CN114149994B - 褐飞虱性别决定调控基因Nlfmd2及其在有害生物防治中的用途 - Google Patents
褐飞虱性别决定调控基因Nlfmd2及其在有害生物防治中的用途 Download PDFInfo
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- CN114149994B CN114149994B CN202111336896.7A CN202111336896A CN114149994B CN 114149994 B CN114149994 B CN 114149994B CN 202111336896 A CN202111336896 A CN 202111336896A CN 114149994 B CN114149994 B CN 114149994B
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Abstract
本发明属于动物基因工程技术领域,本发明提供了褐飞虱性别调控基因Nlfmd2及其在有害生物防治中的用途。所述褐飞虱性别调控基因Nlfmd2的序列如SEQ ID No.1所示。通过RNA干扰技术在褐飞虱体内敲除Nlfmd2后,雌性褐飞虱出现向雄虫转变的嵌合体性状,并失去生殖能力,雄性褐飞虱精子失去活力,从而丧失生殖能力。通过母本RNAi敲低基因Nlfmd2在下一代胚胎中的表达量,则引起胚胎死亡。这表明褐飞虱基因Nlfmd2在褐飞虱性别决定中起到重要作用,鉴于参与昆虫性别决定的基因具有高度特异性,可将该基因用于褐飞虱生物防治中,具有良好的应用场景。
Description
技术领域
本发明属于动物基因工程技术领域,具体涉及一种褐飞虱性别决定调控基因Nlfmd2及其在有害生物防治中的用途。
背景技术
褐飞虱(Nilaparvata lugens(Stal))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等国家水稻的首要害虫。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。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,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。同时除了褐飞虱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生产产量带来灾难性后果。20世纪80年代开始,我国的褐飞虱发生面积约1330-2000万hm2,约为我国种植水稻面积的二分之一,给我国水稻生产造成严重损失,在近三十年来看,褐飞虱危害我国水稻的主要特征有三个:爆发频率增加、危害范围扩大、危害程度加大。
长期以来,大量不合理使用化学防治,已经在成褐飞虱的抗药性激增,并造成褐飞虱大发生,严重威胁我国水稻产区粮食安全。随着生物技术,尤其是基因工程技术的发展,运用生物学手段进行褐飞虱防治已经成为新植物保护的重要手段。昆虫性别决定并保证了个体的发育和物种的延续,不同种类昆虫性别决定通路被报道各不相同,性别通路基因具有成为生物防治的靶标基因的潜力。
发明内容
本发明是针对水稻害虫褐飞虱的生物学防治,提供了影响褐飞虱性别决定通路基因Nlfmd2,及其功能和表达分析。
一方面,本发明提供了一种褐飞虱性别决定通路基因Nlfmd2,该基因是从水稻褐飞虱克隆得到的基因,其核苷酸序列如下(图1):
ATGGCTGATTCCACTCTGGAATACGACATGAGTAACGGAGAATTTCAACAGGGCGGCGAGGATTACACCCAGGACGATCAAATGAACGGATCTGGCGAAAATGGAGGAGCTAACGAAGGAGGCGACAGTGGAAGCGCCGAAGCACCAGGCAAAGATGACGAAAGAAAACTGTTTGTTGGTGGACTCAGCTGGGAAACCTCTGACAAGGAACTCCGTGATCATTTCGGTACATTTGGTGAAATCGAAAGCGTAAATGTGAAGACTGATCCAAATACTGGCAGATCAAGAGGATTCGCGTTCATTGTTTTCAAGTCTGCGGATGCTATCGATAAGGTAGTCAACGCTGGAGACCATGTGATCAACAACAAGAAAATTGACCCAAAGAAAGCCAAGGCCAGACAAGGCAAGATTTTCGTCGGAGGATTGTCGGCTGAATTATCCGATGATGATATCAAGTCATACTTTGCTCAGTATGGAACCATTGTTCAAGTTGATATGCCGTACGACAAGTCAAAGAACCAGAGGAAGGGATTCTGCTTCATCACCTTCGAATCAGAACAGATTGTCACTGAAGTTCTTAAGACACCCAAGGCTTCAATCAAAGGCAAAGAAGTTGACGTGAGGAAAGCGACGCCGAAGCCTGATGGCATGGGCAACACGAGGGGAGGTAGAGGAGCCGGCCGTGGCACTCGAGGCAGGGGCCGTGGAGGATTTGGTCAGGGAGGCTATGGACAGGGAGGTTACGGTAACTACGGAGCTGGTGGCTATGGCACTGGCTACGATTACTATGGCAGTGGTGGCTACGGAGGTGGATACGGCGGAGGCTACGGAACCTACGACTACTCGGGATACGGCGGCTATGGTAGCTATGATGGATATGGAGCTTATGGCAACAACTATGAGACAGGATATGGCACTGGTGGAGGTGGACGAGGAGGCCGAGGAAAAGGTACCGGAGGCGGCTATGGTGGTGGCGGCAAGCAGAGGGGCGGCGGCCGAGGCCAGAGGCACACTCCCTACTGA(SEQ NO.1),或经取代、缺失和/或增加核苷酸与所述核苷酸序列具有至少70%同源性,优选至少80%同源性,更优选至少90%同源性。
一方面,本发明提供了一种由所述核苷酸序列(SEQ NO.1)翻译的氨基酸序列Nlfmd2氨基酸序列,其序列如下(图1):
MADSTLEYDMSNGEFQQGGEDYTQDDQMNGSGENGGANEGGDSGSAEAPGKDDERKLFVGGLSWETSDKELRDHFGTFGEIESVNVKTDPNTGRSRGFAFIVFKSADAIDKVVNAGDHVINNKKIDPKKAKARQGKIFVGGLSAELSDDDIKSYFAQYGTIVQVDMPYDKSKNQRKGFCFITFESEQIVTEVLKTPKASIKGKEVDVRKATPKPDGMGNTRGGRGAGRGTRGRGRGGFGQGGYGQGGYGNYGAGGYGTGYDYYGSGGYGGGYGGGYGTYDYSGYGGYGSYDGYGAYGNNYETGYGTGGGGRGGRGKGTGGGYGGGGKQRGGGRGQRHTPY(SEQ NO.2)或经取代、缺失和/或增加氨基酸与所述氨基酸序列具有至少70%同源性,优选至少80%同源性,更优选至少90%同源性。
本发明提供了褐飞虱性别决定通路基因Nlfmd2在生物学防治褐飞虱中的应用。优选的,用于成虫,若虫和卵期褐飞虱的防治。
本发明提供了Nlfmd2在褐飞虱性别特异性发育中的作用,具体为在褐飞虱三龄若虫期使用RNAi敲除Nlfmd2后,雌性褐飞虱产卵瓣缩短,卵巢不发育,失去生殖能力,与此同时雄性褐飞虱精子发育受到影响,失去活力,从而也失去繁殖能力;在雌成虫第一天使用显微注射将干扰双链注射进褐飞虱体内后,其产下的卵中Nlfmd2也被敲除,同时造成褐飞虱胚胎不发育,没有若虫孵化。
本发明提供的Nlfmd2 RNAi在褐飞虱若虫期造成褐飞虱雌虫和雄虫失去生殖能力,在雌性褐飞虱成虫初期造成后代胚胎期死亡。这表明褐飞虱性别决定通路基因Nlfmd2在褐飞虱性别特异性发育中起到了关键性作用,同时在生物学防治褐飞虱中(转基因水稻等)具有重要的价值,具有良好的应用前景。
附图说明
图1为Nlfmd2基因的核酸和氨基酸序列。
图2基因Nlfmd2在褐飞虱整个发育历期都表达。
图3在褐飞虱2龄期敲除Nlfmd2后,褐飞虱出现明显死亡。
图4在褐飞虱雌虫体内敲除基因Nlfmd2后,没有后代孵化并且产卵量明显下降。
图5为在3龄期使用小RNA干扰敲除基因Nlfmd2后,褐飞虱雌虫出现雄性化现象。
图6为在3龄期使用小RNA干扰敲除基因Nlfmd2后,褐飞虱雌虫卵巢不发育。
具体实施方式
下面通过具体实施例对本发明进行说明,实施例中未作详细描述的技术手段属于本领域专业技术人员熟知的常规技术。实施例只用于说明本发明,但不限制本发明的范围,任何本领域的技术人员在不付出创造性劳动的情况下,以本发明的实施例为基础所获得的其他实施例均属于本发明的保护范围。
实施例1:基因Nlfmd2全长的获得以及干扰RNA双链的合成。
褐飞虱种群在秀水128水稻苗上饲养,养虫室温度设定为26±0.5摄氏度,光照设定为12小时光照,12小时黑暗。总的RNA从10头雌虫和15头雄虫中提取,总量为1微克的RNA被用来反转录,反转录体系为10微升,将反应液稀释10倍(取9.8微升稀释到89.2微升的双蒸水中),被用来作为模板。完整的开放阅读框序列通过特异性引物Nlfmd2-S:ACCCACTATTGCTGCTATTT(SEQ NO.3),Nlfmd2-S:CAACACCCACTGCTAAAGA(SEQ NO.4),经聚合酶链式反应(Polymerase Chain Reaction,PCR)得到,并连接PMD-19T载体上,进行复制筛选,并测序。干扰双链是使用N端带有T7启动子的双向特异性引物dsNlfmd2-S:GATCACTAATACGACTCACTATAGGGAGAATCTTTAGCAGTGGGTGT(SEQ NO.5),dsNlfmd2-AS:GATCACTAATACGACTCACTATAGGGAGATGGTTCGGTGATATTCA(SEQ NO.6)与带有特异性靶序列的PMD-19T质粒的PCR产物为模板,使用MEGAscript T7体外转录试剂盒,合成的双链使用1%的琼脂糖凝胶进行检测,然后稀释1倍,放-80摄氏度冰箱保存。
实施例2:褐飞虱若虫期干扰和母本干扰。
使用拉针仪P-97,使用程序heat=800,pull=150,vel=150,time=80,将毛细管拉成适合显微注射的玻璃针,并将双链注入玻璃针中。选取2龄或3龄褐飞虱幼虫或刚羽化的雌性褐飞虱,使用二氧化碳麻醉1分钟,将褐飞虱置于体视镜下,使用显微注射仪进行注射,其中显微注射仪推荐参数为注射压力(injection pressure,Pi)1000hPa,压力维持时间(injection time,ti)0.5s,补偿压力(compensation pressure,pc)20hpa。
实施例3:死亡率统计和现象记录。
将完成注射的褐飞虱若虫饲养在水稻幼苗上,每24小时统计存活的若虫,并统计现象在表1中。
表1
结果表明:褐飞虱3龄期使用小RNA干扰敲除基因Nlfmd2后,褐飞虱雄虫失去生殖能力,与对照雌虫交配后,没有后代孵化。
以上所述为本发明较佳的具体实施方式,但在其基础上可以进行一些改进或修改,这对任何熟悉本领域的技术人员而言是显而易见的。因此,在本发明基础上所作的这些修改和改进,均属于本发明要求保护的范围。
序列表
<110> 宁波大学
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gattactatg gcagtggtgg ctacggaggt ggatacggcg gaggctacgg aacctacgac 840
tactcgggat acggcggcta tggtagctat gatggatatg gagcttatgg caacaactat 900
gagacaggat atggcactgg tggaggtgga cgaggaggcc gaggaaaagg taccggaggc 960
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Gln Gly Gly Glu Asp Tyr Thr Gln Asp Asp Gln Met Asn Gly Ser Gly
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Glu Asn Gly Gly Ala Asn Glu Gly Gly Asp Ser Gly Ser Ala Glu Ala
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Pro Gly Lys Asp Asp Glu Arg Lys Leu Phe Val Gly Gly Leu Ser Trp
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Glu Thr Ser Asp Lys Glu Leu Arg Asp His Phe Gly Thr Phe Gly Glu
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Ile Glu Ser Val Asn Val Lys Thr Asp Pro Asn Thr Gly Arg Ser Arg
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Gly Phe Ala Phe Ile Val Phe Lys Ser Ala Asp Ala Ile Asp Lys Val
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Val Asn Ala Gly Asp His Val Ile Asn Asn Lys Lys Ile Asp Pro Lys
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Lys Ala Lys Ala Arg Gln Gly Lys Ile Phe Val Gly Gly Leu Ser Ala
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Ile Val Gln Val Asp Met Pro Tyr Asp Lys Ser Lys Asn Gln Arg Lys
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Gly Gly Tyr Gly Gln Gly Gly Tyr Gly Asn Tyr Gly Ala Gly Gly Tyr
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Gly Thr Gly Tyr Asp Tyr Tyr Gly Ser Gly Gly Tyr Gly Gly Gly Tyr
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Gly Gly Gly Tyr Gly Thr Tyr Asp Tyr Ser Gly Tyr Gly Gly Tyr Gly
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Ser Tyr Asp Gly Tyr Gly Ala Tyr Gly Asn Asn Tyr Glu Thr Gly Tyr
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Gly Thr Gly Gly Gly Gly Arg Gly Gly Arg Gly Lys Gly Thr Gly Gly
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Claims (3)
1.一种褐飞虱性别决定通路基因Nlfmd2在生物学防治褐飞虱中的用途,其特征在于:所述褐飞虱性别决定通路基因Nlfmd2序列为SEQ ID NO.1。
2.根据权利要求1所述的用途,其特征在于:其中所述褐飞虱为成虫,若虫和卵期的褐飞虱。
3.权利要求1或2所述的用途,其特征在于:通过RNA干扰技术在褐飞虱体内敲除Nlfmd2后实现。
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