CN116172014A - 沉默caspase5基因在提高防治昆虫效果中的应用 - Google Patents
沉默caspase5基因在提高防治昆虫效果中的应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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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
- A01N47/44—Guanidine; Derivatives thereof
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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Abstract
本发明涉及沉默caspase5基因在提高防治昆虫效果中的应用。沉默靶标昆虫中的caspase5基因可以提高Cry9A蛋白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caspase5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caspase5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caspase5基因表达的效率在60％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，所述靶标昆虫为螟蛾科昆虫。
在一个具体实施方式中，所述靶标昆虫为二化螟(Chilo suppressalis Walker)。
在一个具体实施方式中，沉默所述靶标昆虫中的caspase5基因表达在提高Cry蛋白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，所述Cry蛋白为Cry9Aa蛋白。
在一个具体实施方式中，所述Cry蛋白为Cry9Aa3蛋白，例如其氨基酸序列如Genbank No.ADE60735.1所示。
在一个具体实施方式中，所述Cry蛋白为氨基酸序列如SEQ ID No.11的Cry9Aa655蛋白。
在一个具体实施方式中，沉默所述靶标昆虫中的caspase5基因表达的dsRNA中的正义链的序列如Genbank No.GQ249293.1所示，或如SEQ ID No.3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caspase5基因表达的dsRNA和Cry蛋白。其中，所述dsRNA和所述Cry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Cry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.GQ249293.1所示，或如SEQ ID No.3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Cry9Aa蛋白。
在一个具体实施方式中，所述Cry蛋白为Cry9Aa3蛋白，例如其氨基酸序列如Genbank No.ADE60735.1所示。
在一个具体实施方式中，所述Cry蛋白为氨基酸序列如SEQ ID No.11的Cry9Aa655蛋白。
本发明的有益效果：
本发明首次发现沉默二化螟Cscaspase5基因的表达可以显著提升Cry9A蛋白对二化螟的活性，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
1.二化螟的Cscapase5基因的克隆和dsCscapase5的制备
根据鳞翅目害虫二化螟(Chilo suppressalis Walker)的基因组(NCBI Genomeassemly GCA_004000445.1)设计二化螟的caspase5基因(Cscaspase5，GenbankNo.GQ249293.1)全长的引物对。其中上游引物为Cscaspase5F(SEQ ID No.1)，下游引物为Cscaspase5R(SEQ ID No.2)。
利用RNeasy Mini Kit试剂盒(74106，QIAGEN公司)提取二化螟三龄幼虫的总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Cscaspase5F和Cscaspase5R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Cscaspase5全长基因的pEASYBluntZero-Cscaspase5阳性质粒。
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片段(dsCscapase5，其正义链的碱基序列如SEQ ID No.3所示)。然后利用T7 RiboMAXTM Express RNAi System试剂盒(P1700，Promega)以pEASYBluntZero-Cscaspase5为模板，以dsCscaspase5F(SEQ ID No.4)和dsCscaspase5R(SEQ ID No.5)为引物合成Cscaspase5的dsRNA片段，最后溶于无酶水中，得到dsCscapase5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.6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.7)和gfpR(SEQ ID No.8)为引物，合成gfp的dsRNA片段(dsgfp，其正义链的碱基序列如SEQ ID No.6所示)，最后溶于无酶水中，得到dsgfp水溶液。
实施例2
Cry9Aa655蛋白的制备
1.蛋白提取
以cry9Aa655-F(SEQ ID No.9)和cry9Aa655-R(SEQ ID No.10)为引物，以合成的cry9Aa3基因(Genbank No.GQ249293.1，其氨基酸Genbank No.ADE60735.1)为模板，克隆cry9Aa3基因第1-655位氨基酸(SEQ ID No.11)的DNA序列，并连接到pEB表达载体上，得到pEB-cry9Aa655，并转化到大肠杆菌BL21(DE3)中，得到BL21(DE3)/pEB-cry9Aa655。同时，将pEB空载转化到大肠杆菌BL21(DE3)中，得到BL21(DE3)/pEB。
将大肠杆菌BL21(DE3)/pEB-cry9Aa655接种于5mL含有1‰的氨苄青霉素的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Cry9Aa655成功表达(以相同条件下获得的BL21(DE3)/pEB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Cry9Aa655并收集；将收集到的Cry9Aa655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。
实施例3
对二化螟的生物活性测定
1.dsRNA-SPc复合物的制备
将实施例1制备得到的dsCscapase5水溶液用无酶水梯度稀释为5个浓度；将该5个浓度下的dsCscapase5水溶液与纳米材料SPc均以dsCscapase5与SPc质量比为1:1混合，得到dsCscapase5-SPc溶液；分别各向dsCscapase5-SPc溶液中加入终溶液(即dsCscapase5-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dsCscapase5-SPc复合物。其中，纳米材料SPc的制备按照A facile-synthesized star polycationconstructed as ahighly 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.dsCscaspase5的沉默效率测定
二化螟人工饲料配方：大豆粉40.0g，酵母粉30.0g，干酪素15.0g，蔗糖12.0g，鲜茭白145.0g，琼脂18.0g，抗坏血酸钠(Vc)4.5g，胆固醇0.25g，氯化胆碱0.40g，威氏盐0.15g，复合维生素B 0.020g，山梨酸1.50g，对羟基苯甲酸甲酯1.50g，40％甲醛0.6ml，水731.08m。其中，复合维生素B配方：烟酰胺0.0060g，盐酸硫胺素(VB1)0.0016g，核黄素(VB2)0.0030g，盐酸吡哆醇(VB6)0.0016g，赖氨肌醇(VB12)0.00004g；叶酸0.0016g；泛酸钙0.0060g，生物素0.00016g。其制备过程见CN200910080336.2的实施例1。
使用超纯水将dsCscapase5-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dsCscapase5在人工饲料中的浓度依次为6.25μg/g、12.5μg/g、25μg/g、50μg/g和100μg/g，dsgfp在人工饲料中的浓度为100μg/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头二化螟初孵幼虫，每个处理组3次重复。饲喂48h后，以实施例1相同的操作提取二化螟幼虫的RNA，并反转录获得cDNA，以所获得的cDNA为模板，以Q-Cscaspase5-F(SEQ ID No.12)和Q-Cscaspase5-R(SEQ IDNo.13)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Cscaspase5基因在经dsCscaspase5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dsCscaspase5浓度大于25μg/g时沉默效率达到60％以上，考虑到干扰效率及使用成本，最终选择25μg/g dsRNA作为之后的实验浓度。
3.dsCscaspase5和Cry9A联合使用的生物活性测定
1)dsCscaspase5-Cry9A处理：将用25μg/g dsCscaspase5干扰48h(操作同上述第2小节)的二化螟幼虫转入装有Cry9Aa655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Cry9Aa655蛋白的人工饲料饲喂7天时统计死亡率。
2)dsgfp-Cry9A处理：将用25μg/g dsgfp干扰48h(操作同上述第2小节)的二化螟幼虫转入装有Cry9Aa655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，在用人工饲料饲喂7天时统计死亡率。
3)dsCscaspase5处理：将用25μg/g dsCscaspase5干扰48h(操作同上述第2小节)的二化螟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。
4)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，dsCscaspase5-Cry9A处理的幼虫的死亡率为48.23％，显著高于dsgfp-Cry9A处理的15.38％；此外，dsCscaspase5处理与阴性对照dsgfp处理的二化螟幼虫的死亡率没有显著差异。以上结果说明，虽然仅沉默Cscaspase5基因不能使二化螟死亡，即沉默Cscaspase5基因对二化螟没有杀虫活性；但沉默二化螟的Cscaspase5基因却可以显著提高Cry9Aa655蛋白对二化螟的杀虫效果。
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caspase5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caspase5基因表达的效率在60％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，所述靶标昆虫为螟蛾科昆虫。
5.根据权利要求1所述的应用，其特征在于，所述靶标昆虫为二化螟(Chilosuppressalis Walker)。
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caspase5基因表达在提高Cry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Cry9Aa蛋白；
优选地，所述Cry蛋白为Cry9Aa3蛋白，例如其氨基酸序列如Genbank No.ADE60735.1所示；
优选地，所述Cry蛋白为氨基酸序列如SEQ ID No.11的Cry9Aa655蛋白。
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caspase5基因表达的dsRNA中的正义链的序列如Genbank No.GQ249293.1所示，或如SEQ ID No.3所示。
8.一种联合用药物，其包括用于沉默靶标昆虫中的caspase5基因表达的dsRNA和Cry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.GQ249293.1所示，或如SEQ ID No.3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Cry9Aa蛋白；
优选地，所述Cry蛋白为Cry9Aa3蛋白，例如其氨基酸序列如Genbank No.ADE60735.1所示；
优选地，所述Cry蛋白为氨基酸序列如SEQ ID No.11的Cry9Aa655蛋白。
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