。
图2为PET28A载体图谱。
图3为转基因植株FtASP基因表达量。
图4为FtASP过表达拟南芥抗立枯丝核菌侵染效果。
图5为FtASP过表达拟南芥病菌侵染实验发病率和病情指数统计。
图6为DAB染色观测叶片的受损伤程度。
图7为病原菌侵染的FtASP过表达拟南芥叶片MDA活性。
图8为FtASP编码的蛋白质亲疏水性分析。
图9为FtASP编码的蛋白质三级结构预测图。
图10为重组His-蛋白Western blot鉴定结果。
图11为His-FtASP蛋白对立枯丝核菌生长的影响。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1FtASP基因的抗菌特性
(1)FtASP CDS的克隆
实验用苦荞材料为品苦1号。
选取2周龄-6周龄苗，取植株50-100mg，加液氮充分研磨后，使用Trizol法提取总RNA。以此RNA为模板，使用
III 1st Strand cDNA Synthesis Kit(+gDNA wiper)试剂盒(南京诺唯赞生物技术有限公司)进行反转录获得幼苗的cDNA。
根据FtASP的ORF设计特异性引物FtASP-F：SEQ ID NO.3，FtASP-R：SEQ ID NO.4，以品苦1号幼苗的cDNA为模板进行PCR扩增，获得目的基因的CDS序列。PCR程序为95℃，3min；95℃，30s，57℃，60s，72℃，90s，31个循环。
(2)重组质粒的构建
将PCR纯化产物连接到pTOPO-Blunt Simple平末端克隆载体上，获得FtASP-T载体质粒。经测序、分析、拼接后得到FtASP全长序列，即SEQ ID NO.2。
以FtASP-T载体为模板设计同源重组引物，PCR扩增FtASP的全长序列。上游引物为1307-FtASP-XbaIF：SEQ ID NO.5，下游引物为1307-FtASP-PstIR：SEQ ID NO.6。随后经酶切、回收和连接转化后，将FtASP全长序列正向插入pCAMBIA 1307空载体的CaMV35S启动子下游(图1)，经测序完全后得到过表达载体pCAMBIA 1307-FtASP。
(3)转化农杆菌
pCAMBIA 1307-FtASP重组质粒和pCAMBIA 1307空载体质粒用热激法分别转化农杆菌GV3101感受态细胞，经菌落PCR鉴定后，将得到pCAMBIA1307-FtASP重组质粒阳性菌和pCAMBIA 1307空载体阳性菌。
(4)侵染拟南芥
步骤(3)得到的重组菌分别用蘸花法侵染拟南芥获得转基因植株，获得的转化植株经实时荧光定量PCR(Quantitative Real-time PCR，qPCR)检测FtASP基因表达量。
具体检测方法为：将侵染后的拟南芥种子用10％次氯酸钠消毒8分钟后，用无菌水清洗4-5次，随后将处理过的种子种于含有潮霉素(Hyg)抗性的MS固体培养基(Hyg浓度为100mg/mL)上，培养1-2周，能够长出真叶的拟南芥植株初步认定为候选阳性植株。利用FtASP-EX1：SEQ ID NO.7，FtASP-EX2：SEQ ID NO.8引物，使用Taq Pro Universal SYBRqPCR Master Mix试剂盒(南京诺唯赞生物技术有限公司)检测基因表达，转基因植株FtASP基因表达量高于对照组(图3)，确认获得FtASP过表达植株。
(5)抗病性检测
以野生型(Col-0)和转空载体(Ev)植株为对照，将对照组及过表达(FtASP-OE)阳性植株拟南芥种子于MS固体培养基中培养1-2周后，挑选长势良好且一致的植株移栽到土中，再培养1-2周左右待拟南芥莲座叶长到12片左右，抽薹之前，剪取大小基本一致的叶片放于已有浸湿滤纸的培养皿中，在叶片中间放置一个直径0.5mm的立枯丝核菌菌饼(所用菌株为AG4-HGI 3菌株)，然后用保鲜膜包裹培养皿放置于28℃真菌生化培养箱进行病菌侵染实验，2天后进行发病率和病情指数统计。发病率和病情指数的统计方法具体如下：
立枯丝核菌侵染拟南芥叶片发病分级标准
根据图4和图5可看出，FtASP过表达拟南芥在遭受立枯丝核菌的侵染后，产生的病变面积远小于对照组，其发病率和病情指数也低于对照组，表明FtASP过表达拟南芥拥有更强的立枯病抗性。
拟南芥叶片在被立枯丝核菌侵染后会有过氧化氢酶的富集，用DAB染色可直接观测到过氧化物酶的富集程度，进一步判断叶片的受损伤程度。使用DAB试剂盒(北京索莱宝科技有限公司)对病原菌侵染2天后的叶片进行DAB染色，由图6可看出，FtASP-OE过表达植株的抗病性比对照组更强。
丙二醛MDA可以抑制病原菌的侵入，因其具有强氧化性，所以丙二醛的含量积累会破坏细胞膜的结构，因此MDA含量增加会直接损伤细胞，在植物与病原物互作过程中是植物抗病性反应的重要物质。使用MDA含量检测试剂盒(北京索莱宝科技有限公司)，对病原菌侵染0，16，22和28小时后的叶片进行MDA活性检测，由图7可看出，FtASP-OE过表达植株MDA含量比对照组少，说明FtASP-OE过表达植株抗病性更强。
实施例2FtASP基因编码的蛋白的抗菌性
(1)氨基酸序列分析
将FtASP基因氨基酸序列SEQ ID NO.1在NCBI数据库中BLAST比对。并利用expasy在线数据工作网站https://web.expasy.org/compute_pi/和https://web.expasy.org/protscale/预测该蛋白的等电点和亲疏水性。利用https://swissmodel.expasy.org/interactive预测蛋白的三级结构。
FtASP基因的CDS长度为675bp，编码蛋白SEQ ID NO.2中225个氨基酸，分子量约24.1KDa，所编码蛋白的等电点(pI)为9.10。该蛋白为疏水蛋白(图8)，预测的的三级结构如图9。
(2)重组His-FtASP、His-FtASP-N及His-FtASP-C蛋白的表达纯化
以FtASP基因的CDS为模板，使用PET28A-FtASP-BamHlF：SEQ ID NO.9，PET28A-FtASP-NotlR：SEQ ID NO.10；PET28A-FtASP-N-BamHlF：SEQ ID NO.11，PET28A-FtASP-N-NotlR：SEQ ID NO.12；PET28A-FtASP-C-BamHlF：SEQ ID NO.13，PET28A-FtASP-C-NotlR：SEQ ID NO.14三对引物分别扩增FtASP(全长1-225aa)、FtASP-N(N端1-112aa)及FtASP-C(C端113-225aa)目的片段。将PCR产物用TaKaRa MiniBEST Plasmid Purification Kit Ver4.0进行切胶回收纯化后，连接到His标签载体(图2)，获得His-FtASP、His-FtASP-N及His-FtASP-C重组质粒。
重组质粒转化至宿主菌DE3后筛选阳性克隆进行培养，蛋白纯化采用NEB公司的amylase resin(E8021S)产品说明书上的方法，具体操作如下：取含目的载体的单克隆在5mL液体LB(含50μg/mL卡那霉素)培养基中，37℃，220rpm培养8-12h；将培养完成的菌液全部转入250mL无抗LB液体培养基中，37℃，220rpm培养1-3h使OD600达到0.8左右；将摇床温度调至20℃，转速调至150rpm，待培养基的温度降低至20℃后加入IPTG至终浓度为0.2mM，诱导培养8h；4℃，5,000rpm离心15min收集菌体；加入平衡缓冲液(1M Tris HCl-NaCl,pH8.0)，重悬菌体，超声破碎菌体，至菌液澄清；4℃，12,000rpm离心15min，将上清用0.4μm滤膜过滤后，加入预先用平衡缓冲液平衡的amylase resin柱子中，使蛋白与填料结合；用5-10倍柱体积的平衡缓冲液洗涤层析柱，以除去没有结合的杂蛋白；用5mL的洗脱缓冲液(6.5mM NaH2PO4，3M NaCl，0.25M咪唑，10mM Ni)洗脱，收集流出液；SDS-PAGE、Westernblot检测纯化的蛋白，BCA蛋白定量试剂盒(康为世纪)测定蛋白浓度。
在蛋白样品中加入5×Loading Buffer使其终浓度为1×，沸水浴煮10min，12,000rpm离心10min后吸取适量的上清加入点样孔中，80V恒压电泳，待溴酚蓝进入分离胶后将电压设置为120V继续电泳直至完成；电泳结束后小心剥取凝胶放入考马斯亮蓝R-250染液中，在水平摇床上缓慢摇动室温染色3h以上；染色结束后，将凝胶转移至考马斯亮蓝染色脱色液中，在水平摇床上缓慢摇动4-8h脱色，期间更换脱色液2-3次。
SDS-PAGE电泳结束后，小心取出凝胶并放入预冷的转膜缓冲液中浸泡；夹好凝胶，冰浴中转膜90min；转膜完成后，小心剥取PVDF膜，放入TBST中漂洗3次，每次5min，然后在膜正面均匀滴加TBST稀释好的丽春红染色，观察转膜效果；膜先在TBST中漂洗3-5次，每次5-10min，然后转入5％(W/V)脱脂奶粉的TBST溶液中，室温平缓摇动2-3h或4℃过夜；加一抗孵育：用TBST稀释一抗(根据抗体效价按一定比例稀释)，然后将封闭好的PVDF膜放入稀释好的一抗中，室温下在摇床上缓慢摇动孵育3h左右或4℃过夜；加二抗孵育：在TBST中漂洗3-5次，每次5-10min，用TBST按照比例稀释二抗，然后将一抗孵育完的PVDF膜放入稀释好的二抗中，室温下在摇床上缓慢摇动孵育30-60min；TBST中漂洗3-5次，每次5-10min将膜平铺于透明塑料膜上，滴加ECL显色反应液至覆盖膜表面，然后盖上透明塑料膜，用化学发光成像仪检测。根据图10可看出，重组质粒转化到表达宿主大肠杆菌DE3，经IPTG诱导后，有重组His-蛋白的表达，上清蛋白经Ni柱纯化后得到了较纯的重组蛋白。纯化的蛋白可用于进一步的实验分析。
(3)重组蛋白对立枯丝核菌增殖的影响
将His-FtASP、His-FtASP-N及His-FtASP-C重组蛋白和His空载蛋白液体过滤后加入同样接入了立枯丝核菌菌饼的50mL PDB液体培养基(北京酷来搏科技有限公司)中进行培养，培养一天后发现加入了His-FtASP和His-FtASP-N蛋白的PDB培养基中立枯丝核菌的菌丝球要少于加入了His-FtASP-C和His空载蛋白的PDB培养基中的立枯丝核菌菌丝球(图11)，表明重组FtASP和FtASP-N端蛋白可以抑制立枯丝核菌的生长。综上，天冬氨酸蛋白酶FtASP是苦荞抗立枯病的重要基因。
本发明通过基因克隆获得了一个重要的FtASP基因，对苦荞天冬氨酸的生物代谢过程中所涉及的天冬氨酸的功能进行了较系统的探究，填补了苦荞中天冬氨酸基因功能的空白，深入研究了苦荞天冬氨酸基因对抗立枯丝核菌的作用。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (16)
1.一种氨基酸序列，其特征在于，包括SEQ ID NO.1所示的序列。
2.编码权利要求1所述的氨基酸序列的核苷酸序列。
3.根据权利要求2所述的核苷酸序列，其特征在于，为SEQ ID NO.2，或与SEQ ID NO.2具有93.5％以上相似性的核苷酸序列。
4.包含权利要求2-3任一项所述的核苷酸序列的表达载体。
5.根据权利要求4所述的表达载体，其特征在于，还包括编码组氨酸标签的序列。
6.包含权利要求4-5任一项所述的表达载体的基因工程细胞。
7.权利要求1所述的氨基酸序列的制备方法，其特征在于，通过基因工程表达权利要求2-3任一项所述的核苷酸序列进行制备。
8.根据权利要求7所述的制备方法，其特征在于，包括将权利要求2-3任一项所述的核苷酸序列插入His标签载体后获得His-FtASP重组质粒进行表达。
9.权利要求1所述的氨基酸序列或权利要求2-3任一项所述的核苷酸序列或权利要求4-5任一项所述的表达载体或权利要求6所述的基因工程细胞在抑制立枯丝核菌中的应用。
10.包含权利要求1所述的氨基酸序列或权利要求4-5任一项所述的表达载体或权利要求6所述的基因工程细胞的抑制立枯丝核菌的制剂。
11.权利要求2-3任一项所述的核苷酸序列或权利要求4-5任一项所述的表达载体或权利要求6所述的基因工程细胞在构建抗立枯病的植株中的应用。
12.根据权利要求11所述的应用，其特征在于，所述的立枯病的病原体为立枯丝核菌。
13.一种过表达FtASP基因的植株的构建方法，其特征在于，包括使用权利要求2-3任一项所述的核苷酸序列插入载体构建重组载体，重组载体转染菌株获得重组菌，重组菌株侵染目标植株获得过表达FtASP基因的植株。
14.根据权利要求13所述的构建方法，其特征在于，所述的重组载体为过表达载体pCAMBIA 1307-FtASP。
15.根据权利要求14所述的构建方法，其特征在于，所述的转染菌株为转染农杆菌。
16.用于扩增权利要求2-3任一项所述的核苷酸序列的引物，其特征在于，选自SEQ IDNO.3-6。
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