CN106243199A - 对甜菜夜蛾具有高活性的Vip3Aa11蛋白突变体 - Google Patents
对甜菜夜蛾具有高活性的Vip3Aa11蛋白突变体 Download PDFInfo
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- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
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- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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Abstract
本发明涉及获得了对甜菜夜蛾具有高活性的Vip3Aa11蛋白突变体,属于生物技术领域。本发明将Vip3Aa11蛋白氨基酸序列上686位点的丝氨酸(Ser)突变成精氨酸(Arg),获得的突变体蛋白S686R对甜菜夜蛾的毒杀效果在原有高活力的基础上提高了8.96倍,有效的克服寻找苏云金芽孢杆菌表达蛋白对甜菜夜蛾有高毒力基因的难题、延缓害虫对工程菌和转基因植物的抗药性产生。
Description
技术领域
本发明属于生物防治技术领域,具体涉及对鳞翅目农业害虫具有高毒力的Bt杀虫基因的定点突变及由该基因所编码的蛋白质。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是一种分布广泛的革兰氏阳性细菌,它是一种对人畜无害的昆虫病原微生物,是目前研究最为深入、应用最成功的微生物杀虫剂。营养期杀虫蛋白(VIPs)是一种在Bt在营养期分泌的一类新型杀虫毒蛋白,它对鳞翅目、鞘翅目昆虫具有较广谱的杀虫活性,该蛋白的发现使得Bt杀虫剂在杀虫活性、杀虫范围方面得到很大突破,在一定程度上克服了许多害虫对Bt内毒素低敏感或不敏感的弊端。
近年来一些研究表明,VIP蛋白上某些氨基酸区域与杀虫活性及特异性有关。Selvapandiyan等(Selvapandiyan A,Arora N,Rajagopal R,et al.Applied andEnvironmental Microbiology[J],2001,67(12):5855-5858.)的研究表明缺失Vip蛋白N端的39个氨基酸残基不影响其对蛀茎斑螟Chilo partellus的杀虫活性,但是对斜纹夜蛾Spodoptera litura(Fabricius)的活性显著降低,而缺失C端的154个氨基酸残基导致Vip蛋白对斜纹夜蛾完全丧失活性,对蛀茎斑螟的杀虫活性轻微下降。Chen等(Chen J,Yu J,Tang L,et al.Journal of Applied Microbiology[J],2003,95(2):310-316.)将vip3A-S184基因5′端信号肽编码区的81个核苷酸进行缺失突变后,表达的突变体蛋白在细胞质内形成包涵体,且对甜菜夜蛾、棉铃虫、斜纹夜蛾完全丧失活性。相比之下,Gayen等(Gayen S,Hossain M A,Sen S K.Journal of Plant Biochemistry and Biotechnology[J].2012,21(S1):128-135.)报道Ndv200(缺失Vip3BR N端的200个氨基酸残基)对棉铃虫、小地老虎、海灰翅夜蛾Spodoptera littoralis和三化螟Scirpophaga incertulas的毒力增强了2~3倍,而Cdv200完全失去毒力,Cdv127则维持了对棉铃虫和小地老虎较低水平的杀虫活性。Dong等(Dong F,Zhang S,Shi R,et al.Current Microbiology[J],2012,65(5):583-588.)基于Vip3蛋白在敏感昆虫肠液或Trypsin的作用下能水解成62k Da的毒性核心功能区。将Vip3Aa7蛋白的3个位于62kDa核心功能区的高度保守的半胱氨酸(Cys)残基替换成丝氨酸残基(Ser)(Dong F,Zhang S,Shi R,et al.Current Microbiology[J],2012,65(5):583-588.),结果显示,突变蛋白C292S对小菜蛾的杀虫活性降低、,而C507S和C401S对小菜蛾完全丧失杀虫活性。上述研究结果充分说明了VIP蛋白的N端和C端对于杀虫活性的发挥起到了举足轻重的作用,但VIP蛋白对甜菜夜蛾杀虫特异性相关氨基酸区域尚无明确报道,且多数突变体为多点突变且活性大多丧失,通过单点突变提高新活的成功案例较少。
发明内容
针对上述领域中的缺陷,本发明采用单点突变的方法,将Vip3Aa11蛋白氨基酸序列上686位点的丝氨酸(Ser)突变成精氨酸(Arg),获得的突变体蛋白S686R对甜菜夜蛾的毒杀效果提高了8.96倍,有效的克服寻找苏云金芽孢杆菌表达蛋白对甜菜夜蛾有高毒力基因的难题、延缓害虫对工程菌和转基因植物的抗药性产生。
突变体vip3Aa11-S686S,其具有如SEQ ID NO4所示的氨基酸序列。
编码上述突变体vip3Aa11-S686S的基因序列。
所述的基因序列如SEQ ID NO2所示。
上述突变体vip3Aa11-S686S在杀灭甜菜夜蛾害虫中的应用。
所述应用为将权利要求1所述的突变体vip3Aa11-S686S制成杀虫剂杀灭甜菜夜蛾。
本发明采用定点突变技术将Vip3Aa11蛋白686位点的丝氨酸(Ser)突变成精氨酸(Arg),获得了高活性的Vip蛋白突变体。实验表明,突变体vip3Aa11-S686S对甜菜夜蛾的杀虫活性提高8.9倍左右。
附图说明
图1突变原理示意图,
图2引物设计原理图,
图3 vip3Aa11突变基因的PCR鉴定结果,
其中1:vip3Aa11突变基因;2:vip3Aa11-S686R突变基因;CK-:阴性对照,
图4 vip3Aa11和突变体基因在大肠杆菌(BL21)中表达结果,
其中1:Vip3Aa11原蛋白;2:Vip3Aa11-S686R突变蛋白
具体实施方式
下面结合实施例对本发明做进一步的详细说明。
1.材料与试剂
1.1菌株与质粒
实验所用菌株与质粒详见表1,可以对公众发放。
表1菌株与质粒
33
1.2引物
参考GenBank公布的已知vip3Aa11基因序列,设计全长引物一对V3-F/V3-R。构建单点突变体引物一对Modvip3Aa11-S686S-F/Modvip3Aa11-S686S-R。以上引物均由生工生物工程(上海)股份有限公司合成。
表2 PCR鉴定引物及突变引物序列
1.3供试昆虫
甜菜夜蛾室内敏感种群标准试虫,购自河南省济源白云实业有限公司。
1.4酶与生化试剂
突变试剂盒购自天根生化科技(北京)有限公司;质粒提取试剂盒购自Axygen公司;DNAmarker购自北京康为世纪生物科技有限公司;Taq Mix聚合酶和蛋白Marker均购自TaKaRa公司;抗生素购自纳川生物技术公司;
其它试剂均为市售国产或者进口分析纯或电泳级纯化学试剂。
蛋白高分子量标准:Protein Standard(High range)(kDa):200、116、97、66、44
DM2000DNA Marker(bps):5000、3000、2000、1000、750、500、250、100
1.5培养基与抗生素
液体LB培养基:trytone 1%,yeast extract 0.5%,NaCl 1%,pH 7.0,121℃/20mim灭菌。
固体LB培养基:在液体培养基中加入1.3%琼脂,121℃/20mim灭菌。
抗生素:硫酸卡那霉素水溶液100mg/mL用时稀释1000倍,-20℃保存。
1.6其它试剂
(1)20mM Tris-HCL
Tris-base 2.42g
超纯水定容至1000mL,121℃/20mim灭菌后,用HCL将pH调至8.0
(2)1.0mol/L IPTG
IPTG 1.2g,
超纯水配制x5mL,-20℃保存
(3)0.1g/mL Aps
Aps 0.1g
超纯水定容至1mL,充分混匀,现用现配。
(4)0.1mg/mL SDS
SDS 0.1g
超纯水定容至1mL,充分混匀,现用现配。。
(5)30%丙烯酰胺/甲叉双丙烯酰胺
丙烯酰胺 29.2g
甲叉双丙烯酰胺 0.8g
超纯水配制100mL,4℃保存
(6)SDS-PAGE 3×上样缓冲液
(7)电泳缓冲液
SDS 1.00g
甘氨酸 14.4g
Tris 3.03g
超纯水定容至1000mL,4℃保存
(8)分离胶缓冲液
Tris 27.23g
S DS 0.4g
超纯水定容至150mL,用盐酸调pH 8.8,4℃保存
(9)浓缩胶缓冲液
Tris 6g
SDS 0.4g
超纯水定容至100mL,用盐酸调pH 6.8,4℃保存
(10)50×TAE:
冰乙酸 57.1mL
0.5mol/L EDTA(pH 8.0) 100mL
Tris 242g
纯水定容至1000mL,4℃保存
(11)考马斯亮蓝R250染色法
溶液一:50%乙醇、10%乙酸
溶液二:5%乙醇、7.5%乙酸
溶液三:0.25%R250的95%乙醇溶液
注:50mL溶液二加入200μL溶液三
1.7主要仪器设备
(1)摇床D250,美国NBS公司产品;
(2)恒温培养箱DHP120,上海实验仪器总厂;
(3)Beckman高速离心机Avanti J-26xp;
(4)德国台式Eppenddorf离心机5415C;
(5)PCR仪:TC-XP-G;Multigene Gradient(TC9600-G-230V);
(6)核酸电泳仪:DYY-5型,北京六一厂;
(7)蛋白电泳仪:Mini protein III,美国Bio-Rad公司;
(8)凝胶成像系统Eagle EyeII System:美国STRAGENE公司;
(9)光学显微镜:日本OLYMPUS CX21;
(10)离心机:Z233MK-2;迷你离心机:ABSON MiFly-6型;
(11)高速冷冻离心机:Beckman:A1legraTM64R Centrifuge;
(12)Eppendorf离心机:Centrifuge 5424YK424775;
(13)酸度计:METTLER TOLEDO 320PH Meter;PB-20;
(14)恒温金属浴:CHB-100型;
(15)恒温培养箱:DHP-9162型;
(16)小容量恒温培养摇床:HWY-100B;ZHWY200B;
(17)分析天平:YL-41-160100D;
2实验方法
2.1PCR克隆突变体
突变原理,首先根据原基因序列以及突变位点分别设计正向和反向引物,且两条引物上都要包含突变位点,且除突变位点以外的碱基都要与质粒模板互补配对如图2,以连有载体Pet-3Aa11质粒(本实验室保存)为模板,利用Fast Alteration DNA Polymerase进行PCR扩增将Vip3Aa11基因2056位点的腺嘌呤(A)用胞嘧啶(C)取代,将2056位点的胸腺嘧啶(T)用胞嘧啶(C)取代,获得带有缺口的突变质粒,再用Dpn1消化掉甲基化的模板质粒,留下刚刚扩增得到的突变质粒,最后转入受体菌后突变质粒的缺口得以修复,从而进行复制如图1。(1)PCR克隆突变体的反应体系如下:
(2)扩增条件如下:
2.2消化甲基化模板:
(1)酶切体系:
PCR产物 50μL
Dpn1restriction enzyme 1μL
Total 51μL,
(2)反应条件:
37℃处理1h;
2.3BL21感受态细胞制备及转化
(1)将甘油菌在固体LB培养基上进行活化,37℃培养10小时后,再进行划线培养,37℃培养12小时后,挑取单菌落于5mL液体LB培养基中,37℃,220rpm震荡培养12小时,再按1%接菌量转入100mL液体LB培养基中扩大培养(500mL三角瓶),37℃,220rpm培养,培养至OD600约为0.5。
(2)将菌液转移至2个无菌预冷的50mL离心管中,冰上放置10min,使培养物冷却到0℃;
(3)4℃,4100rpm离心10min;
(4)倒出培养液,将管倒置1min,使最后痕量的培养液流尽;
(5)每50mL初始培养液用30mL预冷的0.1mol/L CaCl2-MgCl2(20mM CaCl2-80mMMgCl2)重悬每份细胞;
(6)4℃,4100rpm离心10min;
(7)每50mL初始培养物用2mL冰预冷的0.1mol/L CaCl2重悬,再加入等体积甘油,分装200μL/管,-70℃保存。
将消化产物10μL加入到大肠杆菌BL21的感受态细胞中,混匀,冰浴30min以上,取出离心管42℃准确热击90s,再冰浴3min,加入900μL LB液体培养基37℃培养1h左右,取200μL于LB固体平板涂布,加入卡纳霉素,4μL,37℃培养过夜,筛选出阳性克隆。
2.4阳性克隆的PCR鉴定
挑取单克隆于5mL LB液体培养基震荡培养,37℃,220rpm,震荡培养6h,取1μL作为模版,进行PCR鉴定,PCR鉴定体系和反应条件如下:
(1)PCR体系:
(2)反应条件:
PCR产物进行琼脂糖凝胶电泳检测。
2.5序列测定及分析
将阳性克隆送往吉林省库美生物科技有限公司进行全长测序,DNAMAN软件分析测序结果正确后,对正确重组转化子进行诱导和表达目的蛋白。
2.6大肠杆菌质粒DNA提取
(1)挑取阳性转化子于LB液体培养基中培养过夜,取1-4ml菌液于12000r/min离心1min,弃去上清;
(2)加入250μL含有50mg/mlRNANase的S1溶液悬浮沉淀;
(3)加入250μL细菌裂解液S2并充分缓慢温和地上下翻转4-6次,直到溶液透明清亮为止,这一步骤不应该超过5分钟;
(4)加入350μL S3中和液并充分温和地上下翻转6-8次,12000r/min下离心10分钟;
(5)随后吸取上清,然后转移到制备管中,在12000r/min条件下离心1分钟并弃掉滤液;
(6)随后加入500μL洗涤液W1,12000r/min条件下离心1分钟并弃掉滤液;
(8)随后加入700μL,W2,洗去多余盐分,12000r/min条件下离心1分钟,之后重复一次;
(9)将收集柱转移到新1.5ml离心管上,收集柱中央滴加60-80μL预热至65℃的TE缓冲液或灭菌水,静止5分钟,12000r/min条件下离心1分钟,风干并保存在-20℃备用。
2.7基因的诱导表达
(1)将大肠杆菌的单菌落接种于LB液体培养基37℃条件下220rpm活化12-16h;
(2)采用1%接种量接种阳性表达菌株于100mL LB液体培养基的锥形瓶中,37℃条件下,220rpm振荡培养大约2h至OD600=0.6左右;之后加入1M的IPTG100μL;放置于摇床上160rpm低速培养,16℃低温条件下诱导大约14h,;
(3)将诱导的培养液在6000rpm的条件下离心5min并收集菌体,用预冷的20mmol/LTris-Cl(pH调至8.0左右)悬浮菌体2-3次;
(3)在冰水混合物中超声波破碎菌体,超3s,停3s,5ml悬浮菌液大约超声8min;
(4)在4℃和15000rpm条件下离心15min分别收集上清和沉淀,沉淀组分依然用20mmol/L Tris-Cl悬浮。之后分别进行蛋白电泳检测即SDS-PAGE。
2.8蛋白电泳检测
电泳:将粗蛋白样品与3x上样Buffer按2:1混匀,沸水浴处理10min,取待测样品液10L上样,电泳仪设置120V预电泳10-15min,然后进行150V恒压电泳1h。
染色:电泳结束后取出蛋白凝胶,放入50mLSI溶液中,置微波炉加热30s,60rpm振荡5min;将胶取出后放入SII和SIII的混合液中(每50mL SII加200L SIII),再微波炉加热30s后取出摇床60rpm振荡至蛋白胶条带清晰可见,凝胶成像系统成像。
2.9甜菜夜蛾生物活性测定
(1)取30g人工饲料置一灭菌培养皿中,加入3.0mL待测样品溶液,用药匙充分搅拌均匀,室温放置,使饲料多余水分蒸发;
(2)将全部饲料分装于3个已消毒的24孔细胞培养板中;
(3)先将幼虫轻轻抖落在一张白纸上,再将白纸倒置,此时幼虫将从白纸上开始拉丝下滑,用毛笔轻轻挑丝将幼虫接于24孔板中,每孔一头;接完后,在24孔板上盖一层吹塑纸,再盖盖子,并用橡皮筋固定,严格密封,防止幼虫逃逸;
(4)放置27℃光照培养箱中培养,光周期为14:10,湿度控制在50%左右。每天观察,检查光照、湿度、温度以及饲料是否霉变,是否有水蒸气凝结;
(5)7天分别调查死、活虫数,计算死亡率和LC50。
3结果与分析
3.1阳性克隆的鉴定
挑取阳性克隆进行PCR鉴定(V3F和V3R为引物),DNA核酸电泳检测结果如图3所示,可以检测到2370bp的目的条带,说明,阳性克隆子中含有vip3Aa11基因或其突变基因。
3.2测序结果比对
利用DNAMAN对测序结果与原序列进行比对,结果显示已经成功获得了连有载体的突变基因。
3.3突变体的表达分析
突变体转入大肠杆菌BL21感受态细胞后,进行IPTG诱导表达,并利用超声波破碎法提取目的蛋白,最后以SDS-PAGE分别检测可溶性蛋白和包涵体蛋白。结果表明:突变体能在大肠杆菌中正常表达分子量为88.6kD的片段(图4)。
3.4甜菜夜蛾生物活性分析
以梯度稀释法将蛋白稀释成相应浓度后,加入到30g人工饲料中,混匀备用。7天分别统计死虫数与活虫数,计算校正死亡率。利用SPSS 17.0软件分析各各蛋白的致死中浓度(LC50),结果显示(表3),Vip3Aa11蛋白对甜菜夜蛾的活性为18.399μg/g,突变体vip3Aa11-S686S对甜菜夜蛾的活性为2.053μg/,比突变前的Vip3Aa11蛋白提高8.9倍左右。
表3突变体对甜菜夜蛾的致死中浓度
4结论
4.1本发明采用定点突变技术将Vip3Aa11蛋白686位点的丝氨酸(Ser)突变成精氨酸(Arg),获得了高活性的Vip蛋白突变体。
4.2突变体vip3Aa11-S686S对甜菜夜蛾的杀虫活性提高8.9倍左右。
Claims (5)
1.突变体vip3Aa11-S686S,其具有如SEQ ID NO4所示的氨基酸序列。
2.编码权利要求1所述的突变体vip3Aa11-S686S的基因序列。
3.权利要求2所述的基因序列如SEQ ID NO2所示。
4.权利要求1所述的突变体vip3Aa11-S686S在杀灭甜菜夜蛾害虫中的应用。
5.根据权利要求4所述应用,为将权利要求1所述的突变体vip3Aa11-S686S制成杀虫剂杀灭甜菜夜蛾。
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CN114907458A (zh) * | 2022-05-10 | 2022-08-16 | 山东大学 | 一种活性提高的Vip3A突变蛋白及其应用 |
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CN107475279B (zh) * | 2017-07-25 | 2020-08-11 | 黑龙江八一农垦大学 | 苏云金芽胞杆菌Vip基因的表达T载体构建方法及应用 |
CN114907458A (zh) * | 2022-05-10 | 2022-08-16 | 山东大学 | 一种活性提高的Vip3A突变蛋白及其应用 |
CN114907458B (zh) * | 2022-05-10 | 2023-12-22 | 山东大学 | 一种活性提高的Vip3A突变蛋白及其应用 |
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