CN116267981A - 沉默stat基因在提高防治昆虫效果中的应用 - Google Patents
沉默stat基因在提高防治昆虫效果中的应用 Download PDFInfo
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Abstract
本发明涉及沉默STAT基因在提高防治昆虫效果中的应用。沉默靶标昆虫中的STAT基因可以提高Cry1F蛋白或Vip3A对所述靶标昆虫的防治效果。
Description
技术领域
本发明涉及生物防治领域,特别涉及沉默STAT基因在提高防治昆虫效果中的应用。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,Bt)表达的杀虫蛋白可以有效防治靶标害虫和减少化学农药的使用。然而单一使用杀虫蛋白用来防治靶标害虫的效果有限。
发明内容
本发明之一提供了沉默靶标昆虫中的STAT基因表达在提高对所述靶标昆虫防治效果中的应用。
在一个具体实施方式中,沉默所述靶标昆虫中的STAT基因表达的效率在50%以上。
在一个具体实施方式中,所述靶标昆虫为鳞翅目昆虫。
在一个具体实施方式中,所述靶标昆虫为夜蛾科昆虫中的至少一种。
在一个具体实施方式中,所述靶标昆虫为草地贪夜蛾(Spodoptera frugiperda)。
在一个具体实施方式中,沉默所述靶标昆虫中的STAT基因表达在提高Cry蛋白和/或Vip蛋白对所述靶标昆虫防治效果中的应用。其中,Cry蛋白和Vip蛋白均为来源于苏云金芽胞杆菌的杀虫蛋白或经来源于苏云金芽胞杆菌的杀虫蛋白突变后氨基酸序列一致性在95%以上的蛋白。
在一个具体实施方式中,所述Cry蛋白为Cry1F蛋白。
在一个具体实施方式中,所述Cry蛋白为Cry1Fa3蛋白,例如其氨基酸序列如Genbank No.AEH31417.1所示。
在一个具体实施方式中,所述Vip蛋白为Vip3A蛋白。
在一个具体实施方式中,所述Vip蛋白为Vip3Aa11蛋白,例如其氨基酸序列如Genbank No.AAR36859.1所示。
在一个具体实施方式中,沉默所述靶标昆虫中的STAT基因表达的dsRNA中的正义链的序列如Genbank No.XP_035457229.1所示,或如SEQ ID No.1所示。其中,在作为RNA时,其中的t替换为u。
本发明之二提供了一种联合用药物,其包括用于沉默靶标昆虫中的STAT基因表达的dsRNA和对所述靶标昆虫具有杀虫活性的蛋白,所述蛋白为Cry蛋白和/或Vip蛋白。其中,所述dsRNA和所述对所述靶标昆虫具有杀虫活性的蛋白可以先后施用,也可以同时施用,优选先后施用;优选在施用所述dsRNA 48小时后再施用所述对所述靶标昆虫具有杀虫活性的蛋白。
在一个具体实施方式中,所述dsRNA中的正义链的序列如Genbank No.XP_035457229.1所示,或如SEQ ID No.1所示。其中,在作为RNA时,其中的t替换为u。
在一个具体实施方式中,所述Cry蛋白为Cry1F蛋白。
在一个具体实施方式中,所述Cry蛋白为Cry1Fa3蛋白,例如其氨基酸序列如Genbank No.AEH31417.1所示。
在一个具体实施方式中,所述Vip蛋白为Vip3A蛋白。
在一个具体实施方式中,所述Vip蛋白为Vip3Aa11蛋白,例如其氨基酸序列如Genbank No.AAR36859.1所示。
本发明的有益效果:
本发明首次发现沉默草地贪夜蛾STAT基因的表达可以显著提升Cry1F蛋白和/或Vip3A蛋白对草地贪夜蛾的活性,这为开发新一代生物杀虫剂和转基因植物奠定了基础,丰富了我国杀虫工程微生物的基因资源库。
具体实施方式
以下通过优选的实施案例的形式对本发明的上述内容作进一步的详细说明,但其不构成对本发明的限制。
如无特别说明,本发明的实施例中的试剂均可通过商业途径购买。
实施例1
1.草地贪夜蛾的SfSTAT基因的克隆和dsSfSTAT的制备
根据草地贪夜蛾的信号传导及转录激活(signal transducer and activator oftranscription,STAT)基因(SfSTAT,Genbank No.XP_035457229.1)序列,在SnapDragon-dsRNA Design网站(https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl)设计用于RNA干扰的dsRNA的DNA片段(dsSfSTAT DNA,其正义链的碱基序列如SEQ ID No.1所示)和dsRNA的引物对,其中上游引物为dsSfSTATF(SEQ ID No.2),下游引物为dsSfSTATR(SEQ IDNo.3)。
利用RNeasy Mini Kit试剂盒(74106,QIAGEN公司)提取草地贪夜蛾三龄幼虫的总RNA,使用反转录试剂盒(R223-01,南京诺唯赞生物公司)进行反转录,得到cDNA,然后以cDNA为模板,以dsSfSTATF和dsSfSTATR为引物,进行PCR,并将PCR产物克隆到pEASYBluntZero质粒(北京全式金生物公司)上,得到含有dsSfSTAT的pEASYBluntZero-SfSTAT阳性质粒。
然后利用T7 RiboMAXTM Express RNAi System试剂盒(P1700,Promega)以pEASYBluntZero-SfSTAT为模板,以dsSfSTATF和dsSfSTATR为引物合成SfSTAT的dsRNA片段,最后溶于无酶水中,得到dsSfSTAT水溶液。
对比例1
阴性对照dsgfp的制备
由上海生工公司合成序列如SEQ ID No.4所示的gfp DNA。
利用T7 RiboMAXTM Express RNAi System试剂盒(P1700,Promega)以gfp DNA为模板,以gfpF(SEQ ID No.5)和gfpR(SEQ ID No.6)为引物,合成gfp的dsRNA片段(dsgfp,其正义链的碱基序列如SEQ ID No.4所示),最后溶于无酶水中,得到dsgfp水溶液。
实施例2
1.Cry1Fa3蛋白的制备
Cry1Fa3基因(Genbank No.HM070028.1,其氨基酸Genbank No.AEH31417.1)连接在pSTK表达载体上,即pSTK-Cry1F,并转化到Bt HD73-中,得到HD73/pSTK-Cry1F。同时,将pSTK空载转化到Bt HD73-中,得到HD73/pSTK。
挑取HD73/pSTK-Cry1F单菌落于5mL LB培养基中(加5μL红霉素),30℃ 230rpm12h;按1%的接菌量转接到1L三角瓶中(每瓶盛有300mL 1/2LB培养基,并加入红霉素),30℃,230rpm,大约22h,镜检观察50%以上的菌体裂解时停止培养,得到发酵液;将发酵液4℃8000rpm离心6min,离心的沉淀物用预冷的1.0mol/L NaCl(50mL/1升菌液)洗涤,4℃8000rpm离心10min后,用预冷的无菌水洗涤一次(50mL/1升菌液);收集沉淀,悬于裂解液中(含3%β-巯基乙醇),pH调到9.5,冰上摇8h;4℃ 14000rpm离心20min,取上清,加入1/7体积的4.0mol/L NaAc-HAc(pH 4.5),调pH 4.5;4℃静止4h(或冰上沉淀2h);4℃ 14000rpm离心15min,沉淀用无菌水洗悬浮后,4℃ 14000rpm离心15min,重复一次,最后收集沉淀,并溶于50mM Na2CO3水溶液中,搅拌至完全溶解,得到Cry1Fa3蛋白溶液。SDS-PAGE检测Cry1Fa3蛋白的提取结果,以确认目标蛋白Cry1Fa3成功表达(以相同条件下获得的HD73/pSTK的蛋白提取液作为阴性对照)。
2.Vip3Aa11蛋白的制备
(1)蛋白提取
Vip3Aa11基因(Genbank No.AY489126.1,其氨基酸Genbank No.AAR36859.1)连接在pET28a表达载体上,得到pET28a-Vip3A,并转化到大肠杆菌BL21(DE3)中,得到BL21(DE3)/pET28a-Vip3A。同时,将pET28a空载转化到大肠杆菌BL21(DE3)中,得到BL21(DE3)/pET28a。
将大肠杆菌BL21(DE3)/pET28a-Vip3A接种于5mL含有1‰的卡那霉素的LB液体培养基中,于37℃ 220r/min培养8h,得到活化培养液;取1%的活化培养液接种于300mL含有1‰的氨苄青霉素的LB液体培养基中,于37℃ 220r/min培养至OD600=0.8;加入IPTG至终浓度为0.5mmol/L,然后于18℃ 150r/min诱导14h,得到诱导培养液;将诱导培养物12000g离心8min收集菌体,将收集到的菌体悬浮于结合缓冲液(50mM咪唑,20mM Tris-HCl,500mMNaCl,pH 8.0),其中结合缓冲液与菌体的用量比以结合缓冲液与诱导培养液的体积比计为7:100;超声破碎细胞5min(超声功率:70%;超声:3s,暂停:5s);13000g离心15min,收集上清液,通过SDS-PAGE分析上清液以确认目标蛋白Vip3Aa11成功表达(以相同条件下获得的BL21(DE3)/pET28a上清液作为阴性对照)。
(2)亲和纯化
将上一步得到的20mL上清液加入2mL镍(Ni)亲和柱料至纯化柱中;以5倍柱体积结合缓冲液洗脱杂蛋白;以10mL洗脱液II洗脱目的蛋白Vip3Aa11并收集;将收集到的Vip3Aa11蛋白使用脱盐柱(HiPrep 26/10,Cytiva)在AKTA-Avant蛋白纯化系统(Cytiva)中进行脱盐纯化,其中缓冲液为20mmol/L Tris-HCl(pH 8.0);流速为8mL/min,检测UV280nm,在蛋白峰处收集脱盐后的蛋白溶液,得到纯化的Vip3Aa11溶液。
实施例3
对草地贪夜蛾的生物活性测定
1.dsRNA-SPc复合物的制备
将实施例1制备得到的dsSfSTAT水溶液用无酶水梯度稀释为5个浓度;将该5个浓度下的dsSfSTAT水溶液与纳米材料SPc均以dsSfSTAT与SPc质量比为1:1混合,得到dsSfSTAT-SPc溶液;分别各向dsSfSTAT-SPc溶液中加入终溶液(即dsSfSTAT-SPc复合物)总体积1%的脂肪醇醚硫酸钠,混合均匀,室温孵育15min得到dsSfSTAT-SPc复合物。其中,纳米材料SPc的制备按照A facile-synthesized star polycation constructed asahighly efficient gene vector in pest management(Li,J.,Qian,J.,Xu,Y.,Yan,S.,Shen,J.,and Yin,M.,ACS Sustainable Chem.Eng.7,6316-6322,2019.)来制备。
以同样的方式制备dsgfp-SPc复合物,作为阴性对照。
2.dsSfSTAT的沉默效率测定
草地贪夜蛾人工饲料配方:琼脂55g,黄豆粉110g,麦胚粉210g,酵母粉40g,山梨酸4g,干酪素55g,抗坏血酸4g,复合维生素3mL,甲醛3mL,乙酸6mL,特克多1mL,蒸馏水1800mL。
使用超纯水将dsSfSTAT-SPc复合物和dsgfp-SPc复合物分别稀释至333μL,然后分别加入到1g人工饲料中,混合均匀,使dsSfSTAT在人工饲料中的浓度为25μg/g,dsgfp在人工饲料中的浓度为25μg/g,将混合均匀的人工饲料分装于指形管中,每个指形管接入15头草地贪夜蛾初孵幼虫,每个处理组3次重复。饲喂48h后,以实施例1相同的操作提取草地贪夜蛾幼虫的RNA,并反转录获得cDNA,以所获得的cDNA为模板,以Q-SfSTAT-F(SEQ ID No.7)和Q-SfSTAT-R(SEQ ID No.8)为引物用qRCR试剂盒(Q131-02,南京诺唯赞生物公司)进行qRCR以检测RNA干扰效率。其中,qRCR的配制体系如表1。
表1
使用QuantStudio 6Flex System(ABI公司)检测,扩增条件为:95℃预变性5min,95℃ 10s,60℃ 30s,40个循环,进行溶解曲线分析。根据2-△△Ct方法,根据Ct值计算SfSTAT基因在经dsSfSTAT处理后与dsgfp对照组相比的相对表达量,并基于相对表达量计算沉默效率(或称之为干扰效率),结果见表2。其中,沉默效率=(1-相对表达量)×100%。
表2
注:同列数据后不同小写字母表示在P<0.001水平上差异显著。
表2的结果显示,当dsSfSTAT浓度为25μg/g时沉默效率达到50%以上。
3.dsSfSTAT和杀虫活性的蛋白联合使用的生物活性测定
1)dsSfSTAT-Cry1F处理:将用25μg/g dsSfSTAT干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有Cry1Fa3蛋白含量为200μg/g的人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用含有Cry1F蛋白的人工饲料饲喂7天时统计死亡率。
2)dsgfp-Cry1F处理:将用25μg/g dsgfp干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有Cry1Fa3蛋白含量为200μg/g的人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用含有Cry1F蛋白的人工饲料饲喂7天时统计死亡率。
3)dsSfSTAT-Vip3A处理:将用25μg/g dsSfSTAT干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有Vip3Aa11蛋白含量为200μg/g的人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用含有Vip3Aa11蛋白的人工饲料饲喂7天时统计死亡率。
4)dsgfp-Vip3A处理:将用25μg/g dsgfp干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有Vip3Aa11蛋白含量为200μg/g的人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用含有Vip3Aa11蛋白的人工饲料饲喂7天时统计死亡率。5)dsSfSTAT处理:将用25μg/gdsSfSTAT干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用人工饲料饲喂7天时统计死亡率。
6)dsgfp处理:将用25μg/g dsgfp干扰48h(操作同上述第2小节)的草地贪夜蛾幼虫转入装有人工饲料的指形管中,每管接32头幼虫,重复3次。将指形管置于温度为27±2℃、湿度为70±5%、14:10h光照周期的培养箱中培养,在用人工饲料饲喂7天时统计死亡率。
结果见表3。
表3的结果显示,dsSfSTAT-Cry1F处理的幼虫的死亡率为69.56%,显著高于dsgfp-Cry1F处理的32.30%;dsSfSTAT-Vip3A处理的幼虫的死亡率为53.26%,显著高于dsgfp-Vip3A处理的30.21%;此外,dsSfSTAT处理与阴性对照dsgfp处理的草地贪夜蛾幼虫的死亡率没有显著差异。以上结果说明,虽然仅沉默SfSTAT基因不能使草地贪夜蛾死亡,即沉默SfSTAT基因对草地贪夜蛾没有生物活性;但沉默草地贪夜蛾的SfSTAT基因却可以显著提高Cry1Fa3蛋白和Vip3Aa11蛋白对草地贪夜蛾的杀虫效果。
表3
注:同列数据后不同小写字母表示在P<0.001水平上差异显著。
Claims (10)
1.沉默靶标昆虫中的STAT基因表达在提高对所述靶标昆虫防治效果中的应用。
2.根据权利要求1所述的应用,其特征在于,沉默所述靶标昆虫中的STAT基因表达的效率在50%以上。
3.根据权利要求1所述的应用,其特征在于,所述靶标昆虫为鳞翅目昆虫。
4.根据权利要求1所述的应用,其特征在于,所述靶标昆虫为夜蛾科昆虫中的至少一种。
5.根据权利要求1所述的应用,其特征在于,所述靶标昆虫为草地贪夜蛾(Spodopterafrugiperda)。
6.根据权利要求1所述的应用,其特征在于,沉默所述靶标昆虫中的STAT基因表达在提高Cry蛋白和/或Vip蛋白对所述靶标昆虫防治效果中的应用;
优选地,所述Cry蛋白为Cry1F蛋白;
优选地,所述Cry蛋白为Cry1Fa3蛋白,例如其氨基酸序列如Genbank No.AEH31417.1所示;
优选地,所述Vip蛋白为Vip3A蛋白;
优选地,所述Vip蛋白为Vip3Aa11蛋白,例如其氨基酸序列如Genbank No.AAR36859.1所示。
7.根据权利要求1所述的应用,其特征在于,沉默所述靶标昆虫中的STAT基因表达的dsRNA中的正义链的序列如Genbank No.XP_035457229.1所示,或如SEQ ID No.1所示。
8.一种联合用药物,其包括用于沉默靶标昆虫中的STAT基因表达的dsRNA和对所述靶标昆虫具有杀虫活性的蛋白,所述蛋白为Cry蛋白和/或Vip蛋白。
9.根据权利要求8所述的联合用药物,其特征在于,所述dsRNA中的正义链的序列如Genbank No.XP_035457229.1所示,或如SEQ ID No.1所示。
10.根据权利要求8所述的联合用药物,其特征在于,所述Cry蛋白为Cry1F蛋白;
优选地,所述Cry蛋白为Cry1Fa3蛋白,例如其氨基酸序列如Genbank No.AEH31417.1所示;
优选地,所述Vip蛋白为Vip3A蛋白;
优选地,所述Vip蛋白为Vip3Aa11蛋白,例如其氨基酸序列如Genbank No.AAR36859.1所示。
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CN117178996A (zh) * | 2023-11-03 | 2023-12-08 | 中国农业大学三亚研究院 | 一种用于防治草地贪夜蛾的多元纳米复合物及其制备方法与应用 |
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