CN108925783A - 烟粉虱manf基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用 - Google Patents
烟粉虱manf基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种烟粉虱MANF基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用;并提供了抑制MANF基因表达的dsRNA,其正义链的核苷酸序列如SEQ IDNO.1所示,反义链的核苷酸序列为SEQ ID NO.1所示的序列的反向互补序列。本发明首次发现MANF基因在防治烟粉虱中的作用,通过RNAi技术获得MANF基因的dsRNA,并通过饲喂方式沉默MANF基因后,能够有效降低对烟粉虱成虫的存活率。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及烟粉虱MANF基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用。
背景技术
烟粉虱属半翅目Hemiptera,是粉虱科Aleyrodidae的一类刺吸式口器的小型昆虫,又名棉粉虱或甘薯粉虱,寄主范围十分广泛,主要危害十字花科、茄科、葫芦科、豆科、菊科、锦葵科等植物,广泛分布于热带、亚热带及低纬度温带地区,是棉花、番茄、烟草等许多经济作物的重要害虫。
烟粉虱不仅通过刺吸植物汁液危害植物,导致植物萎焉枯死;还可以传播200多种植物病毒,导致作物病毒病流行和暴发成灾。由于入侵烟粉虱生活史短、繁殖能力强、适应环境能力强,已经成为我国农业生产上非常难治理的一类害虫,造成农业上巨大损失。
烟粉虱和寄主植物之间的相互作用是非常复杂的。在长期的协同进化过程中,植物逐渐形成了一套组成性和诱导性的防御机制来应对烟粉虱取食,而烟粉虱也进化出多种行为和生理机制以应对植物的防御,从而适应多种寄主植物,顺利成活。
中脑星形胶质细胞衍生神经营养因子基因(MANF),在进化上高度保守,广泛分布于生物体内。在细胞外,MANF作为一种分泌蛋白,具有保护细胞促进神经元修复的功能;在细胞内,MANF主要位于内质网腔,对于维持内质网的稳态具有重要意义。内质网是真核细胞中重要的细胞器,参与蛋白质的合成、加工及转运过程,内质网的稳态在生物体维持正常运转过程中起着非常重要的作用。所述,研究MANF基因在烟粉虱生长发育过程中的作用,能为深入了解烟粉虱和植物互作的分子机制提供基础,为研究烟粉虱防治新策略提供理论依据。
RNA干扰是指内源性或外源性的dsRNA介导的细胞内mRNA发生特异性降解,从而导致靶细胞表达沉默。近些年,RNA干扰(RNA interference)技术在昆虫的研究上得到越来越多的应用,通过RNAi技术沉默昆虫体内某些基因,使昆虫的某些能力增强或者丧失,也能在特定时间抑制其功能基因表达使昆虫的发育停留在某个阶段,进而达到利用或防治其危害的目的。
目前,利用RNAi技术进行害虫防治已有报道,Baum等(2007)证明v-ATPase对玉米根萤叶甲(Western corn rootworm WCR Diabrotica virgifera virgifera LeConte)有致死效果,可以用于田间防治;同年,mao等通过将P450的dsRNA导入棉花中,可以导致棉铃虫(cotton bollworm Helicoverpa armigera)死亡;Tian(2009)证实通过饲喂的方法可以导致甜菜夜蛾(Spodoptera exigua(Hübner))的死亡,一系列的现有技术都表明,RNAi技术作为一种新型的害虫防治方法是可行的。但是,针对于烟粉虱的有效的靶标基因还未见报道。因此,深入研究影响烟粉虱存活的关键基因,以该基因作为防治烟粉虱的靶标基因是非常有必要的。
发明内容
本发明的目的是提供一种烟粉虱MANF基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用以及烟粉虱MANF基因的dsRNA及其应用。
具体内容如下:
烟粉虱MANF基因或其表达的MANF蛋白作为靶点在制备防治烟粉虱的药物中的应用。
本发明所述提供的dsRNA,由两条互补的序列组成,正义链的核苷酸序列如SEQIDNO.1所示,反义链的核苷酸序列为SEQ ID NO.1所示序列的反向互补序列。
上述序列经简单缺失、修饰等变换后,仍能达到本发明所示序列基本相同的抑制效果的核苷酸序列,也应当属于本发明保护范围。
本发明对烟粉虱唾液腺转录组分析表明,MANF是烟粉虱唾液腺中高表达基因之一,基于MANF在生物体内功能,推测其是影响烟粉虱存活的关键基因,并且很可能在烟粉虱应对植物防御反应过程中发挥着重要作用。
编码所述dsRNA的DNA分子也属于本发明的保护范围。
所述DNA分子的核苷酸序列如SEQ ID No.2所示。
所述DNA分子的开放阅读框序列如SEQ ID No.3所示。
包含所述DNA分子的表达盒、重组载体、重组菌或转基因细胞系也应属于本发明的保护范围。
本发明还提供了所述的dsRNA或所述的DNA分子在降低烟粉虱存活率中的应用。
本发明还提供了所述的表达盒、重组载体、重组菌或转基因细胞系在降低烟粉虱存活率中的应用也应属于本发明的保护范围。
更确切地,所述dsRNA,或所述DNA分子,或者表达盒、重组载体、重组菌或转基因细胞系在如下(1)-(3)任一项中的应用也属于本发明的保护范围:
(1)防治烟粉虱或制备防治烟粉虱的产品;
(2)降低烟粉虱存活率或制备降低烟粉虱存活率的产品;
(3)抑制烟粉虱MANF基因表达或制备抑制烟粉虱MANF基因表达的产品。
一种防治烟粉虱的药物,该药物的有效成分为烟粉虱MANF基因或其表达的MANF蛋白的抑制剂。
所述抑制剂可以为dsRNA、shRNA、siRNA、miRNA、cDNA、反义RNA/DNA、低分子化合物、肽、抗体中的至少一种。
本发明提供了一种防治烟粉虱的药物,其活性成分为如下A~C中任一种:
A、权利要求1所述的dsRNA;
B、权利要求2所述的DNA分子;
C、权利要求3所述的表达盒、重组载体、重组菌或转基因细胞系。
所述药物具有如下a~c功能中的至少一种:
a、防治烟粉虱;
b、降低烟粉虱存活率;
c、抑制烟粉虱MANF基因的表达。
本发明还提供了一种防治烟粉虱的方法,将所述dsRNA导入烟粉虱,降低烟粉虱的存活率。
本发明中,所述导入的方式具体为饲喂。
更具体地,所述饲喂的方式为:将权利要求1所述的dsRNA混合于液体饲料中,得到混合液;用所述混合液饲喂烟粉虱。
进一步地,所述dsRNA在所述混合液中的终浓度为100~300ng/ul;所述饲喂的时间为24-~72h。
更优选,所述dsRNA在所述混合液中的终浓度为200ng/μL;所述饲喂的持续时间为48h。
与现有技术相比,本发明具有以下有益效果:
本发明首次发现MANF基因在防治烟粉虱中的作用,通过RNAi技术获得MANF基因的dsRNA,并通过饲喂方式沉默MANF基因后,能够有效降低对烟粉虱成虫的存活率。
附图说明
图1为MANF基因在烟粉虱不同组织中的转录水平。
图2为MANF基因的dsRNA处理对烟粉虱成虫存活率的影响。
图3为MANF基因的经dsRNA处理后的干扰效率。
所有数据均用SPSS20.0统计软件进行数据统计分析,显著性检验水平为P<0.05。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
实施例1MANF基因在烟粉虱不同组织中的分布
1、烟粉虱组织的解剖、不同组织的RNA提取和cDNA合成
解剖烟粉虱成虫的卵巢、中肠、唾液腺和无唾全身,利用Absolutely RNANanoprep Kit(Agilent)试剂盒进行唾液腺总RNA提取,利用Trizol法进行卵巢、中肠和无唾全身的总RNA提取。使用PrimeScript RT reagent Kit(Takara)试剂盒进行cDNA的合成。
2、引物设计
利用Prime primer5.0软件设计引物,由南京金斯瑞公司合成。
MANF基因定量引物:
qRT MANF F:5’-TGCCGGAAACTTAAAGCAGT-3’;
qRT MANF R:5’-TGATGTCAGCTTTCTCAACGC-3’。
内参基因定量引物:
TAF F:5’-TGTGGGACACCCATTATCAG-3’;
TAF R:5’-TGTGCAGCCAAGGAAATAAG-3’。
3、荧光定量PCR监测MANF基因在烟粉虱不同组织中的分布
分别以烟粉虱的卵巢、中肠、唾液腺和无唾全身为模板,使用PremixExTaqTMII(Takara)荧光定量PCR检测MANF基因的表达量。
结果如图1所示,MANF基因在唾液腺的表达量显著高于其他组织。因此,推测MANF基因编码的蛋白在烟粉虱-植物互作中发挥着重要作用。
实施例2烟粉虱MANF基因dsRNA的获得
1、烟粉虱总RNA的提取和cDNA的合成
取烟粉虱的成虫20-30头,放入放在无RNase的1.5mL离心管中,利用Trizol法提取RNA,并在-80℃超低温冰箱保存以备用。在获得烟粉虱总RNA之后,采用PrimeScriptTM RTreagent Kit(Takara)试剂盒进行cDNA的合成。
2、引物设计和基因克隆
利用Prime primer5.0软件设计引物,由南京金斯瑞公司合成。绿色荧光蛋白基因(GFP)片段从浙江大学昆虫科学研究所实验室保存的1305GFP质粒上扩增。加上T7启动子(下划线所示序列)后的引物序列:
T7+MED MANF-F:5’-TAATACGACTCACTATAGGGATGGCAACACCAAGCGATGA-3’;
T7+MED MANF-R:5’-TAATACGACTCACTATAGGGTTCAGGGGGCAATGGAACAG-3’;
T7+GFP F:5’-TAATACGACTCACTATAGGGCTCGTGACCACCCTGACCTAC-3’;
T7+GFP R:5’-TAATACGACTCACTATAGGGTTCACCTTGATGCCGTTCTT-3’。
PCR反应体系:
PCR反应条件:
94℃预变性3分钟;
94℃变性30秒,58℃退火30秒,72℃延伸30秒,35个循环;
72℃延伸7分钟。
以烟粉虱的cDNA为模板,用T7+MED MANF-F/T7+MED MANF-R作为引物进行PCR扩增,电泳后使用AxyPrep凝胶回收试剂盒回收纯化PCR产物,得到240bp的PCR扩增产物,产物经测序确认正确,该240bp的PCR扩增产物具有如SEQ ID NO.2所示的核苷酸序列。
以烟粉虱的cDNA为模板,用T7+GFP F/T7+GFP R作为引物进行PCR扩增,电泳后使用AxyPrep凝胶回收试剂盒回收纯化PCR产物,得到314bp的PCR扩增产物,产物经测序确认正确。
3、MANF基因和GFP的dsRNA合成和纯化
将得到的2种PCR产物,测定浓度,作为体外转录dsRNA的模板;反应体系如下:
反应结束后,反应产物进行电泳检测,加入DNaseI和RNaseA消化残留的模板DNA和单链RNA,用AmpliScribeTMT7High Yield Transcription Kit(Epicentre)试剂盒合成和纯化MANF基因和GFP的dsRNA。测浓度后,即可按需稀释使用。
将MANF基因dsRNA和GFPdsRNA分别测序,结果如下:
MANF基因dsRNA为双链RNA,由正义链和反义链组成,其正义链的核苷酸序列如SEQID NO.1所示。GFPdsRNA的核苷酸序列如SEQ ID NO.4所示。
实施例3MANF基因dsRNA在降低烟粉虱存活率中的作用
1、人工饲料和饲喂装置的准备
取两端通透的玻璃管中,玻璃管上端覆以两层Parafilm膜,并在两膜间的空隙内加入含有质量分数为15%的dsRNA的蔗糖液600μL,玻璃管下端覆以纱布以保持通气,玻璃管周围和下端用锡箔纸包裹有利于烟粉虱向顶端Parafilm膜聚集,从而更好的取食dsRNA混合液。
2、dsRNA在降低烟粉虱存活率中的作用
取初羽化的烟粉虱成虫约200头放入饲喂装置中,将处理好的玻璃管放入人工气候箱中(温度25±0.2℃,24h光照,相对湿度为60-70%),饲喂含MANF基因的蔗糖液(浓度为200ng/μL)的dsRNA 48h,将烟粉虱放置在棉花上恢复12h后,转移至烟草上进行生测。
将夹叶笼夹在烟草叶片背面,每个夹叶笼放10头饲喂过dsRNA的烟粉虱(5雌5雄),每个夹叶笼作为一个重复,每组20个重复。以饲喂含有dsGFP且灭菌处理过的15%蔗糖溶液的烟粉虱作为对照。利用SPSS20.0统计软件分析饲喂不同溶液后烟粉虱死亡率。
结果如图2所示,饲喂MANF基因dsRNA的烟粉虱的存活率显著低于对照组(P<0.001)。
3、dsRNA抑制MANF基因的表达
饲喂48h后,将烟粉虱释放至棉花上,随后在第0、1、3、5、7天收集烟粉虱定量测定干扰效果,MANF基因定量引物及内参基因定量引物同实施例1。
干扰结果如图3所示,由此确保了干扰效果为烟粉虱的MANF基因所产生。因此,本发明说明MANF基因作为唾液腺表达量较高的基因,在烟粉虱取食过程中起着较为关键的作用。
序列表
<110> 浙江大学
<120> 烟粉虱MANF基因或其表达的蛋白作为靶点在制备防治烟粉虱的药物中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 240
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<210> 2
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gatttttgca gcttcatccc tcgttcaggt gctatgctgc tactgttctt tttgtgatat 840
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gtcttgatgc gttaagtcaa gatgcagaaa atgaagttaa actgatcaaa ggtggatgac 1980
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Claims (10)
1.烟粉虱MANF基因或其表达的MANF蛋白作为靶点在制备防治烟粉虱的药物中的应用。
2.一种dsRNA,由两条互补的序列组成,其特征在于,正义链的核苷酸序列如SEQIDNO.1所示,反义链的核苷酸序列为SEQ ID NO.1所示的序列的反向互补序列。
3.编码权利要求2所述dsRNA的DNA分子,其特征在于,其核苷酸序列如SEQ ID No.2所示。
4.包含权利要求3所述DNA分子的表达盒、重组载体、重组菌或转基因细胞系。
5.根据权利要求2所述的dsRNA或权利要求3所述的DNA分子在如(1)~(3)至少一项中的应用;
(1)防治烟粉虱;
(2)抑制烟粉虱MANF基因的表达;
(3)降低烟粉虱存活率。
6.如权利要求4所述的表达盒、重组载体、重组菌或转基因细胞系在如(1)~(3)至少一项中的应用;
(1)防治烟粉虱;
(2)抑制烟粉虱MANF基因的表达;
(3)降低烟粉虱存活率。
7.一种防治烟粉虱的药物,其特征在于,其活性成分为如下A~C中任一种:
A、权利要求2所述的dsRNA;
B、权利要求3所述的DNA分子;
C、权利要求4所述的表达盒、重组载体、重组菌或转基因细胞系。
8.一种防治烟粉虱的方法,其特征在于,将权利要求2所述的dsRNA导入烟粉虱,降低烟粉虱的存活率。
9.如权利要求8所述的方法,其特征在于,所述导入的方式为饲喂。
10.如权利要求9所述的方法,其特征在于,所述饲喂的方式为:将权利要求1所述的dsRNA混合于液体饲料中,得到混合液;用所述混合液饲喂烟粉虱。
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