CN112522132A - 一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用 - Google Patents

一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用 Download PDF

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CN112522132A
CN112522132A CN202011230200.8A CN202011230200A CN112522132A CN 112522132 A CN112522132 A CN 112522132A CN 202011230200 A CN202011230200 A CN 202011230200A CN 112522132 A CN112522132 A CN 112522132A
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张文飞
王俊慧
何佳利
金映虹
赵子君
孙杰
王锐萍
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Haikou Haisenyuan Biotechnology Co ltd
Hainan Normal University
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Abstract

本发明公开了一种芽孢杆菌SJ110、杀虫蛋白、vip3‑like杀虫基因及应用。所述芽孢杆菌SJ110属于东洋芽孢杆菌Bacillus toyonensis，其在中国典型培养物保藏中心的保藏编号CCTCC NO：M2020524。生物活性测定表明菌株SJ110、杀虫蛋白、vip3‑like杀虫基因对甜菜夜蛾、棉铃虫以及秀丽隐杆线虫具有很高的毒性。表明菌株SJ110及其杀虫蛋白、vip3‑like杀虫基因可用于农业害虫的防治。

Description

一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用
技术领域
本发明涉及生物防治技术领域，具体涉及一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用。
背景技术
芽孢杆菌(如苏云金芽孢杆菌)在生长过程中能够生产对多种昆虫、线虫、原生动物和癌细胞等具有生物活性的多种杀虫因子。如杀虫晶体蛋白(Insecticidal CrystalProteins,ICPs)、几丁质酶、苏云金素以及营养期杀虫蛋白(Vegetative InsecticidalProteins,VIPs)等。不同于传统的化学农药，这些杀虫因子具有对人畜无毒害作用、环境友好以及杀虫谱广且易发酵特点，这将非常的有利于农林病虫防治以及转基因作物的应用。
杀虫晶体蛋白(ICPs)是Bt在生长过程中产生的δ-内毒素，是Bt主要的杀虫因子成分之一，以伴胞晶体的形式在菌体内累积。ICPs主要分为晶体蛋白(Crystal protein,Cry)和胞外溶解性蛋白(Crtolytic protein,Cyt)两类蛋白，其中Cry毒素蛋白是最早应用于农作物害虫的防治Bt毒素蛋白。
营养期杀虫蛋白(Vegetative Insecticidal Proteins VIPs)是在Bt菌株生长对数期所产生形成，其与ICPs蛋白氨基酸序列没有同源性，是一种新型的杀虫蛋白。最早是在1996年Estruch 等人发现了Vip3Aa和Vip3Ab蛋白，该蛋白于细菌的营养期生长阶段开始分泌。在四个Vip 蛋白家族中被发现的超过100个中以Vip3的研究最为深入广泛，且只有Vip3被运用于转基因作物中。
目前，少有研究报道新型东洋芽孢杆菌Bacillus toyonensis及其杀虫蛋白、基因在杀虫方面的应用。
发明内容
鉴于现有技术的不足，本发明提供一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用。
本发明技术方案如下：
本发明获得新型芽孢杆菌SJ110及其杀虫蛋白和vip3-like杀虫基因，所述芽孢杆菌SJ110属于东洋芽孢杆菌Bacillus toyonensis，其在中国典型培养物保藏中心的保藏编号CCTCC NO： M2020524，保藏日期2020.09.21，保藏地址为中国·武汉·武汉大学。所述杀虫蛋白的氨基酸序列如SEQ ID NO.1所示。所述vip3-like杀虫基因的核苷酸序列如SEQ IDNO.2所示。
另一方面，本发明还提供了所述芽孢杆菌SJ110、所述杀虫蛋白和所述vip3-like杀虫基因在杀虫方面的应用。
进一步的，所述虫为鳞翅目昆虫和/或小杆线虫目昆虫。
进一步的，所述虫为鳞翅目夜蛾科昆虫和/或小杆线虫目小杆科昆虫。
更进一步的，所述虫为棉铃虫、甜菜夜蛾和/或秀丽隐杆线虫。
本发明的有益效果是：
本发明提供一种新芽孢杆菌SJ110、杀虫蛋白及其vip3-like杀虫基因，生物活性测定表明菌株SJ110、杀虫蛋白、vip3-like杀虫基因对甜菜夜蛾、棉铃虫以及秀丽隐杆线虫具有很高的毒性。表明菌株SJ110及其杀虫蛋白、vip3-like杀虫基因可用于农业害虫的防治。
附图说明
图1：A为考马斯亮蓝染色光学显微镜观察；B为扫描电镜观察；
图2：菌株SJ110蛋白SDS-PAGE分析；注：PM为蛋白分子量标准，Lane1与Lane2为菌株SJ110蛋白，Lane3为苏云金芽胞杆菌YBT1520蛋白；
图3：vip3-like基因PCR克隆出2832bp目的片段；注：Lane1、Lane2、Lane3以及Lane4 为vip3-like基因片段，M为核酸分子标准量。
图4：Vip3-like表达蛋白纯化结果。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
实施例1菌株SJ110的筛选与鉴定
在海南岛热带雨林中选取生态环境人为干预少、土壤腐殖质丰富的地方采集土壤样品。采用Bt菌株的醋酸钠-温度筛选法，称取5g左右土壤放入20mL BPA(牛肉膏0.3％，蛋白胨 0.5％，氯化钠0.5％)培养基中，充分振荡，30℃摇床培养4-5h，75℃水浴15min，取1mL上清稀释至10-3、10-4和10-5，各吸取200μL均匀涂布于NB(牛肉膏0.5％，蛋白胨1％，氯化钠3.4％)培养平板，30℃倒置培养3d，挑取形态各异的单菌落划平板。芽孢杆菌培养3-5d 形成芽孢后，进一步利用考马斯亮蓝染色和光学显微镜观察，观察到无色芽孢和蓝色伴胞晶体。16S rDNA序列同源性分析，认定其为Bacillus toyonensis菌株，菌株编号为SJ110。(图1)
实施例2扫描电镜观察晶体蛋白
菌株SJ110在G-Tris培养基中30℃培养至芽孢完全形成，4℃，12,000g离心10min收集500μL菌体，然后用1M冰冷的NaCl洗涤菌体3次，最后菌体悬浮于500μL冰冷超纯水中。取10μL芽孢和伴胞晶体混合物小心平铺在洁净的盖玻片上，4℃低温真空干燥，然后用1％四氧化锇(OsO4)进行样品固定，样品被置于金属样品台上，放入真空蒸发器中喷镀一层约20nm厚的金属膜。准备好的样品置于S3-400N扫描电镜(HITACHI Ltd，Japan)，20 kV电压条件下进行图像观察记录。
实施例3 SDS-PAGE电泳分析菌株晶体蛋白
一般7.5％或者12％的SDS-PAGE可用于分析晶体蛋白组成，5μL制备好的伴胞晶体蛋白与5μL的2×SDS-PAGE sample Buffer[0.075M Tris·Cl,(pH 6.8)；0.02M EDTA；2％SDS； 0.02％bromophenol blue；0.25M dithiothreitol(DTT)；50％glycerol]混合，100℃沸水中煮5min，然后12,000g离心5min，上清立即上样，在100V电压下，利用Mini-ProteinII,cell slab vertical apparatus装置(BioRad，USA)电泳大约1h，然后用Coomassieblue R250进行染色，蛋白质大小根据标准蛋白质量标准进行判断。
实施例4菌株SJ110基因组DNA提取
单菌落接种于7mL的LB液体培养基中，30℃，220rpm培养过夜，1％转接于盛有50mL的LB培养基的250mL体积的三角瓶中，30℃，220rpm继续培养4小时至OD600＝1.0～2.0；离心收集10mL菌体，2mL J Buffer(0.1M Tris HCl(pH 8.0)、0.1M EDTA(pH 8.0)、0.15M Nacl)洗涤沉淀；将沉淀轻悬于2mL J Buffer中加入80μL新鲜配制的溶菌酶(50mg/mL)，混匀， 37℃温育45min；再加入15μL RNase(10mg/mL)，50℃作用15min；接着加入200μL SDS (20％)，70℃处理20min；缓慢冷却至37℃，用等体积酚：氯仿：异戊醇(25∶24∶1)及氯仿：异戊醇(24∶1)各抽提一次，再加入等体积异戊醇混匀置-20℃沉淀上清20min， 12,000rpm离心10min。70％乙醇洗涤沉淀2次，风干后溶于100μL TE Buffer(10mM Tris HCl (pH 8.0)、1mM EDTA(pH 8.0))中，取5μL DNA样品在0.8％的琼脂糖凝胶进行电泳，EB染色后紫外分析仪检测。
实施例5菌株SJ110基因组序列分析和新型毒素蛋白鉴定
通过Illumina与Pacbio测序技术结合获得SJ110基因组序列，使用Canu v1.5软件对过滤后subreads进行组装，得到最终准确度更高的基因组序列。进一步通过本地blast，发现1 个新型的营养期杀虫蛋白(Vip)，其开放阅读框(ORF)大小为2832bp，编码939个氨基酸，其分子量大小为105kDa，命名为Vip3-like蛋白。
实施例6制备重组蛋白PB6012
根据基因组序列，设计引物6012F(ttccaggggccccctgggatccATGGTTACTACAAAACTGACACCT) /6012R(gtcacgatgcggccgctcgagTTAAATTTGTTCCGATTCGTAAAG)克隆扩增vip3-like基因完整的编码序列，高保真ExTaq酶用于PCR扩增，PCR片段克隆到pGEX6p-1表达载体上，并将正确的重组子PB6012转入表达宿主细胞E.coli BL21(DE3)中诱导蛋白表达。4℃,12,000rpm离心10min收集菌体蛋白，充分悬浮于等体积的TE溶液，加入溶菌酶至终浓度为20ug/mL,37℃,220rpm振荡培养30min，离心收集菌体和蛋白，用等体积冰冷的1M的 NaCl洗涤3次，然后超声波破碎细胞破处理20min(model VC-130,Sonics and Materials Inc,USA)。由于表达的融合蛋白含有GST标签，利用试剂盒Fusion Protein Purification Kit(Sangon, Shanghai,China)在终浓度10mM的还原型谷胱甘肽对重组蛋白PB6012纯化。BSA蛋白作为标准浓度，lowry方法用来测定表达蛋白的浓度。表达的蛋白溶于PBS缓冲液中，然后稀释成8个浓度进行生物活性测定。
实施例7 Vip3-like蛋白对棉铃虫/甜菜夜蛾毒性测定
将重组蛋白PB6012与人工饲料混匀，自然风干，置于24孔细胞培养板。选取爬行较快、活跃初孵幼虫(卵孵化5天)放入细胞培养板，每孔1头。2天、4天分别调查死、活虫数，利用校正死亡率＝[(处理组死亡率-对照组死亡率)/(1-对照率死亡率)]×100％，得出重组蛋白 PB6012表达的Vip3-like蛋白在浓度为0.09mg/mL时对二龄甜菜夜蛾的杀虫活性校正死亡率达到43.33％。
实施例8 Vip3-like蛋白对秀丽隐杆线虫毒性测定
按1∶100的比例接种大肠杆菌OP50菌液于LB培养基，37℃培养6-8h。收集OP50菌体，无菌水洗涤三遍，取适量S medium溶液(1L S basal溶液中加入10mL 1M柠檬酸钾缓冲液 (pH6.0)10mL，3mL 1M CaCL2溶液，3mL 1M MgSO4)，悬浮菌体，最终菌液OD600＝3。同步化处理秀丽隐杆线虫，获取均一化的4龄幼虫(收集的线虫中按1％的量加入0.1％的TritonX-100，可以防止后续实验线虫粘附在枪头或离心管)。取S medium悬浮的E.coliOP50 40μL，M9缓冲液悬浮的4龄幼虫5μL，S medium 48μL，5-氟-2'-脱氧脲核苷5μL，氯霉素2μL，分装于48孔板。另取400μL浓度为0.09mg/mL的PB6012重组蛋白置于48孔板的阳性样品孔中，质粒pET30转BL21(DE3)作为阴性对照。最终将混合均匀的48孔板放入密封的塑料盒中，并放置有湿纸巾以提供湿度。在25℃孵育5d，每天观察生长死亡并记录，根据幼虫是否移动来确定幼虫中毒或死亡。在解剖显微镜下检查每个孔中的蠕虫。明显移动的蠕虫被标记为活着。不移动的蠕虫用铂金轻轻地触摸并观察移动。几次触摸后无法响应的蠕虫标记为死亡。记录每孔死亡幼虫比例，得出表达的Vip3-like蛋白在浓度0.072mg/mL的条件下对秀丽隐杆线虫的杀虫活性校正死亡率为100％。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 海口海森元生物科技有限公司
<120> 一种芽孢杆菌SJ110、杀虫蛋白、vip3-like杀虫基因及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 939
<212> PRT
<213> 东洋芽孢杆菌(Bacillus toyonensi)
<400> 1
Met Val Thr Thr Lys Leu Thr Pro Arg Leu Lys Ala Leu Pro Asp Phe
1 5 10 15
Met Asp Asp Phe Asn Gly Val Tyr Gly Phe Met Asp Asn Ile Ser Asn
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Leu Ile Gly Thr Ile Phe Gly Ile Asn Thr Gly Asp Ser Ser Leu Glu
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Ala Val Leu Glu Asn Gln Glu Leu Leu Gln Gln Met Met Asp Gln Met
50 55 60
Asn Thr Met Gln Gly Thr Leu Asp Asp Val Leu Gln Asn Gln Gly Phe
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Ser Glu Asp Val Leu Leu Gln Leu Arg Ser Leu Ala Asn Glu Gln Leu
85 90 95
Glu Leu Ser Lys Ser Ile Asn Thr Glu Leu Val Gln Ile Glu Gly Ile
100 105 110
Leu Asn Thr Tyr Leu Pro Ala Ile Ser Ser Met Val Asn Lys Val Tyr
115 120 125
Ser Gln Thr Ser Leu Ile Asn Lys Lys Val Asp Lys Leu Leu Gln Met
130 135 140
Met Ala Phe Ala Leu Gln Glu Leu Asp Tyr Ile Lys Asp Asn Val Val
145 150 155 160
Leu Asn Ser Ser Ile Ile Glu Ile Thr Pro His Val Gln Lys Leu Val
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Tyr Val Asn Ser Lys Phe Leu Ser Leu Ser Arg Ser Tyr Leu Gln Gly
180 185 190
Glu Asp Met Ser Ile Asp Arg Met Gln Glu Leu Ile Gln Trp Ala Lys
195 200 205
Ser Ile Val Asp Thr Asp Met Asn Ser Phe Glu Phe Ser Val Asp Thr
210 215 220
Leu His Thr Ile Ile Met Gly Asp Asn Leu Tyr Lys Arg Ser Ala Leu
225 230 235 240
Ala Thr Phe Ala Asp Val Leu Leu Asp Asp Ala Ser Gln Tyr Gly Asp
245 250 255
Phe Gly Thr Pro Val Thr Lys Phe Tyr Thr Phe Phe Ser Ser Leu Ala
260 265 270
Thr Leu Gln Ile Asn Ala Tyr Leu Cys Leu Thr Phe Ala Arg Lys Ile
275 280 285
Leu Gly Leu Thr Gln Val Asp Tyr Gln Ile Thr Met Gln Glu Arg Ile
290 295 300
Ser Lys Gln Asn Gln Leu Phe Ile Asn Leu Ile Glu Asp Lys Asn Val
305 310 315 320
Ser Ser Tyr Leu Glu Val Lys Gly Ile Ala Ala Leu Asp Pro Tyr Thr
325 330 335
Lys Glu Ile Lys Ser Phe Asp Leu Gln Ala Lys Asp Gly Tyr Val Phe
340 345 350
Ile Gly Leu Glu Phe Ile Ser Asp Gly Asn Glu Tyr Lys Val Lys Ala
355 360 365
Tyr Gln Ala Lys Ile Asp Lys Asn Phe Ser Val Phe Ala Asp Thr Val
370 375 380
Glu Glu Ile Ile Ser Asp Asn Leu Met Lys Val Phe Thr Tyr Leu Asn
385 390 395 400
Gly Gly Ser Ser Gly Lys Tyr Val Lys Phe Pro Phe Ser Gly Asn Leu
405 410 415
Thr Gly Ala Thr Asn Thr Leu Ile Thr Arg Ile Gly Phe Gly Cys Lys
420 425 430
Asn Asp Gln Ser Lys Ala Pro Asn Leu Tyr Ala Tyr Ile Asp Ala Asp
435 440 445
Phe Ser Pro Tyr Asn Pro Tyr Thr Gly Glu Ile Met Lys Glu Gly Thr
450 455 460
Gln Thr Ile Ser Leu Glu Gly Asn Lys Asp Thr Val Asn Ala Tyr Gly
465 470 475 480
Ile Trp Pro Ile Gly Leu Leu Gly Asp Ile Tyr Met Ala Pro Leu Lys
485 490 495
Ser Leu Phe Leu Ser Val Asp Ala Asp Ser Ala Ser Tyr Val Asp Ala
500 505 510
Thr Asp Ala Ile Leu Asn Phe Gly Gly Glu Ser Tyr Leu Pro Thr Ile
515 520 525
Leu Ser Lys Glu Tyr Asp Ala Asn Phe Ile Met Tyr Ser His Ile Glu
530 535 540
Asn Thr Asn Pro Val Asn Thr Asn Met Leu Met Asn Gly Asp Phe Glu
545 550 555 560
Glu Gly Asn Lys Tyr Trp Asp Ile Ser Leu Met Pro Ala Tyr Phe Ala
565 570 575
Glu Gly Glu Gly Met Gly Gly Ser Asn Ala Leu Lys Ala Met Glu Tyr
580 585 590
Gly Thr Phe Glu Gln Pro Val Phe Leu Glu Pro Asn Thr Lys Tyr Ile
595 600 605
Leu Gln Ala Phe Gly Lys Val Lys Gly Thr Ser Ser Ser Gly Arg Ile
610 615 620
Gly Ile Arg Asn Pro Arg Gly Glu Tyr Tyr Leu Glu Lys Ile Phe Thr
625 630 635 640
Pro Leu Gln Tyr Glu Leu Ile Glu Ile Glu Phe Gln Thr Gly Asn Asp
645 650 655
Thr Ser Asn Leu Ser Ile Phe Phe Gln Ala Phe Gly Gly Thr Ser Trp
660 665 670
Val Asp Asn Leu Lys Leu Tyr Asp Leu Thr Gln Lys Gly Asn Leu Ile
675 680 685
Ala Asn Pro Asn Phe Lys Ser Gly Asp Leu Ser His Trp Glu Thr Ser
690 695 700
Gly Glu Ala Thr Thr Val Lys Glu Lys Gly Met Phe Asn Ser Tyr Ala
705 710 715 720
Ile Gln Ile Glu Lys Lys Gly Glu Ile Asn Gln Gln Val Thr Met Glu
725 730 735
Pro Asn Thr His Tyr Lys Leu Glu Ala Tyr Val Lys Val Asp Asn Pro
740 745 750
Asn Thr Thr Ala Gln Ile Gly Tyr Gly Gln Asn Tyr Val Thr Cys Ser
755 760 765
Ser Thr Ser Phe Thr Leu Val Thr Val Lys Phe Ser Thr Gly Glu Ser
770 775 780
Pro Leu Asn Thr Glu Asp Leu Val Tyr Cys Thr Asn Ser Ser Asn Gln
785 790 795 800
Gly Thr Val Trp Ala Asp Asn Phe Val Leu Gln Lys Ile Pro Asn Leu
805 810 815
Ile Ala Asn Gly Asp Phe Lys Gln Pro Asn Pro Val Ala Ser Trp Thr
820 825 830
Leu Ser Pro Ser Asp Asn Gly Glu Ile Ser Ile Val Asp Lys Gly Ile
835 840 845
Gly Ile Leu Asn Lys Gly Gln Ile Ser Gln Lys Val Lys Leu Lys Pro
850 855 860
Asn Thr Lys His Thr Leu Thr Ala Tyr Val Arg Val Ala Gly Asn Gly
865 870 875 880
Ser Ala Ser Leu Gly Tyr Gly Asn Thr Ser Thr Thr Cys Thr Ser Gln
885 890 895
Asp Phe Lys Gln Val Ser Val Asp Phe Ile Thr Gly Ser Asn Pro Asn
900 905 910
Asn Asp Ser Leu Tyr Leu Ser Asn Thr Asn Asn Gly Leu Val Ile Gly
915 920 925
Ser Lys Phe Glu Leu Tyr Glu Ser Glu Gln Ile
930 935
<210> 2
<211> 2820
<212> DNA/RNA
<213> 东洋芽孢杆菌(Bacillus toyonensi)
<400> 2
atggttacta caaaactgac acctagacta aaagctcttc cagactttat ggatgacttt 60
aatggtgtat acgggtttat ggataacatt tctaatctaa taggtacaat ttttggaatt 120
aatactggtg attcatctct tgaggctgta ttagagaatc aagagttatt acaacaaatg 180
atggatcaaa tgaatactat gcagggcaca ttggatgacg ttttgcaaaa tcaagggttc 240
tccgaagacg tactactaca gcttcgaagt ttagcaaatg agcagttgga actctcaaag 300
tccattaata cagagcttgt acaaattgaa ggtattctaa atacgtattt acctgcaatt 360
agctctatgg taaataaagt atatagtcaa acgtcactga ttaacaaaaa agtagataaa 420
ttattacaaa tgatggcatt tgctttgcaa gaacttgatt atatcaaaga taatgttgta 480
ctgaattcga gtattataga aatcacaccc catgttcaaa aattggttta tgtgaatagc 540
aagtttcttt cgctatcaag aagttatttg caaggtgaag atatgagtat tgatagaatg 600
caagaactta ttcaatgggc aaaatcaata gttgatacag atatgaacag ctttgaattt 660
tcagtggata cattacacac tattataatg ggggacaatc tatataaaag atctgcttta 720
gcaacttttg ccgatgtttt attggatgat gctagtcaat acggagattt cggtacacca 780
gtaacaaaat tttatacatt tttctcttca ttagcaacgt tacaaataaa tgcttatctt 840
tgtttaactt tcgcaagaaa aattttaggg cttactcaag ttgattatca aataacgatg 900
caagagcgca ttagtaaaca aaatcaattg tttataaatt taattgaaga taaaaatgtt 960
tcctcatatc ttgaggtcaa aggaattgct gctcttgatc cgtatacaaa agagataaaa 1020
tcttttgacc ttcaagcaaa agacggatat gtatttatcg gcttagaatt tatttcggat 1080
ggtaacgaat acaaagtaaa agcctatcaa gctaagatag ataaaaactt ttcagtattt 1140
gctgatacag tagaagaaat cataagtgat aatttaatga aagtgtttac atatcttaat 1200
gggggatcga gtgggaaata tgtgaagttt cctttttcag gaaatctaac tggtgcaact 1260
aatacattga ttactcggat tggttttggt tgtaaaaatg atcaaagcaa agctcctaat 1320
ctatatgcct atatagatgc tgatttttct ccatataatc catacacagg agagataatg 1380
aaagagggca cacaaacaat ttcgttagaa gggaacaagg atactgttaa cgcttatgga 1440
atatggccta ttggtttatt gggagatatt tatatggccc ctttgaaatc tttattttta 1500
agtgttgatg ctgatagtgc ttcgtatgtt gatgccacag acgcaatact taattttggt 1560
ggtgaatctt atcttcctac aatcttgtct aaagaatatg atgctaattt tatcatgtat 1620
tctcatatcg agaatactaa tccagtgaat acgaatatgc ttatgaatgg agatttcgaa 1680
gaaggaaata aatactggga tatctcgtta atgcctgcat attttgctga aggagaaggt 1740
atgggtggtt caaatgcttt aaaagctatg gaatacggta cattcgagca accagtcttc 1800
ttagaaccca atacaaaata tatactacag gcatttggaa aagtaaaagg gacttcttca 1860
tcaggacgta ttggaatacg aaaccctaga ggtgagtact acttggaaaa aattttcaca 1920
ccgttacaat acgagctaat tgaaatagaa tttcaaacag gaaatgatac ttcaaattta 1980
tccatctttt ttcaggcgtt cgggggaact agttgggtag ataaccttaa gttatatgac 2040
ctcacgcaaa aaggaaattt aatagcgaac cctaatttta aatcagggga tctatcccat 2100
tgggaaacca gtggtgaagc aactacagta aaagaaaaag gaatgtttaa ttcgtatgct 2160
atacaaatcg agaaaaaggg agaaattaat caacaagtaa caatggaacc aaatacacat 2220
tacaaattgg aagcatatgt gaaagtagac aatcccaata ctactgcaca aattggatat 2280
ggccaaaatt atgtaacatg tagttcaact tctttcacat tagtgactgt gaaatttagt 2340
acgggtgaaa gccctctcaa tacagaagac ttggtgtact gtacaaattc tagtaatcag 2400
ggtactgttt gggcagataa ctttgtgtta caaaaaatcc ctaatttaat tgccaatgga 2460
gattttaaac agccgaatcc agttgcatct tggacactct ctccctctga taatggtgaa 2520
atttcaatag tagataaagg gattggtata ttaaataagg gtcaaattag tcaaaaagta 2580
aaattaaagc cgaatacaaa acatacattg acagcatatg taagagtagc aggtaatggt 2640
agtgcaagtc ttggatatgg aaatactagt acaacatgca cttcacagga ttttaagcaa 2700
gtgagtgtgg actttataac tggttctaac cctaataatg atagtttata cttgtccaat 2760
acaaataacg gtttggttat tggaagtaaa tttgagcttt acgaatcgga acaaatttaa 2820
<210> 3
<211> 46
<212> DNA/RNA
<213> 东洋芽孢杆菌(Bacillus toyonensi)
<400> 3
ttccaggggc cccctgggat ccatggttac tacaaaactg acacct 46
<210> 4
<211> 45
<212> DNA/RNA
<213> 东洋芽孢杆菌(Bacillus toyonensi)
<400> 4
gtcacgatgc ggccgctcga gttaaatttg ttccgattcg taaag 45

Claims (7)

1.芽孢杆菌SJ110，其特征在于，所述芽孢杆菌SJ110属于东洋芽孢杆菌Bacillustoyonensis，其在中国典型培养物保藏中心的保藏编号CCTCC NO：M2020524。
2.一种杀虫蛋白，其特征在于，所述杀虫蛋白的氨基酸序列如SEQ ID NO.1所示。
3.一种vip3-like杀虫基因，其特征在于，所述vip3-like杀虫基因的核苷酸序列如SEQID NO.2所示。
4.权利要求1所述芽孢杆菌SJ110、权利要求2所述杀虫蛋白和/或权利要求3所述vip3-like杀虫基因在杀虫方面的应用。
5.根据权利要求4所述的应用，其特征在于，所述虫为鳞翅目昆虫和/或小杆线虫目昆虫。
6.根据权利要求5所述的应用，其特征在于，所述虫为鳞翅目夜蛾科昆虫和/或小杆线虫目小杆科昆虫。
7.根据权利要求5所述的应用，其特征在于，所述虫为棉铃虫、甜菜夜蛾和/或秀丽隐杆线虫。
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