CN117025561B - 劳氏粘虫保幼激素酸甲基转移酶、其编码基因及应用 - Google Patents
劳氏粘虫保幼激素酸甲基转移酶、其编码基因及应用 Download PDFInfo
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Abstract
本申请涉及一种劳氏粘虫保幼激素酸甲基转移酶、其编码基因及应用,旨在解决劳氏粘虫缺乏有效的生物防治方法的技术问题。本申请获得一种劳氏粘虫保幼激素酸甲基转移酶,其氨基酸序列如SEQ ID NO:1所示,编码该酶的JHAMT基因核苷酸序列如SEQ ID NO:2所示。本申请研究确认JHAMT基因在劳氏粘虫幼虫生长发育过程中起着关键作用,能够以其作为有效靶点开发劳氏粘虫生长调节剂或杀虫剂;本申请劳氏粘虫保幼激素酸甲基转移酶(JHAMT)为靶标基因开发得到的JHAMT基因的dsRNA对劳氏粘虫幼虫的生长发育具有明显的抑制作用,且具有较高的致死率,无环境毒害,使用安全,可以用于劳氏粘虫的生物防治,对于保障粮食安全具有重要的现实意义。
Description
技术领域
本发明申请涉及害虫生物防治技术领域,具体涉及一种劳氏粘虫保幼激素酸甲基转移酶、其编码基因及应用。
背景技术
劳氏粘虫(Mythimna_loreyi)是一种鳞翅目、夜蛾科的昆虫,主要分布在广东、福建、四川、江西、湖南、湖北、浙江、江苏、山东、河南等地。劳氏粘虫的幼虫具有杂食性,可取食多种植物,尤其喜欢禾本科植物,如苏丹草、羊草、披碱草、黑麦草、冰草、狗尾草等,同时也会危害玉米、小麦和水稻等作物。目前劳氏粘虫已被列入一类农作物病虫害名录。近年来受气候变化和耕作方式改变等多种因素影响,劳氏粘虫在世界上多个国家和地区的发生范围不断扩大,危害程度日益严重,给粮食安全生产带来极大威胁。
已广泛应用的劳氏粘虫的防治方法主要有化学防治、物理防治、农业防治等。当前对劳氏粘虫的防治仍以化学防治为主,而这种防治易产生抗药性,且易产生环境污染。RNA干扰(RNAi)技术是近年来发展起来的一种新型生物防治技术,被称为“农药史上的第三次革命”。利用RNAi技术沉默有害生物生长发育过程中重要基因的表达,导致其生长发育受阻,甚至死亡,从而降低有害生物对农作物的侵害,实现病虫害的防治,达到保障粮食安全的目的。与传统化学农药相比,RNAi技术具有特异性强、无环境毒性、开发周期短、靶标变更灵活和用量低更等优势。但目前有关劳氏粘虫生物防治的报道仍较少,且尚无关于核酸农药在其防治中的应用报道。
公开于该背景技术部分的信息仅仅旨在加深对本发明总体背景技术的理解,而不应当被视为承认或以任何形式暗示该信息构成本领域技术人员所公知的现有技术。
发明内容
研究表明,保幼激素(JH)酸甲基转移酶(JHAMT)是昆虫体内保幼激素生物合成中的一种重要限速酶,在JH生物合成的最后一步将S-腺苷甲硫氨酸的甲基转移到保幼激素酸或法尼酸的羧基上,产生有活性的保幼激素,能够作为开发昆虫生长调节剂或杀虫剂的有效靶点。本申请以劳氏粘虫保幼激素酸甲基转移酶(JHAMT)为靶标基因,开发得到能够用于防治劳氏粘虫的RNAi产品,从而为劳氏粘虫的生物防治提供新的方向和重要依据。
本申请的目的在于提供一种来源于劳氏粘虫酸甲基转移酶(JHAMT)及其编码基因,以解决劳氏粘虫缺乏有效的生物防治方法的技术问题。
为解决上述技术问题,本申请采用如下技术方案:
获得了一种劳氏粘虫保幼激素酸甲基转移酶(JHAMT),其基因编码284个氨基酸,其氨基酸序列如SEQ ID NO:1所示。
克隆得到编码所述劳氏粘虫保幼激素酸甲基转移酶的JHAMT基因,全长cDNA序列为855 bp,其核苷酸序列如SEQ ID NO:2所示。
基于所述劳氏粘虫JHAMT基因核苷酸序列设计合成了一个dsRNA片段, 其模板DNA核苷酸序列如SEQ ID NO:3所示。
所述JHAMT基因或所述dsRNA片段能够有效应用于劳氏粘虫的防治中,或应用于劳氏粘虫防治生物制剂的制备当中。
基于所述JHAMT基因序列设计合成dsRNA或其片段,可使劳氏粘虫摄入dsRNA片段后抑制其生长发育,并且有较高的致死率,从而起到防治劳氏粘虫的作用。
上述编码基因、dsRNA,含有JHAMT基因及其同源基因的重组表达载体、超表达载体、干扰载体、重组病毒、转基因细胞系、转基因植物或组织、重组菌、重组基因表达盒及其应用,均属于本发明构思范畴。
含JHAMT基因的重组表达载体包括双元农杆菌载体,病毒载体,细菌表达载体及酵母表达载体等。含JHAMT基因的载体在构建过程中,可以单独或者多个组合使用诱导型、组成型、增强型、组织特异型等;载体可以包括抗生素或抗化学试剂的抗性筛选标记,也可以含有产生颜色变化的酶,比如GUS、或者红色或绿色荧光蛋白等;构建的载体可以转化单双子叶植物、真菌、细菌等,具体可以为大肠杆菌、酵母、烟草、拟南芥、番茄、小麦、玉米等。
抑制JHAMT基因的表达为本发明的构思范围,本发明构思还涉及用于抑制JHAMT基因的表达的物质在制备产品中的应用。所述产品功能包括抑制劳氏粘虫的生长发育;用于抑制JHAMT基因表达的物质具体可以为抑制JHAMT基因表达的dsRNA,干扰载体,以及病毒载体等。
本申请通过基因沉默,将JHAMTdsRNA导入劳氏粘虫体内,测定了JHAMT基因沉默对劳氏粘虫生长发育的影响,实验结果表明,饲喂JHAMT基因dsRNA的劳氏粘虫幼虫,24 h、48h和72h后的死亡率分别为13.3%、53.3%和86.7%,显著高于饲喂dsGFP组和ddH2O组。
与现有技术相比,本申请的主要有益技术效果在于:
1. 本申请研究确认JHAMT基因在劳氏粘虫幼虫生长发育过程中起着关键作用,能够以其作为有效靶点开发劳氏粘虫生长调节剂或杀虫剂。
2. 本申请劳氏粘虫保幼激素酸甲基转移酶(JHAMT)为靶标基因开发得到的JHAMT基因的dsRNA对劳氏粘虫幼虫的生长发育具有明显的抑制作用,且具有较高的致死率,无环境毒害,使用安全,可以用于劳氏粘虫的生物防治,对于保障粮食安全具有重要的现实意义。
附图说明
图1为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶JHAMT基因 PCR扩增的电泳检测图;其中,1、2:劳氏粘虫JHAMT基因的PCR扩增;3:阴性对照;4:DL2000。
图2为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶氨基酸序列的同源性分析;其中,矩形标注部分(ILDIGCGDG)为S-腺苷甲硫氨酸结合结构域,三角形标注部分为保幼激素类似物或法尼硫酸结合位点。
图3为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶的二级结构图。
图4为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶的三级结构图。
图5为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶的分子进化分析。
图6为本申请一实施例中劳氏粘虫保幼激素酸甲基转移酶经dsRNA处理后的表达情况。
图7为本申请一实施例中劳氏粘虫幼虫经保幼激素酸甲基转移酶dsRNA处理后的死亡率对比图。
具体实施方式
下面结合附图和实施例来说明本申请的具体实施方式,但以下实施例只是用来详细说明本申请,并不以任何方式限制本申请的范围。
下述实施例中所用的试验材料及试剂,如无特别说明,均购买于常规生化试剂公司;所涉及的仪器设备如无特别说明,均为常规的实验室仪器设备;以下实施例中的定量试验,如无特别说明均设置三次重复实验,结果取平均值。
实施例一、劳氏粘虫JHAMT基因全长cDNA的PCR扩增及克隆
(1)总RNA提取:取劳氏粘虫2龄幼虫3头放入1.5 ml离心管中,液氮冷冻后利用研磨棒将其研成粉末,然后参照宝生物RNAiso Plus kit(Total RNA 提取试剂)说明书提取总RNA,电泳检测RNA的完整性后在分光光度计上测定RNA的纯度及浓度。
(2)JHAMT基因全长cDNA序列的PCR扩增及克隆
根据劳氏粘虫转录组数据获得JHAMT全长cDNA序列SEQ ID NO: 2,采用Primer5.0软件设计PCR扩增全长cDNA序列的引物:
JHAMT-F:ATGAACAACGCCGTCCTATACG (SEQ ID NO: 4);
JHAMT-R:CAGCGTCATCTATGAGATTACAC (SEQ ID NO: 5)。
以提取的总RNA为模板,利用cDNA第一链合成试剂盒(cDNA Synthesis Kit,TAKARA)合成cDNA第一链。以合成的cDNA为模板PCR扩增JHAMT全长cDNA序列。反应体系(20μL)为cDNA 3 μL, 10xPCR buffer 2 μL,dNTPs 1.5 μL,rTaq DNA 聚合酶 0.2 μL, 正向引物 1 μL(10 pmol/ul),反向引物 1 μL(10 pmol/ul),超纯水11.3 μL。反应条件为: 95℃预变性 4 min,95℃变性30 s,53℃退火35s,72℃延伸50 s(共35个循环),最后 72℃终止延伸10 min终止反应。
用1%的琼脂糖凝胶对PCR扩增产物进行电泳检测(图1)、利用UNIQ-10柱式DNA胶回收试剂盒回收PCR扩增产物,回收产物连接至pMDTM19-T载体中。连接体系为(10 μL):1 μL T载体、1 μL T4 DNA连接酶、1 μL 10xT4连接酶Buffer、7μL回收产物。将配置好的连接体系放置16℃进行过夜连接后,用热激法转化至大肠杆菌TG1感受态细胞中,后经菌落PCR鉴定和测序分析,获得包含劳氏粘虫JHAMT基因(855bp)的全长cDNA序列(如SEQ ID NO: 2所示)。
实施例二、劳氏粘虫JHAMT氨基酸序列及其同源性比对分析
利用NCBI ORF finder在线预测获得劳氏粘虫JHAMT基因编码的氨基酸序列如SEQID NO: 1所示,由284个氨基酸组成。
利用NCBI在线BLAST和 Clustalx软件对劳氏粘虫JHAMT基因编码的氨基酸序列进行同源性比对分析,结果如图2,劳氏粘虫JHAMT与NCBI上来自东方粘虫Mythimna separata(AJR27469.1)的同源性最高,达92%,与来自棉铃虫Helicoverpa armigera(XP_021182380.1)和美洲棉铃虫Helicoverpa Zea(XP_047019757.1)的同源性均为78%;与来自银纹夜蛾Trichoplusia ni(XP_026733225.1)的同源性为76%,与来自甜菜夜蛾Spodoptera exigua(CAH0702338.1)的同源性为73%,与来自斜纹夜蛾Spodoptera litura(XP_022814811.1)和草地贪夜蛾Spodoptera frugiperda(XP_050561715.1)的同源性为69%。
实施例三、劳氏粘虫JHAMT蛋白的理化性质和结构预测
(1)劳氏粘虫JHAMT蛋白的理化性质
利用在线分析软件ProtParam (https://web.expasy.org/protparam/)对劳氏粘虫JHAMT蛋白理化性质进行分析,结果表明,劳氏粘虫JHAMT的分子量为33428.28,等电点为6.33。TMHMM(http://www.cbs.dtu.dk/services/TMHMM- 2.0/)分析表明,劳氏粘虫JHAMT无跨膜序列。利用SignaIP(http://www.cbs.dtu.dk/services/SignaIP-4.1/)进行信号肽预测结果表明,劳氏粘虫JHAMT无信号肽序列,表明该蛋白为非分泌性蛋白。
(2)劳氏粘虫JHAMT蛋白的二级结构预测
利用InterPro(http://www.ebi.ac.uk/interpro/scan.html) 对劳氏粘虫JHAMT进行结构域分析。结果表明,劳氏粘虫JHAMT属于1型甲基转移酶,在38~46(ILDIGCGDG)处含有SAM结合结构域。同时发现该序列中保幼激素类似物或法尼硫酸的结合位点(见图2 ,三角形标注)比较保守。
利用PSIPRED(http://bioinf.cs.ucl.ac.uk/psipred/)对劳氏粘虫JHAMT二级结构预测的结果表明,其二级结构主要由α-螺旋和β-折叠组成,含有11α-螺旋和9个β-折叠(见图3)。
(3)劳氏粘虫JHAMT蛋白的三级结构预测
利用SWISS-MODEL(https://beta.swissmodel.expasy.org/),以 Heliothis virescens(Tobacco budworm moth))的JHAMT为模板预测劳氏粘虫JHAMT三级结构结果见图4。
实施例四、劳氏粘虫JHAMT蛋白的系统进化分析
利用Mega 5.0软件的NL法对劳氏粘虫JHAMT氨基酸序列构建系统进化树(见图5)。结果表明,在8种昆虫的JHAMT氨基酸序列中,劳氏粘虫的JHAMT与来自棉铃虫、美洲棉铃虫、银纹夜蛾和东方粘虫的聚为一支,并且在该分支中来自劳氏粘虫的和来自东方粘虫的又单独聚为一支,而来自甜菜夜蛾、斜纹夜蛾和草地贪夜蛾的聚为另一支。
实施例五、劳氏粘虫JHAMT基因的RNAi分析
(1)RNAi的引物合成:根据劳氏粘虫JHAMT全长cDNA序列SEQ ID NO: 2,利用e-RNAi在线设计dsRNA引物,并分别在上游和下游引物的5’端加上T7启动子序列taatacgactcactataggg,同时合成绿色荧光蛋白基因GFP引物dsGFP-F和dsGFP-R。
dsRNA-F:taatacgactcactatagggGGGAAAATTCGACCACGTGT (SEQ ID NO:6)
dsRNA-R:taatacgactcactatagggCGCAACACATGTAAGTCTTC(SEQ ID NO:7)
dsGFP-F:taatacgactcactatagggAGAATGAGTAAAGGAGAAGAACTTTTC;
dsGFP-R:taatacgactcactatagggAGATTTGTATAGTTCATCCATGCCATGT。
(2)dsRNA的合成:以实施例一中获得的cDNA为模板,以dsRNA-F和dsRNA-R为引物,采用T7体外转录试剂盒合成dsRNA,具体步骤按照说明书操作,并根据实际情况进行微调。反应体系(50 μL):Ribo MAXTM Express T7 2 x Buffer 25 ul,双链DNA模板 1 ug,EnzymeMix T7Express 5 μL, 加Nuclease-Free Water补齐至50 μL。反应步骤为:37℃水浴过夜,70℃水浴10 min,放至室温自然冷却后加入DNase 1 ul和稀释200倍的RNase 1 μL,然后37℃ 水浴30 min,加入0.1倍体积3mol/L的NaAC和1倍体积的异丙醇,混匀后置于冰上5 min,可见白色絮状沉淀,4℃,12 000 r/min离心30 min,弃上清液,加预冷的75%乙醇500 μL,4℃/12 000 r/min离心10min,弃上清液,干燥5 min后加50~100 μL 无RNA酶污染的水溶液,使用试剂盒纯化dsRNA,按照试剂盒说明书进行操作。
(3)RNAi试验
幼虫的饲喂:剪取新鲜玉米叶片,随后浸泡在含有30 μg/mlJHAMTdsRNA的水溶液中,20 s后取出室温晾30 min,待水分蒸发后饲喂提前饥饿处理4 h的2龄劳氏粘虫幼虫10头,并放于人工气候箱(温度25+1℃,相对湿度70+5%,光照4000 lux,光周期为L14:D10)中饲养。每隔24h观察幼虫状态,记录生长发育情况,统计幼虫死亡率;并于48 h和72 h时随机取2头用于JHAMT基因表达量的检测,以饲喂含有dsGFP的水溶液(dsHGFP终浓度为30ug/ml)(阴性对照)和ddH2O(空白对照)的劳氏粘虫幼虫为对照,各处理均设置4个生物学重复。
(4)基因表达量分析
参照实施例1中(1)提取劳氏粘虫总RNA用于检测JHAMT基因的表达量。 Real-timePCR中JHAMT基因定量分析的特异性引物为SEQ ID NO:8和SEQ ID NO:9,内参基因ribosomal protein L12引物为SEQ ID NO:10和SEQID NO:11。
rJHAMT-F: ACTGCAACGACCAGACTTCC (SEQ ID NO:8);
rJHAMT-R: CGTCCTCACTCAACAGGTTGT(SEQ ID NO:9);
rRNA12L-F: AATGGCAACATCACCCTAGAAGA(SEQ ID NO:10);
rRNA12L-R: TCATCAATGGTAAGAGCACCAGA(SEQ ID NO:11)。
荧光PCR反应体系为:10 μL SYBR Green Master Mix,0.5 μmol/L上/下游引物,0.5 μL cDNA,用ddH2O补足至20 μL。荧光PCR程序为:95℃预变性 5 min,95℃变性 15s,54℃退火10 s,72℃延伸20 s,40个循环。每个样品进行4个生物学重复,用无模板的ddH2O做阴性对照。
通过2-△△Ct方法计算基因的相对表达量,以最低表达量为1,用Excel 2010对数据进行处理。结果如图6所示,结果表明饲喂dsJHAMT可以显著降低劳氏粘虫JHAMT基因的表达水平。
(5)幼虫死亡率
饲喂不同溶液后劳氏粘虫幼虫的死亡率差异明显,结果如图7所示。饲喂JHAMT基因dsRNA的劳氏粘虫幼虫,24 h、48 h和72h后的死亡率分别为13.3%、53.3%和86.7%,显著高于饲喂dsGFP组和ddH2O组。
本申请所饲喂的靶序列片段为JHAMT基因特有的序列,由此确保了干扰效果为劳氏粘虫JHAMT基因所产生,说明JHAMT基因在劳氏粘虫幼虫生长发育过程中起着关键作用。
上面结合附图和实施例对本申请作了详细的说明;但是,所属技术领域的技术人员能够理解,在不脱离其发明构思的前提下,所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,从而形成多个具体的实施例,均为本申请的常见变化范围,在此不再赘述。
Claims (8)
1.一种劳氏粘虫保幼激素酸甲基转移酶,其特征在于,其氨基酸序列如SEQ ID NO: 1所示。
2.编码权利要求1所述劳氏粘虫保幼激素酸甲基转移酶的JHAMT基因,其特征在于,其cDNA序列如SEQ ID NO: 2所示。
3.一种用于抑制权利要求2所述JHAMT基因表达的dsRNA,其对应的模板DNA核苷酸序列如SEQ ID NO: 3所示。
4.含有权利要求2所述JHAMT基因的重组表达载体、超表达载体、干扰载体、重组病毒、转基因细胞系、重组菌或重组基因表达盒。
5.权利要求2所述JHAMT基因或权利要求3所述dsRNA在制备防治劳氏粘虫的生物制剂中的应用。
6.根据权利要求5所述的应用,其特征在于,以如SEQ ID NO: 2所示的cDNA为模板,以如下RNAi引物体外转录合成dsRNA:
dsRNA-F:taatacgactcactatagggGGGAAAATTCGACCACGTGT,
dsRNA-R:taatacgactcactatagggCGCAACACATGTAAGTCTTC。
7.权利要求2所述JHAMT基因或权利要求3所述dsRNA在防治劳氏粘虫中的应用。
8.一种劳氏粘虫防治方法,其特征在于,包括如下步骤:
(1)基于权利要求2所述JHAMT基因的cDNA序列设计合成JHAMT基因特异的dsRNA,所述dsRNA对应的模板DNA核苷酸序列如SEQ ID NO: 3所示;
(2)使劳氏粘虫摄入所述dsRNA,以致死劳氏粘虫或抑制其生长发育。
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