CN116622714B - 褐飞虱NlA7基因及其dsRNA在防治褐飞虱的应用 - Google Patents
褐飞虱NlA7基因及其dsRNA在防治褐飞虱的应用 Download PDFInfo
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Abstract
本发明公开了褐飞虱NlA7基因及其dsRNA在防治褐飞虱的应用。NlA7基因的核苷酸序列如SEQ ID NO.1的第1642位到第3003位所示,其编码的氨基酸序列如SEQ ID NO.2所示。褐飞虱NlA7基因的dsRNA能抑制褐飞虱NlA7基因表达、降低褐飞虱对水稻的取食以及降低褐飞虱的存活率。本发明得到了褐飞虱NlA7基因的cDNA序列,采用显微注射dsRNA的RNAi技术,沉默褐飞虱NlA7基因,导致褐飞虱产生拒食效应,显然该基因的dsRNA在褐飞虱防治领域有着重要的应用价值。
Description
技术领域
本发明涉及生物技术领域,具体涉及褐飞虱NlA7基因及其dsRNA和针对该基因的RNA干扰在控制褐飞虱方面的应用。
背景技术
褐飞虱[Nilaparvata lugens]属于半翅目飞虱科(Hemiptera:Delphacidae),是一种可以远距离迁飞危害的水稻害虫,对环境有极强的适应性,是目前危害我国和许多东南亚国家水稻的首要害虫(Sogawa,1982;Velusamy et al.,1986)。褐飞虱对寄主植物选择单一,只在水稻或野生稻上取食和产卵(Dyck et al.,1979;洪晓月等,2007;Wang et al.,2008)。褐飞虱主要通过刺吸寄主水稻韧皮部汁液给水稻生长发育造成严重影响,褐飞虱取食时形成的唾液鞘会阻塞维管束,而褐飞虱在取食后又分泌大量的“蜜露”,“蜜露”中含有大量的氨基酸和糖,可以使寄主水稻感染细菌(李春凤等,2019),受褐飞虱危害严重的水稻会引起瘫痪倒伏,引起水稻“虱烧”现象,导致大田水稻严重减产甚至绝收。同时褐飞虱除了自身需求对水稻植株造成影响外,也作为媒介传播水稻病毒病如草状丛矮病和齿叶矮缩病,水稻病毒病会给水稻产量带来灾难性后果(Hibino,1996;程遐年等,2003)。
RNAi技术即RNA干扰技术,RNAi主要指的是由双链RNA(dsRNA)诱导的对靶标基因mRNA进行沉默的技术,进而影响靶标生物的正常生理活动的现象。RNAi的研究开始于1995年,康奈尔大学Guo等在实验中对线虫Caenorhabditiselegans进行正义RNA和反义RNA干扰后,发现正义RNA或反义RNA都会引起线虫Caenorhabditiselegans的Parl基因表达下调(Guo et al.,1995)。1998年,为了揭示该现象的出现,华盛顿卡耐基研究院Fire等将纯化后的单链RNA注入线虫Caenorhabditiselegans,发现只有微弱的抑制效果,而将纯化后的dsRNA注入线虫Caenorhabditiselegans中,发现纯化后的dsRNA能对相关基因的表达有较强的抑制效果,由此证实Guo和Kemphues的实验中正义RNA抑制Parl基因表达是因为有微量的dsRNA污染,导致实验结果与理论不符,成为最早的RNAi现象(Fire et al.,1998)。到目前为止,由于RNAi技术操作简便、成本相对较低,更重要的是RNAi技术的特异性以及高效性的特点,已经广泛应用到了有关昆虫研究的各个领域(Bellés,2010),如昆虫基因功能的验证、害虫防控防治、农药开发新靶标等(田宏刚等,2012;王会冬等,2012)。
目前利用RNAi手段对昆虫进行干扰试验的技术已经比较成熟,在与昆虫有关的研究之中利用RNAi技术的领域主要集中在基因功能、RNAi诱导转基因抗虫植物以及益虫疾病控制等方面(Baum et al.,2007;Tian et al.,2009;Chen et al.,2010;Hunter et al.,2010;Yao et al.,2010),其主要的导入昆虫体内的手段有三种,即饲喂、注射以及组织培养,需针对不同实验需求选择最佳的导入方式。例如沉默豌豆蚜(Acyrthosiphonpisum)唾液蛋白C002的实验是第一个对刺吸式昆虫唾液蛋白功能研究的实验,通过实验发现豌豆蚜的取食和C002唾液蛋白有很重要的关系(Mutti et al.,2006)。纪锐(2013)通过RNA干扰技术沉默唾液蛋白Nl1860后,发现蜜露量显著减少,死亡率显著增加等。但另一方面,利用RNA干扰研究唾液蛋白也有一定的局限性,例如有些昆虫某些基因只有在昆虫特定龄期、特定时期或者特定组织中出现;如烟草天蛾(Manduca sextai)和家蚕(Bombyx mori)除了血淋巴以外的其他生物组织难以被基因沉默(Eleftherianos et al.,2007;Miller et al.,2008;黄晓慧,2016);而另一方面有些唾液蛋白基因是昆虫正常发育不可缺少的部分,如果对这些基因进行RNA干扰会使昆虫迅速死亡,难以对后续该基因在昆虫取食中的作用进行实验。
发明内容
本发明的目的是提供一种从褐飞虱中克隆的NlA7基因的cDNA序列以及蛋白质序列。通过显微注射法和助剂介导的RNAi沉默褐飞虱NlA7基因,降低褐飞虱对水稻的取食,从而保护水稻。
本发明的第一个目的是提供一种褐飞虱NlA7基因,核苷酸序列如SEQ ID NO.1的第1642位到第3003位所示。
本发明的第二个目的是提供褐飞虱NlA7基因编码的蛋白质,其氨基酸序列如SEQIDNO.2所示。
本发明的第三个目的是提供一种褐飞虱NlA7基因的dsRNA,是由SEQ ID NO.1所示序列的能达到抑制NlA7基因表达效果的核苷酸和其反向互补序列的核苷酸组成的双链RNA。
优选地,所述dsRNA的核苷酸序列如SEQ ID NO.3所示。
本发明的第四个目的是提供抑制褐飞虱NlA7基因表达的制剂在制备防治褐飞虱产品中的应用。
优选地,所述的防治褐飞虱产品是褐飞虱NlA7基因的dsRNA。
优选地,所述的防治褐飞虱产品是将dsRNA导入褐飞虱,抑制褐飞虱NlA7基因的表达,使褐飞虱取食降低、存活率降低,从而实现防治褐飞虱。
本发明的第五个目的是提供一种使用抑制褐飞虱NlA7基因表达的dsRNA防治褐飞虱的方法,其特征在于,将dsRNA导入褐飞虱体内。
优选地,是将dsRNA制备成纳米载体dsRNA并喷洒到有褐飞虱的水稻上。
优选地,所述的将dsRNA导入褐飞虱体内是通过注射导入,例如显微注射。
本发明具有的积极有益的技术效果:
实验证明,本发明得到了褐飞虱NlA7基因的cDNA序列,采用显微注射dsRNA的RNAi技术,沉默褐飞虱NlA7基因,导致褐飞虱产生拒食效应;显然该基因的dsRNA在褐飞虱防治领域有着重要的应用价值。
附图说明
图1为褐飞虱不同龄期、不同组织表达模式,A:褐飞虱若虫1-5龄,成虫1、2、3、4、5、6、7、8、9、10、11、12、13、14、15天虫体NlA7表达量;B:褐飞虱卵巢(Ov)、头部(Hd)、中肠(Mg)、表皮(In)、脂肪体(Fb)、足中(Lg)NlA7表达量。
图2为显微注射后NlA7基因相对表达量分析。
图3为褐飞虱取食孔测定。
图4为不同处理褐飞虱取食水稻的蜜露量和获得性体重,A:不同处理褐飞虱取食水稻的蜜露量;B:不同处理褐飞虱取食水稻的获得性体重。
图5为不同处理褐飞虱的死亡率。
具体实施方式
以下的实施例进一步说明本发明的内容,但不应该理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。下述实施例中所用的材料、试剂等,如无特殊说明,均可市购。
下述实例中所用褐飞虱,饲养于广东省农业科学院植物保护研究所,饲养温度26±2℃。
实施例1:褐飞虱NlA7基因的克隆
提取褐飞虱总RNA,并反转为cDNA。通过该序列设计引物,使用TaKaRa公司5’与3’Full RACE试剂盒,获得了该候选基因5’末端及3’末端序列,确定了候选基因的转录起始位点及终止位点,并拼接出该基因的全长cDNA序列。根据全长cDNA序列重新合成引物NlA7-F,NlA7-R,扩增获得NlA7的全长cDNA(如SEQ ID NO.1所示),预测ORF,其ORF序列如序列表SEQID NO.1的第1642位到第3003位所示,编码的氨基酸序列如SEQ ID NO.2所示。
NlA7-F:GCCAGCCACTCACTACTATC;
NlA7-R:ATTGCCGTATTCTGCATTTT。
实施例2:褐飞虱NlA7基因在不同龄期、不同组织的表达模式
在无RNA酶的环境下利用Trizol提取褐飞虱6种不同组织(包括卵巢、头部、中肠、表皮、脂肪体、足)的总RNA,反转录为第一链的cDNA后,使用荧光定量PCR技术进行检测,结果如图1B所示:NlA7在褐飞虱头部中表达量最高,其次为中肠,而在卵巢、表皮、脂肪体、足中少量表达。
提取不同龄期虫态褐飞虱(1-5龄若虫以及羽化后1、2、3、4、5、6、7、8、9、10、11、12、13、14、15d的短翅型雌性褐飞虱)的总RNA,然后反转录成cDNA,使用荧光定量PCR技术进行检测。结果如图1A所示,通过定量结果NlA7在1-5龄若虫表达量较高,成虫期第一天表达最高,随后表达量逐步下降。
内参基因Nlactin的荧光定量PCR引物:
QNlactin-F:ATGAAACCGTCTACAACTCG;
QNlactin-R:GCATCCTGTCGGCAATAC。
NlA7的荧光定量PCR引物:
QNlA7-F:AAATGTTGATAGCGATGGTGAC;
QNlA7-R:GCGTAAAGGAATGGATTGTAGC。
实施例3:用于沉默褐飞虱NlA7基因的dsRNA(dsNlA7)以及用于对照的绿色荧光蛋白GFP基因dsGFP的制备
1.以实施例1得到的cDNA为模板,用dsNlA7-F,dsNlA7-R为引物进行PCR扩增,得到PCR扩增产物。
dsNlA7-F(正向引物):
GGATCCTAATACGACTCACTATAGGTCAGCATGATGGCAACTCTC;
dsNlA7-R(反向引物):
GGATCCTAATACGACTCACTATAGGAGACATCGTTCAGCAGATTT,
下划线标注的区域为T7 RNA聚合酶启动子序列。
2.以实验室已有的含GFP的质粒(Takara,Japan,Code No.3270)为模板,用dsGFP-F、dsGFP-R为引物进行PCR扩增,得到PCR扩增产物。
dsGFP-F(正向引物):
GGATCCTAATACGACTCACTATAGGTGAATTAGATGGTGATGTTA;
dsGFP-R(反向引物):
GGATCCTAATACGACTCACTATAGGTCACCTTCAAACTTGACTTC,
下划线标注的区域为T7 RNA聚合酶启动子序列。
3.将步骤1、步骤2扩增产物回收,加A,连pMD18-T(TAKARA)载体,阳性克隆送测序。得到正确的克隆,以该克隆质粒为模板,同样用上述引物扩增,扩增产物纯化,浓缩,使其浓度为lμg/μL,该产物为dsRNA合成的模板。使用T7 RiboMAXTM Express RNAi System(Promega,USA,P1700)试剂盒合成dsRNA。
4.根据上述试剂盒的说明书,按以下比例加入Transcription buffer 10μL,ATP、CTP、GTP、UTP(100mM)各lμL,RNA酶抑制剂1.25μL,T7 RNAPolymerase lμL,模板1.5μg,DEPCH2O定容至49μL。轻弹混匀,瞬时离心。放入PCR中,程序为37℃,4h;75℃,5min;16℃保存。吸出1μL,凝胶电泳检测,检测到目的条带后继续下一步操作。
5.按照说明书内容加入10×Reactionbuffer 6μL,DNase 12μL,RNase 0.5μL,DEPC H2O1.5μL,充分混匀,瞬时离心,放入PCR仪中37℃,30min。之后取出加入EDTA(Fementas)lμL,继续放回PCR仪中65℃,5min终止反应。吸出1μL,稀释10倍,2μL凝胶电泳检测,2μL用NANO drop紫外分光光度计检测浓度。如果检测条带是单一明亮的条带,且OD260/280在1.8-2.0之间,则说明dsRNA质量良好,可以进行RNA酚氯仿抽提。
6.常规酚氯仿抽提,去除蛋白质,根据浓度将dsRNA调至5μg/μL每管10μL分装好,放入-80℃保存。dsNlA7的核苷酸序列如SEQ ID NO.3所示;dsGFP的核苷酸序列如SEQIDNO.4所示。
实施例4:dsNlA7的显微注射及效果检测
1.平板准备:称取1.5g的琼脂粉加入到100mL水中,煮沸,倒入玻璃平皿中,待其凝固备用。
2.注射:取长势相似的褐飞虱初羽化第一天雌成虫于试管中,通入CO2麻醉20s,通入CO2时不要对着虫吹,避免虫乱飞造成碰撞损伤。再将虫倒于1.5%的琼脂粉平板上,腹部朝上。依照说明书用Nanoliter 2010显微注射仪进行注射dsRNA。注射位置在前中胸之间,注射量为46μL(5μg/μL)。
3.褐飞虱注射dsRNA后,从注射后12h开始取样,每12h取样三头,取样36h,同时取没有注射的以及注射dsGFP的褐飞虱作为对照,用荧光定量PCR验证基因表达量变化。
具体操作如下:取样后,提RNA并反转为cDNA,用TAKARA的PrimeScript RTreagentKit with gDNAEraser(货号RR047A)按照说明书进行反转,得反转产物。将反转得到的cDNA取5μL,稀释10倍,按以下操作进行实时定量PCR。PCR反应在CFX96 TouchTM Real-TimePCRDetection System(Bio-Rad)仪器上进行,遵循以下反应体系:DNase/RNase-FreeddH2O2.9μL,2×Supermix 4μL,Primers(5mM)0.6μL,cDNA模板0.5μL反应条件:95℃预变性2min,95℃变性5-10s,65℃退火延伸30s,重复后两步骤40个循环,最后65℃-95℃,每步增加0.5℃,5s做溶解曲线以确定扩增产物的特异性。用2-△△Ct法进行数据分析。
内参基因Nlactin引物:
QNlactin-F:ATGAAACCGTCTACAACTCG;
QNlactin-R:GCATCCTGTCGGCAATAC。
NlA7定量引物:
QNlA7-F:AAATGTTGATAGCGATGGTGAC;
QNlA7-R:GCGTAAAGGAATGGATTGTAGC。
4.结果附图2所示,显微注射dsNlA7的实验组,与未注射的褐飞虱以及注射dsGFP的对照组相比,从注射12h起,褐飞虱体内NlA7基因的相对表达量显著降低,与对照相比有显著差异(P<0.05)。结果表明,通过显微注射dsNlA7能够引起褐飞虱体内NlA7基因的RNAi效应,导致基因表达量明显降低。
实施例5:显微注射后褐飞虱取食行为检测
为了进一步验证NlA7基因对褐飞虱取食的影响,我们对褐飞虱24h内固定位置的取食痕数量进行了统计。首先将注射NlA7基因的dsRNA 24h后的褐飞虱放于30天苗龄的水稻上取食,一株水稻放置一头褐飞虱,20个重复。24h后去除褐飞虱,用0.04%曙红Y浸泡褐飞虱取食水稻茎秆部位12h,12h后取出放到体视镜下观察取食痕,以正常褐飞虱作为control组,以注射dsGFP 24h后的褐飞虱作为dsGFP组。结果如图3所示,dsNlA7组的取食痕数量相比于control组和dsGFP组显著增加,这表明dsNlA7对褐飞虱取食具有抑制作用。
实施例6:显微注射后褐飞虱取食量检测
将注射NlA7基因的dsRNA 24h后的褐飞虱接到30天苗龄的水稻上取食,一株水稻放置一头褐飞虱,20个重复。统计24h的蜜露量和获得性体重,以正常褐飞虱作为control组,以注射dsGFP 24h后的褐飞虱作为dsGFP组。结果如图4所示,dsNlA7组在水稻上不能正常取食,其取食量显著低于control组和dsGFP组在水稻上的取食量。将注射NlA7基因的dsRNA24h后的褐飞虱转移到水稻上统计其24h获得性体重。dsNlA7组体重比control组和dsGFP组显著下降。这表明沉默褐飞虱NlA7后,褐飞虱的取食量显著降低;并且dsGFP组与control组没有显著差异。综合以上结果,NlA7的表达量与褐飞虱的取食量正相关。
实施例7:显微注射后褐飞虱的死亡率
将注射NlA7基因的dsRNA后的褐飞虱接到30天苗龄的水稻上取食,一株水稻放置一头褐飞虱,30个重复。每隔一天统计褐飞虱死亡率,共统计15天,以正常褐飞虱作为control组,以注射dsGFP 24h后的褐飞虱作为dsGFP组。结果如图5所示。dsNlA7组的死亡率显著高于control组和dsGFP组,并且在第4天就超过了50%。
SEQ ID NO.1(NlA7基因的cDNA序列)
GCCAGCCACTCACTACTATCGTCCATCTACTTGTTCCTCAGCGGCCACCTGATCACAGCAACATATGCGAAAATCTCTCCACTTCTATGGAATGCTGTCGTATATGAACTGCTGCTTCTCTGAAGCTTATAATTTCTGGGAGTCATTGGGGAACGACCGTCTTGATTGAGATCGCTCGGAGTATTGGATGAATCACGTTCTACGAGACTTAATTGTATTACAAGTTCCTCCATAGCTTACTAAAAACGTGACAAAATATAAGTTCGGTGTGAGAGACGTCAAATTGAATTTCCAGCTCTGTGATTATCAAGAGAATCGAATTGATGGAGCTCCTTGGAGAGTGGTTGCCATGCTGTGAAATGCAGCCTTCAGTACTCCTAGATTCGACAAGTGAATAATTTCTCATCGATTGATCTTTTAGCATTCTTTGAACCAAATGAACCACTTGTTACGAAGCTTTATAGAACCAATCTGCATGATAATATCAAAAGTGTAGGAAAGGAATCCCACTTCAATAACTGTGGAGCTGCAACTCAATTTAGGCTATAACGCTACGTATTGGGCTCTGGTTAGCAGATACAAGCTTGATCGTGCTATGTTATCATTATTGAGTTGTTTGGCTCATCACTCTATCAGCAATAATTAGCTACATAATTTGTGATCACAGAGTCAAGAAATTTTCAGTTCAGTTGAAGAAATGATATTCATTTATTAGTAGCCGGTAGAAATGTAATGGATGGAGTGACTCACCAAATGGTTGTCATTTGAAAGAAAGCAGTCCATAATCATTGACTGCCAAATTCATGAGAAACGTTCAACCAAGCAATCGATCGATTCAAAAAGCTCATTCATTCAGTATAGGACCAAATGCTGAGTGATTTTCAAAATGGGAAACTATTCTTTTGAAACGTAATGGAAAACTAAGTCCATTCCCTGAAACAGATTACGGTTGATAGCATTCAATGAGGGGAACTACTATTATTACCAGATCACTGTATGTTTGTAGTGGTTAACTGCTATCGAAAATTGATTTCCTGGCTGCTGTTGCTATAAGCGTTTTGTATTGCAAAAAGGAGATTGAATGGAATCTGTTCCACATTAACAGCTTCAATCATGCAATGATTACACAATAATCACACAATCATGTAATACTCAATTCAATAGAATCAATCATTCTTGTGAAACAAATCAAAATGGATTTTTGCCCAAGCAGAATATAGTGGAGATTTGATGCGAATATGATTCTCATAGAGAGTTGAAAAAAATATAAATCAAATAGACTCGATTTTTACATCGTTAGTAGATGACAGCTACTATTCAGGCTTAGTAAATAGTTTGAGTTTCAAGAACTAATTTACAATTTATCCAGTTCCTCATCATCGAACATTCATCGGTGTTATTTTATGAATTAAAATAATAGACATTGGTCTTCGTAGAGAGAAAACCTGTGAGTTGAGAAGTCTAGAATGCAGTTCAGCCTTTCCTAAATCGAAATCTGAGCTGAATCAGATCAGTACAGAAATGTAAAATAAGGCACCTGAGAGCTATCAAGATCTGTCCTAACCGGCCATCAGTTCGTTATCGACTGGGGAACAATTCCTGCATTGAATAATCGTATGATTATGGTGATATCATGACGGTATGAATATGAATGAGTCAACAAGCACAGCTGTTGAAGGCGCTTCAGACAGGAACGTTTCCAACTTAACGACGGCAAATCCCGTGCTAGACGCCACCGACACTTCTGTTTTCAACGAAACGCTCTCGGATATCATTCAGGACCCGGCCGTGCAGGGCATATTCTGCGTGCTCTACACCACCATCTGCCTCCTGGGACTTTTCGGCAACGTTCTCGTCTGCTACGTGGTTGGCCGCAACCGCGCCATGCACACTGTCACCAATTGCTTCATCACCAACCTGGCGCTGTCCGACATTCTGCTCTGCGTGCTCGCCGTTCCTTTCACGCCTCTCTACACATTCGCCAGTACTTGGATGTTCGGTCGAGCCCTCTGTCACCTCGTCGCCTACGCCCAGGGCACCAGCGTCTACATCTCGACTCTGACCCTCACCTCCATCGCAATAGACCGATTTTTTGTCATCATCTACCCGTTTCAACCGCGAATGAAGCTGTCAACCTGTCTCTCCATCATTCTTCTTATTTGGATATTCAGCATGATGGCAACTCTCCCCTATGGTATATACATGCATCACGGACCCATGACAGGACATGAAGACAGAGTTTTCATCTGCGATGAGAACTGGCCCTCTGAATCATTCAGACAGGTGTATGGACTGCTGACAGCGGCTCTCCAGTTTCTGCTGCCTTTCTTCATAATCGCCTTCTGCTATGTGAGGGTGTCACTCAAGTTGAATGACAGAGCGCGATCGAAGCCCGGCTCGAAGAACACGAGGAAGGAGGAGGCTGATCGGGAGCGCAAGAGACGCACCAACAGGTATATCAAAATGTTGATAGCGATGGTGACAATCTTTGGAGTTTCGTGGCTGCCACTGACTGTGATAAATCTGCTGAACGATGTCTACATTCCCACAGGCAGTTGGCGATATTACAACCTGTGTTTCTTCATCGTTCACGCGCTAGCTATGTCCTCAACATGCTACAATCCATTCCTTTACGCATGGCTCAATGATAACTTCAGGAAAGAGTTCAAACAGGTGCTTCCATGCTTTGACTCGACGGGTGGCCCGGGAGGGAGCAGCGGATGCGGGGCGGGCGGTCGCCTGGGCAATTGGCGGTCGGAGCGCACCTGCAACGGTCAGGAGACCTGCCAGGAAACACTGCTGCCAACCTCCGTAGTCCTGCCGGGAAAGACTGGAGACTCGCCACCTACCGTTCCAGAACTGGACCCGAGCTGGCTGAAGGCGAACAAGTCGAGCGACTCGGTGGAGGTTGTGTTGGTGGCGTACACGGCAGGAGAGGACATGGTTCGTATCGATGATGCTGCAGCCACGGCAACCAGGCGAGCCTCACGCCAGGACGTCTAGCAGGGGATACCCAAGTCAGTAGTGTGGCCTGCTGACAAGACAGTGACTACATCATTCTAGAGCACAGGAATGGAATTTAGTGTGCATAACGGAAACGATGAGGGTAGCATACAAGCATGATGTATGAGCACCACGCACAACCCACTACTGCAATGGAACGTCGTGAGGCGGTAAAGGCATCGTTAGTCGCTATCAATCATTAGCAGTGCTTGTACTAAGTTTGAATTCTATGTAGGGAAGAATATTGAGTCAAACGTTATCCTATCATTTGCGATCGATTTGGAAATTCTTATCAGATCCATTCAGGTCACATTTATTATTGTAAGTGAGAATGAAAAAGCAAAATACTGAAGACATTCATCAAATACTGTACTAATCTAGAATGATGTATGGTAACGTATGTAACTTCTATGAAAGAAATCAGACATTATTCATTGGAAATTCAATTTTGTAGTATGACATGAAATTTATAGGAAATAAAATGTCTTGTAAATAACATTTAATCGTTAAGTACATTCCTCCTTGAAGTGTATTGCATGGATGACCAACCTTGCACACAATCCAGTCAGCGGCACACACAAAATTACAAAAATACGTAGTGGAAGAAAAACAACGTAGTCATCGTTAATAATATTGATATACTGTTCTATAAAATATCACAGTATTTTACATTTTTATGAATCTGAAATAAATTATTATCTGATGTCATTCCGACGTTGCTGATGATTTAAGTTTTTCATTGTTCACTGGGCACAATTACAATATTAACGTTGATTTTAAGCCACAATAACTGTTGTTAATCAAATTTAAAATGCAGAATACGGCAATSEQ ID NO.2(NlA7的氨基酸序列)
MNMNESTSTAVEGASDRNVSNLTTANPVLDATDTSVFNETLSDIIQDPAVQGIFCVLYTTICLLGLFGNVLVCYVVGRNRAMHTVTNCFITNLALSDILLCVLAVPFTPLYTFASTWMFGRALCHLVAYAQGTSVYISTLTLTSIAIDRFFVIIYPFQPRMKLSTCLSIILLIWIFSMMATLPYGIYMHHGPMTGHEDRVFICDENWPSESFRQVYGLLTAALQFLLPFFIIAFCYVRVSLKLNDRARSKPGSKNTRKEEADRERKRRTNRYIKMLIAMVTIFGVSWLPLTVINLLNDVYIPTGSWRYYNLCFFIVHALAMSSTCYNPFLYAWLNDNFRKEFKQVLPCFDSTGGPGGSSGCGAGGRLGNWRSERTCNGQETCQETLLPTSVVLPGKTGDSPPTVPELDPSWLKANKSSDSVEVVLVAYTAGEDMVRIDDAAATATRRASRQDVSEQ ID NO.3(dsNlA7的核苷酸序列)
TCAGCATGATGGCAACTCTCCCCTATGGTATATACATGCATCACGGACCCATGACAGGACATGAAGACAGAGTTTTCATCTGCGATGAGAACTGGCCCTCTGAATCATTCAGACAGGTGTATGGACTGCTGACAGCGGCTCTCCAGTTTCTGCTGCCTTTCTTCATAATCGCCTTCTGCTATGTGAGGGTGTCACTCAAGTTGAATGACAGAGCGCGATCGAAGCCCGGCTCGAAGAACACGAGGAAGGAGGAGGCTGATCGGGAGCGCAAGAGACGCACCAACAGGTATATCAAAATGTTGATAGCGATGGTGACAATCTTTGGAGTTTCGTGGCTGCCACTGACTGTGATAAATCTGCTGAACGATGTCTSEQ ID NO.4(dsGFP的核苷酸序列)
TGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGCCTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGA
Claims (5)
1.抑制褐飞虱NlA7基因表达的制剂在制备防治褐飞虱产品中的应用,所述褐飞虱NlA7基因的核苷酸序列如SEQ ID NO.1的第1642位到第3003位所示,所述制剂是所述褐飞虱NlA7基因的dsRNA,所述dsRNA的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的应用,其特征在于,所述的防治褐飞虱产品是将所述dsRNA导入褐飞虱,抑制所述褐飞虱NlA7基因的表达,使褐飞虱取食降低、存活率降低,从而实现防治褐飞虱。
3.一种使用抑制褐飞虱NlA7基因表达的dsRNA防治褐飞虱的方法,其特征在于,将所述dsRNA导入褐飞虱体内,所述dsRNA的核苷酸序列如SEQ ID NO.3所示。
4.根据权利要求3所述的方法,其特征在于,将所述dsRNA制备成纳米载体dsRNA并喷洒到有褐飞虱的水稻上。
5.根据权利要求3所述的方法,其特征在于,将所述dsRNA导入褐飞虱体内是通过注射导入。
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