CN115029369B - 一种防治松材线虫病的苏云金芽孢杆菌工程菌制备方法与应用 - Google Patents
一种防治松材线虫病的苏云金芽孢杆菌工程菌制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种苏云金芽孢杆菌高效表达载体、工程菌及其制备方法与应用。所构建苏云金芽孢杆菌表达载体在宿主细胞中高效表达外源蛋白。所构建工程菌株兼具杀线虫能力和马尾松树体内定殖能力。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种苏云金芽孢杆菌高效表达载体、工程菌及其制备方法与应用。
背景技术
松材线虫病(Pine wilt disease)是国际重大检疫性病害，是我国林业上的首要病害，其病原是松材线虫(Bursaphelenchus xylophilus)(理永霞和张星耀，2018)，自1982年入侵我国以来，现已蔓延至17个省(区、市)(国家林业和草原局公告(2021年第5号))，造成大面积松树死亡，带来了巨大的经济和生态损失，严重威胁我国生态系统安全。目前，利用微生物农药对松材线虫进行生物防治，不但适用于航空喷洒施药，而且对环境友好，可以很好的满足大规模防控需求，因此受到越来越多的关注。
苏云金芽胞杆菌(Bacillus thuringiensis，Bt)产品是目前世界上应用最为广泛的微生物农药。在菌体形成芽胞的同时，菌体的一端或两端产生伴胞晶体(InsecticidalCrystal Proteins，ICPs)，伴胞晶体随芽胞的释放而释放到胞外。伴胞晶体主要由Cry毒素形成，是芽胞杆菌产生的最大的一类杀虫蛋白。杀线虫伴胞晶体蛋白主要包括三个亚类：Cry5亚类(Cry5B、Cry5C、Cry5E、Cryl2A、Cryl3A、Cryl4A、Cry21A和Cry21B)，Cry6亚类(Cry6A和Cry6B)以及Cry55亚类(Cry55A)(Pinos et al.,2021,Yu et al.,2015,Xiang-Qian et al.,2008)。Cry被线虫吞食后破坏其肠道，在肠道细胞中形成空泡结构，导致肠道收缩与体壁分离，也能在细胞膜上形成穿孔而导致线虫死亡(Pinos et al.,2021)。基于伴胞晶体线虫的特异性杀虫活性，Bt菌剂和Cry转基因植物在根结线虫、包囊线虫和松材线虫的防治中表现出较好的防治效果，具有广泛的应用前景(Yu et al.,2015,Li et al.,2007)。目前已经报道多个Bt菌株对松材线虫具有显著的致死效果(罗兰等,2007，徐华潮等，2010)，且与伊氏杀线真菌菌株为Esteya vermicola联用，表现出显著的增效作用(李恩杰等，2019)。Cry1Ea、Cry5Ba和Cry6A三种Cry晶体蛋白对松材线虫具有较高的毒力(Huanget al.,2018,雷梦英，2015，王殿东等,2013)，通过基因工程手段，转Cry6A基因的灰葡萄孢菌对松材线虫表现出显著的抑制作用。
松材线虫松树茎干内寄生危害，因此将Bt应用于松材线虫的防治，不但要具备杀线虫活性，还要Bt能够适应林间生态系统，定殖于松树体内。Bt工程菌的研发为该问题的解决提供了途径，苏云金芽胞杆菌G033A是国内第一个获得农药登记证的工程菌，也是第一个防治鞘翅目害虫的Bt产品，同时对鳞翅目害虫也具有高毒力。将含有靶标鞘翅目害虫的cry3Aa7基因启动子和编码区构建至重组质粒Pstk-3A，将该质粒导入对鳞翅目害虫高毒力的菌株G03中获得了Bt工程菌G033A，获得了可以同时防治鳞翅目和鞘翅目害虫的工程菌G033A，扩大了杀虫谱(王宇航等，2020)。苏云金芽胞杆菌G033A的研发和产业化为利用Bt防治松材线虫病的研究提供了重要借鉴。利用基因工程手段，以松树体内广泛存在的Bt为出发菌株，使其表达杀线虫Cry蛋白，从而获得兼具杀死松材线虫能力和松树体内定殖能力的苏云金芽孢杆菌工程菌，在松材线虫病防治中具有广阔的应用前景。
发明内容
针对上述存在的问题，本申请一方面提供一种或多种苏云金芽孢杆菌表达载体。
另一方面，本申请提供包含所述表达载体的苏云金芽孢杆工程菌株。
另一方面，本申请提供所述表达载体或工程菌株的制备方法。
另一方面，本申请提供所述表达载体或工程菌株以下一项或多项的应用：
(1)表达绿色荧光蛋白sfGFP的应用；
(2)在表达杀线虫Cry蛋白中的应用；
(3)在杀灭线虫中的应用；
(4)在预防和/或治疗松材线虫病中的应用。
本申请所达到的效果包括但不限于：
本申请所构建苏云金芽孢杆菌表达载体在宿主细胞中高效表达外源蛋白，例如绿色荧光蛋白sfGFP，或杀线虫蛋白Cry6A；
本申请利用所述高效表达载体，在野生型Bxy19菌株中构建工程菌株Bxy19P3C6，表达杀线虫蛋白Cry6A，使其具备显著杀线虫能力，且通过喷洒和注射两种接种手段，可以在马尾松体内定殖并进行一定距离的传播。
本申请工程菌株兼具杀线虫能力和马尾松树体内定殖能力。
附图说明
图1：Bxy19sfGFP、Bxy19P3C6的PCR检测
图2：pP3A-sfgfp表达蛋白荧光显微观察
图3：SDS-PAGE检测蛋白表达
图4：Bxy19P3C6营养期晶体蛋白的形态观察
图5：工程菌Bxy19P3C6杀线虫活性测定
图6：松材线虫形态观察
图7：本申请菌株定殖能力测定
具体实施方式
为使本发明的技术方案和有益效果能够更加明显易懂，下面通过列举具体实施例的方式进行详细说明。、除非另有定义，本文所使用的技术和科学术语与本申请所属的技术领域中的技术和科学术语的含义相同。
一方面，本申请提供一种或多种表达载体。
在一些实施方式中，所述表达载体是指导绿色荧光蛋白sfGFP表达的载体pP3A-sfgfp，其序列如SEQ ID NO:1所示。
在一些实施方式中，所述表达载体pP3A-sfgfp通过如下方式制备：
利用调控Cry蛋白在苏云金芽孢杆菌营养期表达的cry3Aa基因的启动子(GenBank：AJ237900.1)，构建指导绿色荧光蛋白sfGFP在苏云金芽孢杆菌体内高效表达的重组质粒pP3A-sfgfp。
在一些实施方式中，所述表达载体是指导杀线虫蛋白Cry6A表达的载体pP3A-cry6a，其序列如SEQ ID NO:2所示。
在一些实施方式中，所述表达载体pP3A-cry6a通过如下方式制备：
(1)利用调控Cry蛋白在苏云金芽孢杆菌营养期表达的cry3Aa基因的启动子(GenBank：AJ237900.1)，构建指导绿色荧光蛋白sfGFP在苏云金芽孢杆菌体内高效表达的重组质粒pP3A-sfgfp；
(2)通过同源重组手段，将sfgfp基因替换为杀线蛋白Cry6A基因，得到SEQ ID NO:2所示表达载体。
另一方面本申请提供包含所述一种或多种表达载体的工程菌株。
在一些实施方式中，所述工程菌株通过将本申请所述的表达载体导入宿主细胞后得到。
在一些实施方式中，所述宿主细胞为苏云金芽孢杆菌，例如野生型Bxy19苏云金芽孢杆菌，如保藏于中国微生物菌种保藏管理委员会普通微生物中心、保藏编号为CGMCCNO.24656的苏云金芽孢杆菌Bxy19。
在一些实施方式中，所述工程菌株是包含表达载体pP3A-sfgfp的工程菌株Bxy19sfGFP。
在一些实施方式中，所述工程菌株是包含表达载体pP3A-cry6a的工程菌株Bxy19P3C6。
另一方面，本申请提供所述表达载体或工程菌株以下一项或多项的应用：
(1)表达绿色荧光蛋白sfGFP的应用；
(2)在表达杀线虫Cry蛋白中的应用；
(3)在杀灭线虫中的应用；
(4)在预防和/或治疗松材线虫病中的应用。
在一些实施方式中，所述表达载体pP3A-sfgfp或工程菌株Bxy19sfGFP能够高效表达绿色荧光蛋白sfGFP。
在一些实施方式中，所述表达载体pP3A-cry6a或工程菌株Bxy19P3C6能够高效表达杀线虫Cry蛋白，例如杀线虫Cry6A蛋白。
在一些实施方式中，所述表达载体pP3A-cry6a或工程菌株Bxy19P3C6用于杀灭线虫。
在一些实施方式中，所述表达载体pP3A-cry6a或工程菌株Bxy19P3C6用于预防和/或治疗松材线虫病。
另一方面本申请提供一种苏云金芽孢杆菌Bxy19，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24656，保藏时间为2022年04月08日。
本申请所涉及序列信息如下：
SEQ ID NO:1：
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatccagcttaattaaagataatatctttgaattgtaacgcccctcaaaagtaagaactacaaaaaaagaatacgttatatagaaatatgtttgaaccttcttcagattacaaatatattcggacggactctacctcaaatgcttatctaactatagaatgacatacaagcacaaccttgaaaatttgaaaatataactaccaatgaacttgttcatgtgaattatcgctgtatttaattttctcaattcaatatataatatgccaatacattgttacaagtagaaattaagacacccttgatagccttactatacctaacatgatgtagtattaaatgaatatgtaaatatatttatgataagaagcgacttatttataatcattacatatttttctattggaatgattaagattccaatagaatagtgtataaattatttatcttgaaaggagggatgcctaaaaacgaagaacattaaaaacatatatttgcaccgtctaatggatttatgaaaaatcattttatcagtttgaaaattatgtattatgataagaaagggaggaagaaaaatgagcaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtccgtggagagggtgaaggtgatgctacaaacggaaaactcacccttaaatttatttgcactactggaaaactacctgttccgtggccaacacttgtcactactctgacctatggtgttcaatgcttttcccgttatccggatcacatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaacgcactatatctttcaaagatgacgggacctacaagacgcgtgctgaagtcaagtttgaaggtgatacccttgttaatcgtatcgagttaaagggtattgattttaaagaagatggaaacattcttggacacaaactcgagtacaactttaactcacacaatgtatacatcacggcagacaaacaaaagaatggaatcaaagctaacttcaaaattcgccacaacgttgaagatggttccgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtcgacacaatctgtcctttcgaaagatcccaacgaaaagcgtgaccacatggtccttcttgagtttgtaactgctgctgggattacacatggatgagctctacaaatagaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc
SEQ ID NO:2：
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatccagcttaattaaagataatatctttgaattgtaacgcccctcaaaagtaagaactacaaaaaaagaatacgttatatagaaatatgtttgaaccttcttcagattacaaatatattcggacggactctacctcaaatgcttatctaactatagaatgacatacaagcacaaccttgaaaatttgaaaatataactaccaatgaacttgttcatgtgaattatcgctgtatttaattttctcaattcaatatataatatgccaatacattgttacaagtagaaattaagacacccttgatagccttactatacctaacatgatgtagtattaaatgaatatgtaaatatatttatgataagaagcgacttatttataatcattacatatttttctattggaatgattaagattccaatagaatagtgtataaattatttatcttgaaaggagggatgcctaaaaacgaagaacattaaaaacatatatttgcaccgtctaatggatttatgaaaaatcattttatcagtttgaaaattatgtattatgataagaaagggaggaagaaaaggtaccattataggtaagagaggaatgtacacatgattattgatagtaaaacgactttacctagacattcacttattcatacaattaaattaaattctaataagaaatatggtcctggtgatatgaccaatggaaatcaatttattatttcaaaacaagaatgggctactattggagcatatattcagactggattaggtttaccagtaaatgaacaacaattaagaacacatgttaatttaagtcaggatatatcaatacctagtgatttttctcaattatatgatgtttattgttctgataaaacttcagcagaatggtggaataaaaatttatatcctttaattattaaatctgctaatgatattgcttcatatggttttaaagttgctggtgatccttctattaagaaagatggatattttaaaaaattgcaagatgaattagataatattgttgataataattccgatgatgatgcaatagctaaagctattaaagattttaaagcgcgatgtggtattttaattaaagaagctaaacaatatgaagaagctgcaaaaaatattgtaacatctttagatcaatttttacatggtgatcagaaaaaattagaaggtgttatcaatattcaaaaacgtttaaaagaagttcaaacagctcttaatcaagcccatggggaaagtagtccagctcataaagagttattagaaaaagtaaaaaatttaaaaacaacattagaaaggactattaaagctgaacaagatttagagaaaaaagtagaatatagttttctattaggaccattgttaggatttgttgtttatgaaattcttgaaaatactgctgttcagcatataaaaaatcaaattgatgagataaagaaacaattagattctgctcagcatgatttggatagagatgttaaaattataggaatgttaaatagtattaatacagatattgataatttatatagtcaaggacaagaagcaattaaagttttccaaaagttacaaggtatttgggctactattggagctcaaatagaaaatcttagaacaacgtcgttacaagaagttcaagattctgatgatgctgatgagatacaaattgaacttgaggacgcttctgatgcttggttagttgtggctcaagaagctcgtgattttacactaaatgcttattcaactaatagtagacaaaatttaccgattaatgttatatcagattcatgtaattgttcaacaacaaatatgacatcaaatcaatacagtaatccaacaacaaatatgacatcaaatcaatatatgatttcacatgaatatacaagtttaccaaataattttatgttatcaagaaatagtaatttagaatataaatgtcctgaaagtaattttatgatatattggtataataattcggattggtataataattcggattggtataataattaaaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc
实施例1：苏云金芽孢杆菌高效表达载体的构建
利用调控Cry蛋白在苏云金芽孢杆菌营养期表达的cry3Aa基因的启动子(GenBank：AJ237900.1)，构建指导绿色荧光蛋白sfGFP在苏云金芽孢杆菌体内高效表达的重组质粒pP3A-sfgfp，经质粒测序，确认质粒构建正确，序列如SEQ ID NO:1所示。
以质粒pP3A-sfgfp为出发质粒，通过同源重组手段，将sfgfp基因替换为杀线蛋白Cry6A基因(GenBank：DQ835612.1)，获得指导杀线虫蛋白Cry6A表达的重组质粒pP3A-cry6a。经质粒测序，确认质粒构建正确，序列如SEQ ID NO:2所示。
实施例2：荧光标记菌株Bxy19sfGFP和杀线工程菌株Bxy19P3C6的获得
将鉴定正确的质粒pP3A-sfgfp导入已经获得的松树内生菌苏云金芽孢杆菌Bxy19(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24656，保藏时间为2022年04月08日)中，经PCR检测，获得荧光标记菌株Bxy19sfGFP(图1)。荧光显微观察表明，启动子Pcry3a在苏云金芽孢杆菌Bxy19中具有较高的转录活性(图2)，重组质粒pP3A-sfgfp可以成功在苏云金芽孢杆菌Bxy19中表达目标蛋白。
将鉴定正确的质粒pP3A-cry6a导入松树内生菌苏云金芽孢杆菌Bxy19中，经PCR检测，获得工程菌株Bxy19P3C6(图1)。
实施例3：杀线蛋白的表达鉴定和晶体形态观察
将野生菌Bxy19(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24656，保藏时间为2022年04月08日)和工程菌Bxy19P3C6接种于牛肉膏蛋白胨液体培养基(NA)(蛋白胨10g/L、牛肉粉3g/L、氯化钠5g/L、琼脂20g/L)中，置于28℃摇床中220rpm培养3d后，离心收集菌体，并用无菌水冲洗3遍后重悬。通过OD600测定菌体浓度，并用无菌水稀释调整菌体浓度一致。利用SDS-PAGE检测两个样品中晶体蛋白的表达。由图3可知，工程菌Bxy19P3C6产生分子量约54kDa的Cry6A蛋白。同时，取少量收集到的工程菌Bxy19P3C6涂布于载玻片上，室温自然干燥，用酒精灯轻烤玻片6-8次，石炭酸复红染料1-2min，用无菌水冲洗干净染色液，晾干玻片后进行油镜观察，可观察到在菌体两端存在米粒状晶体(图4)。
实施例4：杀虫活性测定
将野生菌Bxy19(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24656，保藏时间为2022年04月08日)和工程菌Bxy19P3C6接种于NB培养基中，置于28℃摇床中220rpm培养24h后，离心收集菌体，并用无菌水冲洗3遍后重悬。通过OD600测定菌体浓度，并用无菌水稀释调整菌体浓度一致，离心去除菌体获得发酵上清液用于杀线虫活性检测。向96孔板中每孔加200μL上清液、10μL线虫液(约1000头线虫)、4μL氯霉素溶液(1mg/mL)，并置于25℃下暗培养24h，统计线虫死亡率。并将实验前后的线虫置于显微镜下观察线虫形态。两种菌株分别进行10个生物学重复。结果表明，工程菌Bxy19P3C6的上清液具有显著的杀线虫活性，其活性显著高于野生型菌株(图5)。显微镜下观察发现松材线虫表皮完整，但是肠道细胞溶解，同已报道的Cry6A穿孔机制相似(图6)。
实施例5：定殖能力测定
将荧光标记菌株Bxy19sfGFP接种于NB培养基中，置于28℃摇床中220rpm培养24h后，离心收集菌体，并用无菌水冲洗3遍后重悬。通过OD600测定菌体浓度，并用无菌水稀释调整菌体浓度至OD600＝1.0用于定殖能力检测实验。通过喷洒接种和注射接种两种手段，将Bxy19sfGFP接种在温室内3年生马尾松松苗。喷洒实验中，将稀释后的菌体利用喷壶均匀喷洒至马尾松苗表面，每棵松苗喷洒100mL，在喷洒后5天、10天、20天和30天，对叶部取样，同时钻孔茎部获得茎干表皮及茎干内部木屑，通过PCR技术检测菌株在叶部、茎表面和茎内部的定殖情况。注射实验中，利用电转在马尾松苗茎干钻孔，孔径约2mm，塞入脱脂棉，使用移液枪注射40ul稀释后的菌体，用封口膜封住孔口，在注射接种5天、10天、20天和30天，对接种点、距离接种点5cm和10cm进行钻孔取样，取木屑进行PCR实验检测菌株定殖情况。结果表明(图7)，通过喷洒接种，荧光标记的Bxy19sfGFP，可以在叶表面存活20天，茎表面存活30天以上，通过喷洒可以进入茎内部，存活10天左右。通过注射接种，在接种点处可以存活30天以上，同时可以进行短距离的运输传播，在注射接种5天后，可以传播至距离接种点5cm处，在注射接种10天后，可传播至距离接种点10cm处。短距离传播的菌株可以存活20天以上。
应当理解，以上实施例均为示例性的，不用于包含权利要求所包含的所有可能的实施方式。在不脱离本公开的范围的情况下，还可以在以上实施例的基础上做出各种变形和改变。同样的，也可以对以上实施例的各个技术特征进行任意组合，以形成可能没有被明确描述的本发明的另外的实施例。因此，上述实施例仅表达了本发明的几种实施方式，不对本发明专利的保护范围进行限制。
序列表
<110> 中国林业科学研究院生态保护与修复研究所
<120> 一种高效表达载体、工程菌及其制备方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7678
<212> DNA
<213> Artificial Sequence
<400> 1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420
gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480
cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540
accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600
gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660
tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720
atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780
aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840
cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900
ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960
catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020
aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080
ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140
gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200
aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260
cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320
gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380
gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440
ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500
ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560
gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620
ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680
cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740
gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800
tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860
tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920
ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980
agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040
attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100
tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160
acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220
tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280
acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340
gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400
atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460
acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520
ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580
tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640
gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700
gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760
aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820
atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880
ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940
agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000
ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060
catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120
agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180
ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240
atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300
aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360
aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420
cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480
cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540
gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600
aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660
aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720
aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780
aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840
atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900
aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960
gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020
aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080
gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140
tttttgagca actggatcca gcttaattaa agataatatc tttgaattgt aacgcccctc 4200
aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa ccttcttcag 4260
attacaaata tattcggacg gactctacct caaatgctta tctaactata gaatgacata 4320
caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca tgtgaattat 4380
cgctgtattt aattttctca attcaatata taatatgcca atacattgtt acaagtagaa 4440
attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa atgaatatgt 4500
aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt ctattggaat 4560
gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg gatgcctaaa 4620
aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatttatgaa aaatcatttt 4680
atcagtttga aaattatgta ttatgataag aaagggagga agaaaaatga gcaaaggaga 4740
agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca 4800
caaattttct gtccgtggag agggtgaagg tgatgctaca aacggaaaac tcacccttaa 4860
atttatttgc actactggaa aactacctgt tccgtggcca acacttgtca ctactctgac 4920
ctatggtgtt caatgctttt cccgttatcc ggatcacatg aaacggcatg actttttcaa 4980
gagtgccatg cccgaaggtt atgtacagga acgcactata tctttcaaag atgacgggac 5040
ctacaagacg cgtgctgaag tcaagtttga aggtgatacc cttgttaatc gtatcgagtt 5100
aaagggtatt gattttaaag aagatggaaa cattcttgga cacaaactcg agtacaactt 5160
taactcacac aatgtataca tcacggcaga caaacaaaag aatggaatca aagctaactt 5220
caaaattcgc cacaacgttg aagatggttc cgttcaacta gcagaccatt atcaacaaaa 5280
tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt cgacacaatc 5340
tgtcctttcg aaagatccca acgaaaagcg tgaccacatg gtccttcttg agtttgtaac 5400
tgctgctggg attacacatg gatgagctct acaaatagaa gcttggcgta atcatggtca 5460
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5520
agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5580
cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5640
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5700
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5760
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5820
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5880
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5940
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6000
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 6060
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6120
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6180
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6240
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 6300
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6360
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6420
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6480
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6540
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6600
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6660
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6720
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6780
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6840
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6900
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6960
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 7020
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 7080
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 7140
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 7200
atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 7260
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7320
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7380
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7440
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7500
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7560
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7620
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 7678
<210> 2
<211> 8426
<212> DNA
<213> Artificial Sequence
<400> 2
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420
gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480
cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540
accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600
gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660
tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720
atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780
aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840
cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900
ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960
catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020
aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080
ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140
gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200
aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260
cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320
gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380
gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440
ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500
ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560
gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620
ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680
cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740
gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800
tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860
tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920
ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980
agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040
attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100
tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160
acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220
tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280
acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340
gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400
atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460
acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520
ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580
tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640
gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700
gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760
aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820
atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880
ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940
agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000
ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060
catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120
agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180
ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240
atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300
aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360
aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420
cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480
cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540
gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600
aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660
aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720
aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780
aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840
atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900
aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960
gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020
aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080
gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140
tttttgagca actggatcca gcttaattaa agataatatc tttgaattgt aacgcccctc 4200
aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa ccttcttcag 4260
attacaaata tattcggacg gactctacct caaatgctta tctaactata gaatgacata 4320
caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca tgtgaattat 4380
cgctgtattt aattttctca attcaatata taatatgcca atacattgtt acaagtagaa 4440
attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa atgaatatgt 4500
aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt ctattggaat 4560
gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg gatgcctaaa 4620
aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatttatgaa aaatcatttt 4680
atcagtttga aaattatgta ttatgataag aaagggagga agaaaaggta ccattatagg 4740
taagagagga atgtacacat gattattgat agtaaaacga ctttacctag acattcactt 4800
attcatacaa ttaaattaaa ttctaataag aaatatggtc ctggtgatat gaccaatgga 4860
aatcaattta ttatttcaaa acaagaatgg gctactattg gagcatatat tcagactgga 4920
ttaggtttac cagtaaatga acaacaatta agaacacatg ttaatttaag tcaggatata 4980
tcaataccta gtgatttttc tcaattatat gatgtttatt gttctgataa aacttcagca 5040
gaatggtgga ataaaaattt atatccttta attattaaat ctgctaatga tattgcttca 5100
tatggtttta aagttgctgg tgatccttct attaagaaag atggatattt taaaaaattg 5160
caagatgaat tagataatat tgttgataat aattccgatg atgatgcaat agctaaagct 5220
attaaagatt ttaaagcgcg atgtggtatt ttaattaaag aagctaaaca atatgaagaa 5280
gctgcaaaaa atattgtaac atctttagat caatttttac atggtgatca gaaaaaatta 5340
gaaggtgtta tcaatattca aaaacgttta aaagaagttc aaacagctct taatcaagcc 5400
catggggaaa gtagtccagc tcataaagag ttattagaaa aagtaaaaaa tttaaaaaca 5460
acattagaaa ggactattaa agctgaacaa gatttagaga aaaaagtaga atatagtttt 5520
ctattaggac cattgttagg atttgttgtt tatgaaattc ttgaaaatac tgctgttcag 5580
catataaaaa atcaaattga tgagataaag aaacaattag attctgctca gcatgatttg 5640
gatagagatg ttaaaattat aggaatgtta aatagtatta atacagatat tgataattta 5700
tatagtcaag gacaagaagc aattaaagtt ttccaaaagt tacaaggtat ttgggctact 5760
attggagctc aaatagaaaa tcttagaaca acgtcgttac aagaagttca agattctgat 5820
gatgctgatg agatacaaat tgaacttgag gacgcttctg atgcttggtt agttgtggct 5880
caagaagctc gtgattttac actaaatgct tattcaacta atagtagaca aaatttaccg 5940
attaatgtta tatcagattc atgtaattgt tcaacaacaa atatgacatc aaatcaatac 6000
agtaatccaa caacaaatat gacatcaaat caatatatga tttcacatga atatacaagt 6060
ttaccaaata attttatgtt atcaagaaat agtaatttag aatataaatg tcctgaaagt 6120
aattttatga tatattggta taataattcg gattggtata ataattcgga ttggtataat 6180
aattaaaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 6240
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 6300
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 6360
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6420
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6480
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6540
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6600
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6660
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6720
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6780
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6840
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6900
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6960
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7020
gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 7080
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7140
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 7200
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 7260
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 7320
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 7380
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 7440
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 7500
accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 7560
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7620
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 7680
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 7740
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 7800
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 7860
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7920
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 7980
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 8040
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 8100
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 8160
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 8220
actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 8280
cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 8340
ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 8400
taggcgtatc acgaggccct ttcgtc 8426
Claims (7)
1.一种表达载体，其序列如SEQ ID NO:2所示。
2.如权利要求1所述表达载体的制备方法，其包括如下制备步骤：
(1)利用调控Cry蛋白在苏云金芽孢杆菌营养期表达的cry3Aa基因的启动子，构建指导绿色荧光蛋白sfGFP在苏云金芽孢杆菌体内高效表达的重组质粒pP3A-sfgfp；
(2)通过同源重组手段，将sfgfp基因替换为杀线蛋白Cry6A基因，得到SEQ ID NO:2所示表达载体。
3.一种苏云金芽孢杆菌工程菌株，其包含权利要求1所述的表达载体。
4.如权利要求3所述的工程菌株，其特征在于，其通过将权利要求1所述的表达载体导入宿主细胞后得到，所述宿主细胞为野生型Bxy19苏云金芽孢杆菌，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24656，保藏时间为2022年04月08日。
5.权利要求1所述表达载体，或权利要求3-4任一项所述工程菌株在表达杀线虫Cry蛋白中的应用。
6.如权利要求5所述的应用，其特征在于，所述杀线虫Cry蛋白为杀线虫Cry6A蛋白。
7.权利要求1所述表达载体，或权利要求3-4任一项所述工程菌株在以下一项或多项中的应用：
(1)在杀灭线虫中的应用；
(2)在预防和/或治疗松材线虫病中的应用。
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| US6063756A (en) * | 1996-09-24 | 2000-05-16 | Monsanto Company | Bacillus thuringiensis cryET33 and cryET34 compositions and uses therefor |
| CN1318584C (zh) * | 2003-10-17 | 2007-05-30 | 中国农业科学院植物保护研究所 | 苏云金芽孢杆菌工程菌g033a及其制备方法 |
| US20110225681A1 (en) * | 2008-08-25 | 2011-09-15 | Dow Agrosciences Llc | Modified Bacillus Thuringiensis Cry6 Proteins For Nematode Control |
| UA112516C2 (uk) * | 2010-04-23 | 2016-09-26 | Дау Аґросаєнсиз Елелсі | ТРАНСГЕННА РОСЛИНА, ЯКА ПРОДУКУЄ БІЛОК Сry34Ab1, БІЛОК Сry35Ab1 І ІНСЕКТИЦИДНИЙ БІЛОК Сry3Ba1, ДЛЯ ЗАПОБІГАННЯ РОЗВИТКУ СТІЙКОСТІ В КУКУРУДЗЯНИХ КОРЕНЕВИХ ЖУКІВ (Diabrotica spp.) |
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