CN117417921A - 蛋白酶在水解制备根皮素及植物抗虫中的应用 - Google Patents
蛋白酶在水解制备根皮素及植物抗虫中的应用 Download PDFInfo
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Abstract
本申请属于分子检测及植物育种领域,具体涉及一种蛋白酶在水解制备根皮素及植物抗虫中应用,本申请通过调控BGLU13.1‑A或BGLU13.1‑a的表达或酶活来生成根皮素,可用于植物虫害防控及抗性育种。
Description
技术领域
本申请属于生物分子检测及育种技术领域,具体涉及一种蛋白酶在水解制备根皮素及在植物抗虫中应用。
背景技术
根皮素属于类黄酮苷元,对植物生长发育有害,无法在植物体内大量积累(Dareet al.2017;Gutierrez et al.2018)。因此,植物体内,根皮素在相应的糖基转移酶的催化下进行糖基化,形成毒性较低的根皮苷或三叶苷,然后转运到液泡中贮存起来(Gosch etal.,2010;Dare et al.2017;Wang et al.,2020)。然而,在植物遭受逆境胁迫时,其体内贮存的根皮苷和三叶苷会被释放出来,在相应的水解酶的催化下发生水解,产生根皮素。
虫害是制约作物产量和品质的重要因素之一。我国目前对虫害的防治主要依赖化学农药,但化学防治成本高,而且给生态环境带来不可忽视的负面影响。因此,如果可以利用天然产物根皮素,或通过基因工程手段调控植物体围绕根皮素的代谢,提高植物的抗虫性,则对于植物虫害的绿色生物防控或生物育种等具有重要意义和应用价值。
有鉴于此,提出本申请。
发明内容
针对上述技术问题,本申请通过锐意研究发现蛋白酶BGLU13.1-A和BGLU13.1-a具有植物抗虫活性。其中,BGLU13.1-A可将根皮苷水解生成根皮素,BGLU13.1-a可将三叶苷水解生成根皮素。本申请还发现对于不同植物而言,与根皮苷和三叶苷相比,根皮素具有着显著的抗虫活性。因此,本申请通过调控BGLU13.1-A和BGLU13.1-a的表达或酶活性来水解根皮苷和三叶苷进而生成根皮素,或者直接外施根皮素或含根皮素的生物农药,对于植物虫害防控及抗性育种等具有重要的应用价值。
本申请具体提供如下技术方案:
本申请首先提供一种蛋白酶,该蛋白酶具有如下氨基酸序列为:
与SEQ ID NO.3或4具有至少90%同源性,且来源于苹果属。
在一些方式中,所述蛋白酶分别为BGLU13.1-A和BGLU13.1-a,序列分别如与SEQID NO.3或4所示。
在一些方式中,所述蛋白酶序列可通过突变实现催化底物的改变,比如当BGLU13.1-A中的Asn346Phe350Gln356Ala414Ala415Ala615六个氨基酸全部突变为BGLU13.1-a中的Thr322Leu326Lys332Val390Thr391Glu591中的六个氨基酸,或BGLU13.1-a中的Thr322Leu326Lys332Val390Thr391Glu591六个氨基酸全部突变为BGLU13.1-A中的Asn346Phe350Gln356Ala414Ala415Ala615六个氨基酸时,两个蛋白的活性也会随之发生交换,即MBGLU13.1-A(突变后的BGLU13.1-A)失去水解根皮苷的活性而获得水解三叶苷的活性,MBGLU13.1-a(突变后的BGLU13.1-a)也由水解三叶苷转变为水解根皮苷。
因此,本申请的蛋白酶还包括如下序列:
一些方式中,所述蛋白酶序列如与SEQ ID NO.3所示,且SEQ ID NO.3的346Asn350Phe 356Gln 414Ala 415Ala 615Ala六个氨基酸分别替换为Thr Leu Lys ValThr Glu。
另一些方式中,所述蛋白酶序列如与SEQ ID NO.4所示,且SEQ ID NO.4的322Thr326Leu 332Lys 390Val 391Thr 591Glu六个氨基酸分别替换为Asn Phe Gln AlaAla Ala。
本申请还提供一种分离的核酸,其编码上述任蛋白酶的核酸。
在一些优选方式中,所述核酸序列如SEQ ID NO.1或2所示。
本申请还提供一种重组载体,该重组载体包含上述的核酸。
在一些方式中,所述载体为表达载体。
在一些优选方式中,所述表达载体包括原核表达载体或真核表达载体;
在一些特定优选的方式中,所述表达载体包括但不限于:原核表达载体Pet28a,过表达载体pCambia 2300或沉默载体PK7WIWG2D。
本申请还提供一种重组细胞或重组微生物,包含上述的重组载体。
在一些特定方式中,所述重组细胞或重组微生物为大肠杆菌BL21(DE3)或农杆菌GV3101。
本申请还提供上述蛋白酶的如下任一应用:
1)在水解制备根皮素中的应用;
2)在植物抗虫中的应用;
3)在植物育种中的应用。
进一步的,所述抗虫是包括抗二斑叶螨、抗卷叶蛾和抗红蜘蛛等。
另外,鉴于本申请首次发现了根皮素在植物抗中方面的应用,因此本申请还提供了根皮素的如下任一应用:
1)在植物抗虫中的应用;
2)在植物育种中的应用。
进一步的,所述抗虫包括但不限于抗二斑叶螨、抗卷叶蛾和抗红蜘蛛等。
本申请还提供一种改善植物抗虫活性的方法,该方法包括如下任一步骤:
1)通过在植物体中调控上述蛋白酶的表达量或酶活性,通过改善根皮苷和三叶苷水解生成根皮素来实现抗虫;
或者,
2)直接对植物体施加根皮素或包含根皮素的生物农药。
优选的,上述所述改善植物抗虫活性为提高植物抗虫活性;所述调控上述蛋白酶的表达量或酶活性为提高调控上述蛋白酶的表达量或酶活性。
与现有技术相比,本申请至少具有如下优势:
1)本申请发现蛋白酶BGLU13.1-A和BGLU13.1-a可将根皮苷和三叶苷水解为根皮素。
2)本申请将蛋白酶在苹果GL3中稳定表达,过表达转基因植株对害虫表现出比野生型更强的抗性,而沉默植株相比野生型降低了对害虫的抗性,因此其在植物虫害防治及抗性育种等方面具有重要的应用价值。
3)本申请通过外施根皮素处理,发现与根皮苷和三叶苷相比,根皮素能显著提高植物的抗虫性。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本申请的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1所述的从富士花瓣中提取粗蛋白经离子层析的结果;
图2为实施例1所述的从富士花瓣中提取粗蛋白经疏水层析的结果;
图3为实施例1所述的从富士花瓣中提取粗蛋白经凝胶过滤层析的结果;
图4为实施例1所述的SDS-PAGE凝胶电泳结果;
图5为实施例1所述的BGLU13.1-A和BGLU13.1-a重组蛋白对根皮苷和三叶苷水解活性的测定结果;
图6为实施例2所述的BGLU13.1-A和BGLU13.1-a的蛋白同源建模;
图7为实施例2所述的突变后BGLU13.1-A和BGLU13.1-a重组蛋白对根皮苷和三叶苷水解活性的测定结果;
图8为实施例3所述的过表达和沉默BGLU13.1-A转基因株系RNA水平的检测;
图9为实施例3所述的二斑叶螨侵染后的二斑叶螨的数量;
图10为实施例3所述的红蜘蛛侵染后的红蜘蛛的数量;
图11为实施例3所述的卷叶蛾啃食叶片的消耗量;
图12为实施例4所述的外源施加化合物对二斑叶螨存活率的影响;
图13为实施例4所述的外源施加化合物对红蜘蛛存活率的影响;
图14为实施例4所述的外源施加化合物对卷叶蛾啃食叶片消耗量的影响。
具体实施方式
下面将结合附图对本申请的技术方案进行清楚、完整地描述,显然,所描述的实施例是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
以下术语或定义仅仅是为了帮助理解本申请而提供。这些定义不应被理解为具有小于本领域技术人员所理解的范围。
除非在下文中另有定义,本申请具体实施方式中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。虽然相信以下术语对于本领域技术人员很好理解,但仍然阐述以下定义以更好地解释本申请。
如本申请中所使用,术语“包括”、“包含”、“具有”、“含有”或“涉及”为包含性的(inclusive)或开放式的,且不排除其它未列举的元素或方法步骤。术语“由…组成”被认为是术语“包含”的优选实施方案。如果在下文中某一组被定义为包含至少一定数目的实施方案,这也应被理解为揭示了一个优选地仅由这些实施方案组成的组。
在提及单数形式名词时使用的不定冠词或定冠词例如“一个”或“一种”,“所述”,包括该名词的复数形式。
本申请中的术语“大约”、“大体”表示本领域技术人员能够理解的仍可保证论及特征的技术效果的准确度区间。该术语通常表示偏离指示数值的±10%,优选±5%。
此外,说明书和权利要求书中的术语第一、第二、第三、(a)、(b)、(c)以及诸如此类,是用于区分相似的元素,不是描述顺序或时间次序必须的。应理解,如此应用的术语在适当的环境下可互换,并且本申请描述的实施方案能以不同于本申请描述或举例说明的其它顺序实施。
下面为具体的实施例。
实施例1蛋白酶等位基因克隆发现及原核表达
提取富士(Malus domestica Fuji)花瓣粗蛋白,然后粗蛋白先后经离子层析(图1)、疏水层析(图2)和凝胶过滤层析(图3)进行分离,期间检测分离的粗蛋白各组分水解根皮苷的活性,逐步分离纯化有活性的目的蛋白。通过SDS-PAGE凝胶电泳检测发现,红色虚线标注的蛋白条带(图4)和根皮苷水解活性趋势一致(图3),故作为目的蛋白进行鉴定。
选取杂蛋白相对较少而目的蛋白相对高的5-6泳道蛋白条带进行QE质谱分析,根据苹果基因组相关信息鉴定到目的蛋白MD03G1069500,功能注释后命名为BGLU13.1。以苹果属不同种质资源的cDNA为模板进行扩增,共得到两种序列,通过扩增基因上下游序列证明两种序列互为等位基因,分别命名为BGLU13.1-A和BGLU13.1-a。具体的:
设计扩增目的基因BGLU13.1-A和BGLU13.1-a的引物,BGLU13.1-A-F:ATGTTCAGACTTAACTCG;BGLU13.1-A-R:TTATCCAAGGAAATATTTGAA;BGLU13.1-a-F:ATGGCAGCCATCCACTC;BGLU13.1-a-R:TTATCCAAGGAAATATTTGAA,以苹果属不同种质资源的cDNA为模板进行扩增并连接到PMD-19T(Takara)载体上进行测序,得到两种序列BGLU13.1-A和BGLU13.1-a,具体得到基因序列如SEQ ID NO.1-2所述,编码氨基酸序列如SEQ IDNO.3-4所示。
SEQ ID NO.1(BGLU13.1-A)
ATGTTCAGACTTAACTCGGTGCCCCTCCCGACTCTGTCCCAACGGTCAGCCATGGCCGCCATCCACTCCATAGCAGCTCAGAGCTTCCTCTCTCTCCCGAACCCCAAACCCAGAAAACGCATTGCCGCTCACCCAAAACCCATCTTGACCTCCAAACTCCCAAAAAACCCAACTTTCCCCAGAAACAAACAGAAAGGCAGGAGGGTTTTGAACTCGGCGGTCAAGCGAGAAGAGTGCGATGTGATTCCGGTTCAGAGCTCCGACAGTACGGACCAGCAGGAAGGGGTGGTGGCCAGCAGGGTGGAGAGCGAAGCCGGGGATCAGGGGGAGTTGGTGAGTCAGGTGGGCGGGTTCGGAGCGAGTGAGGGGAGGCTTTCGTTTGAAGGGGCTGGTGGGTTTGGGTCTACTGGGGTTGGAAGTGAGAGGGAGAGTGAGGAATTCGAGAGGCCGGTGGAAAAACTACTAACTAATATACCTAGTCGTTATAGCAGTGCTTCCCTGAACAGAAGCAGTGCTTCCCTGAACAGGAGCAGTTTTCCATCAGGCTTTGTATTTGGTTCAGCTTCAGCATCTTACCAATATGAAGGTGCATGGAATGAAGGTGGTAAAGGACCAAGCATATGGGATAACTTCACCCACCAGTATCCAGAAAAAATCAGTGATGGAAGCAATGGGGACGTGGCTAATGATCAATACCACCGCTATAAGGAAGATGTAAAGATTATGAAGGATATGGGATTGGATGCTTATAGGTTCTCTATCTCATGGTCCAGATTGTTACCAAATGGAAAGCTAAGTGGGGGTGTGAGCAAGGAAGGAGT ACAATACTACAACAATCTCATCAATGAACTCCAAAACAAAGGTATAGCGCCATATGTGACAATCTTTCATTGGGATCTTCCTCAAGCTTTAGAAGAAGAATATGGTGGTTTCTTAAACCGTCAAATTGTCAATCATTTTCGAGACTATGCAGAACTTTGCTTTAAGTTATTTGGCGATCGGGTAAAGCATTGGATCACACTAAATGAGCCATATAATTTTATTAATTTTGGCTATGCATCAGGACAATTAGCACCCGGGCGATGTTCTGCTTGGCAGAACTTAAACTGCACCGGCGGTGATTCGGCTACCGAACCCTATATAGTAGCACACCACTTCCTCCTCGCTCATGCACATGCTGTTGAAGTGTACAAGACTAAATATCAGGCATCTCAAGAAGGCGTGATAGGAATAACATTAGCGGCAAATTGGTTTGTGCCAATTTCTAACGAAACTCGTCATCAGAATGCTGCAAATCGATCTTTGGATTTTATGTTTGGATGGTTTATGGAGCCATTGACAAGCGGCCAATATCCGCACAGTATGCAAGTTCTTGTTAAAGAAAGATTACCTAAATTTACAGAAGAAGAATCCAAGTTAATAAAAGGGTCATTTGATTTTGTTGGAATGAATTATTATACTACTCACTATTCAAGCGATCAACCTGATAATAATTCTGCAAATCCAAGCTTCTTGACCGATGCTTGCGTTTTTGAATCAACCGAGCTTAATGGAGTCCCCATTGGTCCTCCGGCTGCTTCAAGCTGGCTAGTTATTTATCCAAAAGGCATTCGAGAGATTTTACTTTACGCAAAGCACAAATATAATAATCCGGTCATTTACATTACTGAGAACGGCATGGATGAGTTCGATGATCCCAAATTGTCACTTCCGCAATCCCTCAATGATACCCACAGAATTGATTACCACTACCACCACCTCGACTATCTTCGAAAAGCAATCGATGATGGTGTAAATGTGAAGGGATACTTTGCATGGTCATTGACGGACAATTTTGAATGGGCTGCTGGATACACCTTACGATTTGGTTTCGTCTATATAGATTACAATGATGGACTTAAGAGGCACCCAAAACTCTCAGCAAGCTGGTTCAAATATTTCCTTGGATAA
SEQ ID NO.2(BGLU13.1-a)
ATGGCAGCCATCCACTCCATAGCAGCTCAGAGCTTCCTCTCTCTCCTGAACCCCAAACCCAGAAAACCCATTGCCGCTCACCCAAAACCCATCTTGACCTCCAAACTCCCAAAAAACCCAACTTTCCCCAGAAACAAACAGAAAAGCAGGTGGGTTTCGAACTCGGCGGTCGAGCGAGAAGAGTGCGATGTGATTCCGGTTCAGAGCTCCGACCGTACGGACCTGCAGGAAGGGGTGGTGGCCAGCAGGGTGGAGAGCGAAGGCGGGGATCAGGGGGAGTTGGTGAGTCAGGTGGGCGGGTTCGGAGCGAGTGAGGGGAGGCTTTCGTTTGAAGGGGCTGGTGGGTTTGGGTCTTCTGGGGTTGGAAGTGAGAGGGAGAGTGAGGAATTCGAGAGGCTGGTGGTAAAACCACTAACTGCTATACCTAGTCGTTATAGCAGTGCTTCCCTGAACAGAAGCAGTTTTCCATCAGGCTTTGTATTTGGTTCATCTTCATCATCTTACCAATATGAAGGTGCATGGAATGAAGGTGGTAAAGGACCAAGCATATGGGATAACTTCACCCACCAGTATCCAGAAAAAATCAGTGATGGACGCAATGGGGACGTGGCTGATGATCAATACCACCGCTATAAGGAAGATGTAAAGATTATGAAGGATATGGGATTGGATGCTTATAGGTTCTCTATCTCATGGTCCAGATTGTTACCAAATGGAAAGCTAAGTGGGGGTGTGAACAAGGACGGAGTACATCACTACAACAATCTCATCAATGAACTCCTAAACAAAGGTGTAACGCCATATGTGACAATCTTTCATTGGGATCTTCCTCAAGCTTTAGAAGAAGAATATGGCGGTTTCTTAAACCGTCAAATTGTCAATCATTTTCAAGACTATGCAGAACTTTGCTTTAAGTTATTTGGCGATCGGGTAAAGCATTGGATCACGTTAAATGAGCCATATACTTTTGTTACTTTAGGCTATGCATCAGGAAAATTAGCACCCGGACGATGTTCTGCTTGGCAGAACTTAAACTGCACCGGCGG TGATTCGGCTACCGAACCCTATATAGTAGCACACCACTTCCTCCTCGCTCATGCACATGCTGTTAAAGTGTACAAGACTAAATATCAGGCATCTCAAGAAGGCGTGATAGGGATAACATTAGTGACAAATTGGTTTGTGCCAGTTTCTAACGCAACGCGTCATCAGAATGCTGCAAATCGATCTTTGGATTTTATGTTTGGATGGTTTATGGAGCCATTGACAAGCGGCCAATATCCGCACAGTATGCAAGTTCTTGTTAAAGAAAGATTACCTAAATTTACAGAAGAAGAATCCAAGTTAATAAAAGGGTCATTTGATTTTGTTGGAATGAATTATTATACTACTCACTATTCAAGAGATCAACCTCATAATAATTCTGCAAATCCAAGCTTCTTGACCGATGCTCGCGTTTCTGAATCAACCGAGCTTAATGGAGTCCCCCTTGGTCCTCCGGCTGCTTCAAGCTGGCTAGTTGTTTATCCAAAAGGCATTCGAGAGATTTTACTGTACGCAAAGCACAAATATAATGATCCGCTCATTTACATTACTGAGAACGGCGTTGATGAGTTCGATGATCCCACATTGTCACTTCCGCAATCCCTCGATGATACCCACAGAATTGATTACCACTACCACCACCTCGACTATCTTCGAAAAGCAATCAATGATGGTGTAAATGTGAAGGGATACTTTGCATGGTCATTGATGGACAATTTTGAATGGGAATCTGGATACACCTTACGATTTGGTTTCGTCTATATAGATTACAATGATGGACTTAAGAGGCACCCAAAACTCTCAGCAAGCTGGTTCAAATATTTCCTTGGATAA
SEQ ID NO.3(BGLU13.1-A)
MFRLNSVPLPTLSQRSAMAAIHSIAAQSFLSLPNPKPRKRIAAHPKPILTSKLPKNPTFPRNKQKGRRVLNSAVKREECDVIPVQSSDSTDQQEGVVASRVESEAGDQGELVSQVGGFGASEGRLSFEGAGGFGSTGVGSERESEEFERPVEKLLTNIPSRYSSASLNRSSASLNRSSFPSGFVFGSASASYQYEGAWNEGGKGPSIWDNFTHQYPEKISDGSNGDVANDQYHRYKEDVKIMKDMGLDAYRFSISWSRLLPNGKLSGGVSKEGVQYYNNLINELQNKGIAPYVTIFHWDLPQALEEEYGGFLNRQIVNHFRDYAELCFKLFGDRVKHWITLNEPYNFINFGYASGQLAPGRCSAWQNLNCTGGDSATEPYIVAHHFLLAHAHAVEVYKTKYQASQEGVIGITLAANWFVPISNETRHQNAANRSLDFMFGWFMEPLTSGQYPHSMQVLVKERLPKFTEEESKLIKGSFDFVGMNYYTTHYSSDQPDNNSANPSFLTDACVFESTELNGVPIGPPAASSWLVIYPKGIREILLYAKHKYNNPVIYITENGMDEFDDPKLSLPQSLNDTHRIDYHYHHLDYLRKAIDDGVNVKGYFAWSLTDNFEWAAGYTLRFGFVYIDYNDGLKRHPKLSASWFKYFLG
SEQ ID NO.4(BGLU13.1-a)
MAAIHSIAAQSFLSLLNPKPRKPIAAHPKPILTSKLPKNPTFPRNKQKSRWVSNSAVEREECDVIPVQSSDRTDLQEGVVASRVESEGGDQGELVSQVGGFGASEGRLSFEGAGGFGSSGVGSERESEEFERLVVKPLTAIPSRYSSASLNRSSFPSGFVFGSSSSSYQYEGAWNEGGKGPSIWDNFTHQYPEKISDGRNGDVADDQYHRYKEDVKIMKDMGLDAYRFSISWSRLLPNGKLSGGVNKDGVHHYNNLINELLNKGVTPYVTIFHWDLPQALEEEYGGFLNRQIVNHFQDYAELCFKLFGDRVKHWITLNEPYTFVTLGYASGKLAPGRCSAWQNLNCTGGDSATEPYIVAHHFLLAHAHAVKVYKTKYQASQEGVIGITLVTNWFVPVSNATRHQNAANRSLDFMFGWFMEPLTSGQYPHSMQVLVKERLPKFTEEESKLIKGSFDFVGMNYYTTHYSRDQPHNNSANPSFLTDARVSESTELNGVPLGPPAASSWLVVYPKGIREILLYAK HKYNDPLIYITENGVDEFDDPTLSLPQSLDDTHRIDYHYHHLDYLRKAINDGVNVKGYFAWSLMDNFEWESGYTLRFGFVYIDYNDGLKRHPKLSASWFKYFLG。
进一步将BGLU13.1的CDS全长序列通过一步克隆(Vazyme非连接酶依赖型单片段快速克隆试剂盒)的方法连接到Pet28a载体上,转化至大肠杆菌BL21(DE3)菌株中,将阳性菌落活化并用0.5mM的IPTG诱导24h(18℃,120rpm),将菌液离心去上清,使用缓冲液(50mMNaH2PO4,30mM NaCl,20mM咪唑,用NaOH调节pH 8.0)重悬后加入2mg L-1的溶菌酶在37℃孵育30min,液氮反复冻融3次,4℃,12000g离心10min,上清用来测酶活。
利用原核表达的BGLU13.1-A和BGLU13.1-a的重组蛋白,在酶促反应液加入终浓度为0.5mM的底物根皮苷或三叶苷,酶液100μL,柠檬酸缓冲液(100mM,pH 6.0)90μL。各组分混匀后37℃孵育20min,加入400μL甲醇溶液终止反应。然后反应液经离心和滤膜过滤后,利用高效液相色谱分析产物根皮素的产生量。
结果发现,BGLU13.1-A可将根皮苷水解为根皮素,其不水解三叶苷;而BGLU13.1-a不水解根皮苷,但可将三叶苷水解为根皮素(图5)。
实施例2蛋白酶同源建模和点突变试验
利用在线软件HHPred(https://toolkit.tuebingen.mpg.de/tools/hhpred)完成BGLU13.1-A和BGLU13.1-a蛋白同源建模。通过蛋白三维结构模型找到底物结合的关键活性中心,比较找出BGLU13.1-A和BGLU13.1-a在活性中心附近的氨基酸差异位点作为点突变的对象。
对BGLU13.1-A和BGLU13.1-a进行蛋白三维结构进行预测,发现在两个蛋白的活性中心位置附近存在6个氨基酸的差异,分别对应BGLU13.1-A中的Asn346Phe350Gln356Ala414Ala415Ala615和BGLU13.1-a中的Thr322Leu326Lys332Val390Thr391Glu591(图6)。
通过点突变试验,将两条等位基因之间的6个氨基酸突变位点进行定向突变,即BGLU13.1-A中的6个氨基酸突变为BGLU13.1-a对应的6个氨基酸,或将BGLU13.1-a中的6个氨基酸突变为BGLU13.1-A对应的6个氨基酸,发现突变后两个蛋白的活性也会随之发生交换,即MBGLU13.1-A(突变后的BGLU13.1-A)失去水解根皮苷的活性而获得水解三叶苷的活性,MBGLU13.1-a也由水解三叶苷转变为水解根皮苷(图7)。
实施例3转基因苹果抗虫活性试验
1)苹果遗传转化载体的构建
过表达载体构建:将BGLU13.1-A的CDS全长序列分别通过一步克隆(Vazyme非连接酶依赖型单片段快速克隆试剂盒)连接到pCambia 2300载体上,目的基因由35S启动子启动转录。
沉默载体构建:RNAi使用的载体是PK7WIWG2D,在NCBI数据库中找到BGLU13.1-A的特异片段,设计引物克隆特异片段,通过Gate-way的方法将特异片段通过BP反应(GatewayBP Clonase,Invitrogen)先连接到中间载体pDONR 222,再通过LR反应(Gateway LRClonase,Invitrogen)连接到目的载体上。
通过农杆菌介导的叶盘转化法对目的基因BGLU13.1-A在GL-3苹果苗中进行过表达和RNA干扰沉默。结果表明,RNA水平上的6个过表达株系中目的基因都有一定程度的上调,沉默株系中目的基因的表达也显著降低(图8)。
2)改变基因表达影响植物抗虫性实验
1)抗二斑叶螨实验
将二斑叶螨饲养在蚕豆苗上2-3周,然后将长有二斑叶螨的蚕豆苗放置于野生型和转基因苹果苗中间。10d后,统计每棵苹果苗上的二斑叶螨成螨的头数,以平均每个叶片上二斑叶螨数量作为评价指标。
2)抗红蜘蛛实验
将红蜘蛛饲养在蚕豆苗上2-3周,然后将长有红蜘蛛的蚕豆苗放置于野生型和转基因苹果苗中间。10d后,统计每棵苹果苗上的红蜘蛛的数量,以平均每个叶片上二斑叶螨数量作为评价指标。
3)抗卷叶蛾实验
选取野生型苹果苗和转基因苗顶部第3-4片成熟叶,每片叶子称重后叶柄处用湿棉花包住放置在玻璃培养皿,将饥饿处理2h的二至三龄幼虫卷叶蛾放置其中,6h后观察植株的受伤害程度,拍照记录并称取叶片重量计算消耗量。
结果表明,在被二斑叶螨、红蜘蛛和卷叶蛾侵染的过程中,过表达BGLU13.1-A的转基因植株对二斑叶螨、红蜘蛛和卷叶蛾表现出比野生型更强的抗性,而沉默植株相比野生型降低了对二斑叶螨、红蜘蛛和卷叶蛾的抗性。统计不同转基因植株和野生型植株上二斑叶螨、红蜘蛛数量以及卷叶蛾啃食前后叶片消耗量,与植株受伤害程度的趋势一致,过表达材料二斑叶螨数量、红蜘蛛数量和卷叶蛾啃食叶片消耗量明显低于野生型和沉默植株(图9-11)。
实施例4外施根皮素的抗虫实验
1)抗二斑叶螨实验
利用含有20%甲醇的50mM Tris-HCl(pH 7.4)缓冲液溶解化合物,将溶解后的化合物用零号毛笔均匀涂抹在健康的蚕豆叶片上,最终达到根皮素1μg每毫克鲜叶,根皮苷1.5μg每毫克鲜叶,三叶苷1.5μg每毫克鲜叶,叶片的叶柄处用湿棉花包住,放在超净台中吹干涂抹的溶液。从前期养殖二斑叶螨的蚕豆叶片上用零号毛笔轻轻把螨转移至涂有化合物的蚕豆叶片上,每个叶片接20头螨,叶片放在一次性培养皿中置于光照培养箱中,3d后统计二斑叶螨的存活率,对照涂抹相同体积的缓冲液。
统计发现,和对照相比,几种化合物对二斑叶螨数量都有一定程度的影响,而且根皮素处理使二斑叶螨的成活率显著降低(图12)。
2)抗红蜘蛛实验
与抗二斑叶螨实验类似。
结果与二斑叶螨结果一致,几种化合物对红蜘蛛数量都有一定程度的影响,而且根皮素处理使红蜘蛛的成活率显著降低(图13)。
3)抗卷叶蛾实验
健康的蚕豆叶片称重后涂抹上述化合物,对照涂抹相同体积的缓冲液。叶柄处用湿棉花包住放置在玻璃培养皿,吹干后将饥饿处理2h的三龄幼虫卷叶蛾放置其中,6h后观察植株的受伤害程度拍照记录并称取叶片重量计算消耗量。
结果与二斑叶螨和红蜘蛛结果一致,针对三龄幼虫卷叶蛾,根皮素处理使蚕豆叶片消耗量显著降低(图14),
上述结果说明,根皮素能够用于苹果和大豆的抗虫,在苹果和大豆的抗虫方面发挥着重要作用。
前述对本申请的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本申请限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本申请的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本申请的各种不同的示例性实施方案以及各种不同的选择和改变。本申请的范围意在由权利要求书及其等同形式所限定。
Claims (10)
1.一种蛋白酶,其特征在于,所述蛋白酶序列与SEQ ID NO.3或4具有至少90%同源性,且来源于苹果属。
2.如权利要求1所述的蛋白酶,其特征在于,所述蛋白酶序列如与SEQ ID NO.3或4所示。
3.如权利要求1所述的蛋白酶,其特征在于,所述蛋白酶序列如SEQ ID NO.3所示,且SEQ ID NO.3的346Asn、350Phe、356Gln、414Ala、415Ala、615Ala六个氨基酸分别替换为Thr、Leu、Lys、Val、Thr、Glu。
4.如权利要求1所述的蛋白酶,其特征在于,所述蛋白酶序列如与SEQ ID NO.4所示,且SEQ ID NO.4的322Thr、326Leu、332Lys、390Val、391Thr、591Glu六个氨基酸分别替换为Asn、Phe、Gln、Ala、Ala、Ala。
5.分离的核酸,其特征在于,所述核酸编码权利要求1-4任一所述蛋白酶。
6.重组载体,其特征在于,包含权利要求5所述的核酸;优选的,所述载体为表达载体;更优选的,所述表达载体包括原核表达载体Pet28a,过表达载体pCambia 2300或沉默载体PK7WIWG2D。
7.重组细胞,其特征在于,包含权利要求6所述的重组载体:优选的,所述重组细胞包括大肠杆菌BL21(DE3)或农杆菌GV3101。
8.权利要求1所述蛋白酶的如下任一应用:
1)在水解制备根皮素中的应用;
2)在植物抗虫中的应用;
3)在植物育种中的应用。
9.根皮素的如下任一应用:
1)在植物抗虫中的应用;
2)在植物育种中的应用。
10.一种改善植物抗虫活性的方法,其特征在于,包括如下任一步骤:
a)通过在植物体中调控权利要求1-4任一所述蛋白酶的表达量或酶活性,改善根皮苷或三叶苷水解生成根皮素;
或,b)对植物体外施根皮素或包含根皮素的生物农药。
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