CN111606989B - 生殖发育相关基因UCP及其dsRNA在防治桔小实蝇中的应用 - Google Patents
生殖发育相关基因UCP及其dsRNA在防治桔小实蝇中的应用 Download PDFInfo
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Abstract
本发明公开了生殖发育相关基因UCP及其dsRNA在防治桔小实蝇中的应用,新基因UCP参与调节桔小实蝇雌虫的卵巢发育和繁殖力,设计靶向基因UCP的dsRNA沉默UCP后,桔小实蝇雌虫出现极明显的卵巢发育迟缓现象,产卵量极显著低于对照组,因此基因UCP可以作为桔小实蝇防治的新靶标。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种与桔小实蝇生殖发育相关的基因UCP及其dsRNA在防治桔小实蝇中的应用。
背景技术
桔小实蝇Bactrocera dorsalis(Hendel),又称东方果实蝇(Oriental fruitfly),俗称果蛆,属双翅目Diptera,实蝇科Tephritidae,果实蝇属Bactrocera,是我国一种毁灭性果蔬害虫,严重危害柑橘、芒果等在内的250多种水果和蔬菜。该虫主要以幼虫取食危害果实,不转果危害;幼虫潜居危害的特性使其难以从果实外观察觉,常随被害果、包装物、运输工具等远距离传播;传入非疫区后遇到适宜环境条件即可迅速蔓延危害,对当地果蔬产业发展构成严重威胁,故被列为世界性检疫害虫。因此,桔小实蝇的防治依旧是我们需要面对的一个重要问题。
近年来,有关桔小实蝇的研究日趋深入,已经从最初的生物学基础、生态学,逐步过渡到分子生物学领域。昆虫繁殖对其物种繁衍至关重要。在种群繁衍和扩增过程中,生殖***对于维持其种群数量及延续至关重要。两***配是昆虫种群繁衍的一个重要的环节,在受到一些昆虫的生理因素和外部生态因素的干扰后,生殖适合度会受到影响。雌性昆虫的生殖发育主要包括卵巢发育、生殖行为和繁殖力。由于桔小实蝇成虫羽化后需要经历一段时间的生殖发育才能达到性成熟进而交配产卵,所以从防治害虫的角度出发,鉴定并研究桔小实蝇雌成虫生殖发育相关基因,明确其对卵巢发育与繁殖力的调控能力,有利于开发桔小实蝇绿色可持续治理措施。
研究表明,多个基因及多种信号途径参与调控雌虫生殖发育过程。雌性昆虫的生殖受多种激素调控,在果蝇Drosophila、德国小蠊Blattella germanica、埃及伊蚊Aedesaegypti等模式昆虫中,胰岛素、蜕皮激素信号通路均可通过影响卵巢卵黄形成从而调控昆虫的生殖。对果蝇卵子发生前后的对比研究证明,胰岛素信号通路调控脂质储备并抑制生长转变。果蝇敲除Hr39、Beadex基因以及神经肽相关基因Nep1、Nep2、Nep4,都会导致生殖力和产卵量的显著下降。在埃及伊蚊中,***的形成受取食血液触发的通路的调节,该通路负责Vg基因的转录,其中20E信号是主要的调节因子。抑制基质金属蛋白酶Mmps活性会导致家蚕Bombyx mori的卵巢发育迟缓,而Mmp的过表达促进卵巢发育。除了对果蝇等模式物种的生殖发育相关基因的研究以外,近年来,桔小实蝇生殖发育也涌现了较大进展。桔小实蝇BdRab40、csn3、csn5和pts基因的沉默均可抑制卵巢发育,显著降低产卵量。在对桔小实蝇卵黄蛋白受体VgR的研究中,发现VgR在卵巢组织中特异性表达,并且在Vg的运输和吸收中起重要作用,通过RNAi技术沉默VgR可显著抑制卵巢发育。将卵黄蛋白基因(Bdyp1)抑制导致卵巢发育迟缓,产卵量显著下降,雌虫产卵器畸形。综上所述,有关生殖发育的研究主要集中于转录水平的卵黄沉积相关基因或信号通路,因而深入研究桔小实蝇雌成虫生殖发育相关基因,不仅可以为阐明生殖发育的复杂机制提供全面的资料,而且有助于开发高效绿色的害虫防控策略。
发明内容
为了解决上述问题,本发明提供了一种参与调控桔小实蝇生殖发育的新基因UCP,沉默该基因后,桔小实蝇卵巢发育被抑制,产卵量显著降低,基因UCP可以作为桔小实蝇防治的新靶标。
为了实现上述目的,本发明采用以下技术方案:
桔小实蝇生殖发育相关基因UCP,核苷酸序列如SEQ ID NO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示。
桔小实蝇生殖发育相关基因UCP的dsRNA,由序列如SEQ ID NO.3所述的核苷酸和与其反向互补的核苷酸组成,通过下述方式获得:
以桔小实蝇9日龄雌成虫卵巢RNA(1μg)为模板进行去基因组反转录,得到cDNA;以cDNA为模板,进行普通PCR扩增(引物UCP-T7 F:GGATCC TAATACGACTCACTATAGGCTGCTTGGCGTCTGCTTA,UCP-T7 R:GGATC CTAATACGACTCACTATAGGTTGCTGCTTACATTCACCTT),并用DNA凝胶回收试剂盒回收目标基因片段,回收产物用于进一步合成dsRNA。也可以通过人工合成的方法获得。
桔小实蝇基因UCP作为靶标基因在调节桔小实蝇雌虫的卵巢发育和繁殖力中的应用,通过以下方法验证:
1)采用初羽化3d的桔小实蝇雌成虫作为实验用虫,每头试虫腹部注射1μl 2000ng的目的基因dsRNA,以注射相同量的dsEgfp为对照,每个处理两个生物学重复,每个处理虫数为180头,共进行三次重复实验;
2)对RNAi桔小实蝇的卵巢形态、产卵量进行测定,结果表明,沉默UCP后出现约60%极明显的卵巢发育迟缓现象;处理组单雌日均产卵量1-15d均低于对照组,且第1、3、5、7、9、11天具有显著性差异;处理组(28粒)产卵总量也极显著低于对照组(130粒)。
3)分别解剖dsUCP注射后24h雌成虫卵巢、脂肪体的样品,提取总RNA反转录为cDNA,并通过qPCR进行检测。qPCR检测发现沉默UCP后,Vg1、Vg2和VgR的表达量均显著上调,胞吞作用相关基因Endophilin B1显著下调。以上结果说明,沉默UCP后可能通过影响胞吞作用进而调节桔小实蝇雌虫的卵巢发育和繁殖力。
因此,UCP基因参与调控雌成虫的卵巢发育和产卵量,在桔小实蝇雌虫生殖发育中发挥重要作用,通过dsRNA沉默UCP后,卵巢发育被抑制,产卵量显著下调,基因UCP可以作为桔小实蝇防治的新靶标,dsRNA通过调节雌虫生殖发育可用于防治桔小实蝇,或用于制备桔小实蝇杀虫剂。
与现有技术相比,本发明具有以下优点及有益效果:
本发明是国内外首次公开桔小实蝇生殖发育相关基因UCP序列,目前该类基因可调控桔小实蝇雌虫生殖发育的功能没有相关报道。利用桔小实蝇作为受体的RNAi实验证实,与对照组相比,处理组出现极明显的卵巢发育迟缓现象,产卵量极显著低于对照组。桔小实蝇雌成虫沉默UCP可有效抑制桔小实蝇卵巢发育和产卵量,从而有助于桔小实蝇绿色可持续防控。
附图说明
图1:注射dsUCP对桔小实蝇卵巢发育和产卵量的影响。A-C:注射dsEgfp的卵巢形态;D-F:注射dsUCP的卵巢形态;比例尺为500μm,7d:羽化后七天,10d:羽化后十天,13d:羽化后十三天;G:卵巢平均直径(μm);H:单雌分别在交配后15天的产卵量;I:单只桔小实蝇交配后15天产卵总量;*代表在P<0.05水平上有显著性差异,**代表在P<0.01水平上有显著性差异,***代表在P<0.001水平上有显著性差异。
图2:雌成虫注射dsUCP24小时后检测其对基因UCP表达量的影响。
图3:雌成虫注射dsUCP 24小时后检测其对相关基因(Vg1、Vg2、VgR、EndophilinB1)表达量的影响。
具体实施方式
以下实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均可通过购买获得。
实施例1:桔小实蝇生殖发育相关基因UCP的克隆及其dsRNA的体外合成
我们从桔小实蝇雌成虫转录组文库中鉴定获得UCP基因,其核苷酸序列如SEQ IDNO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示。
首先根据转录组序列信息利用Primer Premier 5软件设计含T7启动子的基因片段引物(表1),以桔小实蝇9日龄雌成虫cDNA为模板,进行普通PCR扩增,并用DNA凝胶回收试剂盒回收目标基因片段,回收产物用于进一步合成dsRNA。同时合成Egfp基因的dsRNA作为对照组。
表1桔小实蝇靶基因dsRNA合成引物序列
(注:黑体加粗部分为T7启动子。)
dsRNA合成步骤如下:
第一步:分别合成两个单链RNA(ssRNA),反应前5×Transcript Aid peactionBuffer中有沉淀时先于37℃溶解后使用。
反应体系如下:
反应条件:42℃水浴30min
第二步:将合成的两个单链RNA(ssRNA)混合,72℃水浴10min,并在水中自然冷却至室温。
第三步:消化反应,反应体系如下(其中Rnase solution当场稀释200倍后使用)。
反应条件:37℃处理30min
第四步:加0.1个体积的醋酸钠,1个体积的异丙醇,混匀冰浴5min,4℃13000×g离心10min。
第五步:弃上清,用500μl 75%的乙醇洗涤沉淀,4℃13000×g离心5min。并在室温下干燥15min,加2-5倍体积的RNase Free dH2O溶解沉淀,取少量dsRNA进行电泳检测,并用Nanodrop2000测定其浓度,记录OD260/OD280,OD260/OD230的值,置于超低温冰箱-80℃保存。
将该dsRNA命名为dsUCP,由正义链和反义链组成,其正义链的核苷酸序列如SEQID NO.3所示,其反义链的核苷酸序列与SEQ ID NO.3所示的序列反向互补,dsUCP也可以通过人工合成的方法获得。
实施例2:生殖发育相关基因UCP的dsRNA在防治桔小实蝇中的应用与验证
一、显微注射dsUCP
显微注射用针使用武汉微探科学仪器有限公司的微电极玻璃毛细管,采用日本Narishige公司拉针仪在60.4℃下制成,用Narishige公司的微型磨针仪磨成注射用针,注射压强300-400hPa,注射时间0.2s。采用初羽化3d的桔小实蝇雌成虫作为实验用虫,每头试虫腹部注射1μl 2000ng的目的基因dsUCP,以注射相同量的dsEgfp为对照,每个处理两个生物学重复,每个处理虫数为180头,共进行三次重复实验。
二、卵巢形态观察及卵巢直径统计
分别在注射dsUCP后解剖第7、10、13日龄雌成虫的卵巢组织,与dsEgfp的显微镜图像相比,注射dsUCP雌虫卵巢发育在7日龄时形态无显著差异(图1A和D),10和13日龄的桔小实蝇卵巢图片处理组较对照组出现约60%极明显的发育迟缓现象(图1B-C、E-F);统一比例尺下的卵巢直径统计比较发现,第7、10、13日龄雌成虫的dsUCP组相较于dsEgfp组均有显著的卵巢发育被抑制,分别为20%、63%和23%左右的抑制率(图1G)。
三、注射dsRNA对桔小实蝇产卵量的影响
将UCP注射后第12日龄的雌虫与正常未交配的第12日龄雄虫以1:1的比例放置于14.4cm×8.5cm×8.5cm(长×宽×高,口径为6.6cm)的透明盒子中,每盒2对,每个处理15盒,统计交配后15天雌虫的产卵情况。处理方法:首先将小纸杯壁上扎孔以便雌虫产卵,然后将扎孔后的小纸杯置于透明盒子内,盒子口处密封,纸杯内放置块状香蕉,使香蕉的气味只能通过纸杯上的小孔散发出去,从而引诱雌虫在纸杯上产卵。分别统计处理组和对照组中产卵数量,并计算单雌日均产卵量和单雌产卵总量,并用Graph Pad Primer7软件进行t测验来分析干扰组(注射UCP-dsRNA的雌虫)与对照组(注射EGFP-dsRNA的雌虫)之间交配率的差异。注射12d后观察对照组和处理组雌虫的单雌日均产卵量和产卵总量,结果表明处理组单雌日平均产卵量1-15d处理组均低于对照组,且第1、3、5、7、9、11天具有显著性差异(图1H);处理组产卵总量也极显著低于对照组,处理组产卵总量为28粒,对照组130粒(图1I)。因此,UCP基因参与调控雌成虫的卵巢发育和产卵量,在桔小实蝇雌虫生殖发育中发挥重要作用。
四、实时荧光定量PCR
实时荧光定量方法采用2×T5 Fast qPCR Mix(SYBR Green I)试剂盒在Bio-RadCFX96实时荧光定量PCR仪上进行。每个样品设三个技术重复,根据需要设置相应空白对照,PCR总体系为20μl,如下所示。荧光定量PCR结束后分析溶解曲线,以确保特异性扩增。实时荧光定量PCR结果利用2-ΔCt方法分析,相关引物合成由南京金斯瑞生物科技有限公司完成,序列如下表。
表2实时荧光定量PCR引物序列
具体步骤如下:
实时荧光定量PCR反应程序:
Stage 1:预变性
95℃ 3min
Stage 2:PCR反应
Stage 3:融解曲线分析温度以0.5℃/s的速率从55℃缓慢递增到94℃,连续测定样品的荧光强度以获取溶解曲线,借此判断扩增反应的特异性。
注射dsUCP 24h后,分别提取卵巢和脂肪体组织RNA,反转录为cDNA并进行qPCR检测后,结果表明:dsUCP处理组相对表达量相较于对照组有明显下调,卵巢中UCP相对表达量下降约49%左右,脂肪体中UCP相对表达量下降约22%左右(图2)。桔小实蝇雌成虫沉默UCP后发现Vg1、Vg2、VgR的表达量显著上调(图3A、B、C),胞吞作用相关基因Endophilin B1在卵巢和脂肪体中均显著下调(图3D)。
序列表
<110> 华中农业大学
<120> 生殖发育相关基因UCP及其dsRNA在防治桔小实蝇中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3549
<212> DNA
<213> Artificial Sequence
<400> 1
atgatgtaca atcaaaaatg ggcacgtaca attgagctga tagtagtagt gcttttattc 60
acagttttta gcagtagcaa tggcaaaaac ttagttatcc ctcagctagg taaaagcgat 120
atatttaata gtgaatatga acgcgtgcaa caaaaatcaa tgccaatgga atttgatgtt 180
tctgaagctg aacgcggtaa aaaaattaat cagcggttgc aaccccgcag tgttaaatat 240
aaatattttg ccttggaaag cgggacagat gtatcgatat cgtcaacaat agtaccactt 300
caaatctcta ccgaactgga taccgacgag aaaacttcag gagatacgct aagtatggtt 360
gaagctctct cggaaacgtc gagtatcact ccaacaacgc atcgaccagt acgactttcc 420
gtttcgtcaa atatatcctc acaggagagg gagcggagac tgatgatggt tctggctgcc 480
cgtcgtaatg cattacctcg tgtgagtgtc cgaacacgcc ttggcaccac cggacgatcc 540
tcactcttag agacaacatc taaagcacca agtgatgtac gatcagattg tttaaaaaac 600
tgtacaaaga actttacgcg tcgtacgact gctagttgta tgcgaaagtg ctctaattta 660
acacgtacaa tgaatggtac ggtcatcatt cacgaatcat ctctaaccgg ttcagctacg 720
ctgacctccg atcgagacaa caacgagata ctctttacgg acgaatcttc ccatggacta 780
tttattgttg caaagggaca aaatgataca tcaaatcata tacaagatgt tatgaaaaat 840
gtgaaagtgc ggcctaaaaa taaggattta tcatcagtaa ccattacttc cagttccatt 900
gaggaagatg atatccaacc gtacttgtac ttcggtgaaa aacttccacc tattaagccc 960
ggtacattga caattacttc cgttagcgag ggacttccca ggctattgga gatttctgca 1020
aataaattag gtcctactac tcctacctca cgcgtctctt catcaacttt aaccgctgtt 1080
gagcgcagcc gatcaagata taaatataga gatcgaggat acaactattc tagacgttcg 1140
cctccgcatg cgagtgcaat aacttccgtg gaaactaaga gtggactttc ttttaacaac 1200
gaaagtatag catctttcat caatgaaaat aaagaacaga atatggacca aagagttgtt 1260
ttacaattaa caaaactacc atcagttaca tctacagttg tttcttctac accattatta 1320
ataacggttt ccgacgagag taatgaaact tctgggccta ttcagaccac cacagaattc 1380
actaaaaact ttgtgtctga ttcacccatt attttgtacc ctacaacact tcgcgttcat 1440
tcaaccaacg ctacaatgaa aacatttgtc agaaatacaa ctgtaccaaa tgactcaatc 1500
ataaaaatca cctcttctgc gttcagaaaa ttatcaccag tttcacctac tgttcgacca 1560
agaacagttg atgaagaggg gattagaaac cctgattttc tgaaccaaaa agtaacacaa 1620
gaaattaata gtcacgaaac tgtaaaacca actacaaata atacaaattc aagggccatt 1680
gttaaagagg acattgagaa acttcgagct ctaggcccaa gtctcactca agaaataaaa 1740
aatgatgaaa atatagttat atttcttaac cagtcttctc cagatcgatc aaatggggaa 1800
atagaaatca tcgacaaacc aggtgcaata ttacacccga ctttgaaatc gagacactcc 1860
tatgagaatc caatcactga aacaccatct gtgccgttag agtcatcttc tcccaattca 1920
aatgaagata ttgaaccctc tcttgtaggt gttatagaaa cagccccaat aaaaaaacag 1980
actcagcttg agcaaacgaa tataaataga ggagatatta agtatacttt cgtcgccaat 2040
gatagagaag tatctttaga tatgcggcgc gttaatattg ccaccatggt gctggctgga 2100
atcggaataa tacctctttg tgcacttacc ctttaccttg tgcgatcata catatttcgt 2160
cgcaccgtta aaattgagga agatttcgat gtatgcattg gtgaccaaca gcctattagt 2220
cctgtaaaga aacttgattc gaagtttcaa aacaaaggcg aaaaggaaaa ctatgaaatc 2280
gaccatcacc gatccgatca tccgaaagca attaacaaag ttacctctcg gaaaacaaat 2340
gatggcatgc gatatgatga acaatgctca gtgactagtg agcaagaatt tgatcgtagc 2400
aatattcgcc tgaagagtct actaggtgag ggtaactttg gtcaagtgtg gaaagctgag 2460
gcagataatc ttagcggcca ttttggagct acacgtattg ttgccgttaa aacgatccgg 2520
aaattcagtc ctcaatccag tttgaaagaa gaagcagaca ttatgcgaaa actgggttca 2580
catcaaaacg ttgtgactct gcttggcgtc tgcttagaaa ttgagccaca catgctaatt 2640
atggagtatg caatgcgtgg acgattgttg tcattactgc gcgctgcgcg tagtgctgta 2700
aatattttac cagcctcagt gcctggtggt cgcagttcca cacctctgtc gccacggacg 2760
ctaggtggat ttgcattgga cattgcttgt ggaatggagt acattgctga aaagaggatt 2820
gtacatcgtg atcttgccgc acgcaacgtc cttctggatc ataatggtgt ttgtaaaatt 2880
tgtgattttg gaatgtcaat tgatttagaa ggtgaatgta agcagcaaga ggttgaccta 2940
aagagtgcta acaaaataat aagtagcaat acgaagagga aattagattt cggcagccgt 3000
ttcataataa accattggaa caataacttc aatacaaacc aaatcccaac aaaagatgct 3060
attgaaaaaa gaacccattc tcatgatcac agtcatggac atgctcatga ttcgattaat 3120
cgtaggcctg cattaccaat acgatggatg gccccagaag cgttacaata tcatattttt 3180
acgcatgaga ctgatgtatg ggcttttggc atagtattgt gggaaatatc tacattggga 3240
tctacaccat atgccaatct aacaggacgc gaagttatcc gacgagttcc aaatggtctt 3300
aggccagagc tgccaaagga atcgcgttta gaattttaca atttaatgac tcgatgttgg 3360
cataaggatt cccatttacg accttcgttt tcatatgcga gacaggaaat atctcgttcc 3420
ctccacaaat gggatgagga tgactcggca gaatcggact atatggatgt tagcggattt 3480
agtgaagatc tggaacatgg catggtttat ttcaatcaac gaatatccga gtttgaatgc 3540
gaaatatga 3549
<210> 2
<211> 1182
<212> PRT
<213> Artificial Sequence
<400> 2
Met Met Tyr Asn Gln Lys Trp Ala Arg Thr Ile Glu Leu Ile Val Val
1 5 10 15
Val Leu Leu Phe Thr Val Phe Ser Ser Ser Asn Gly Lys Asn Leu Val
20 25 30
Ile Pro Gln Leu Gly Lys Ser Asp Ile Phe Asn Ser Glu Tyr Glu Arg
35 40 45
Val Gln Gln Lys Ser Met Pro Met Glu Phe Asp Val Ser Glu Ala Glu
50 55 60
Arg Gly Lys Lys Ile Asn Gln Arg Leu Gln Pro Arg Ser Val Lys Tyr
65 70 75 80
Lys Tyr Phe Ala Leu Glu Ser Gly Thr Asp Val Ser Ile Ser Ser Thr
85 90 95
Ile Val Pro Leu Gln Ile Ser Thr Glu Leu Asp Thr Asp Glu Lys Thr
100 105 110
Ser Gly Asp Thr Leu Ser Met Val Glu Ala Leu Ser Glu Thr Ser Ser
115 120 125
Ile Thr Pro Thr Thr His Arg Pro Val Arg Leu Ser Val Ser Ser Asn
130 135 140
Ile Ser Ser Gln Glu Arg Glu Arg Arg Leu Met Met Val Leu Ala Ala
145 150 155 160
Arg Arg Asn Ala Leu Pro Arg Val Ser Val Arg Thr Arg Leu Gly Thr
165 170 175
Thr Gly Arg Ser Ser Leu Leu Glu Thr Thr Ser Lys Ala Pro Ser Asp
180 185 190
Val Arg Ser Asp Cys Leu Lys Asn Cys Thr Lys Asn Phe Thr Arg Arg
195 200 205
Thr Thr Ala Ser Cys Met Arg Lys Cys Ser Asn Leu Thr Arg Thr Met
210 215 220
Asn Gly Thr Val Ile Ile His Glu Ser Ser Leu Thr Gly Ser Ala Thr
225 230 235 240
Leu Thr Ser Asp Arg Asp Asn Asn Glu Ile Leu Phe Thr Asp Glu Ser
245 250 255
Ser His Gly Leu Phe Ile Val Ala Lys Gly Gln Asn Asp Thr Ser Asn
260 265 270
His Ile Gln Asp Val Met Lys Asn Val Lys Val Arg Pro Lys Asn Lys
275 280 285
Asp Leu Ser Ser Val Thr Ile Thr Ser Ser Ser Ile Glu Glu Asp Asp
290 295 300
Ile Gln Pro Tyr Leu Tyr Phe Gly Glu Lys Leu Pro Pro Ile Lys Pro
305 310 315 320
Gly Thr Leu Thr Ile Thr Ser Val Ser Glu Gly Leu Pro Arg Leu Leu
325 330 335
Glu Ile Ser Ala Asn Lys Leu Gly Pro Thr Thr Pro Thr Ser Arg Val
340 345 350
Ser Ser Ser Thr Leu Thr Ala Val Glu Arg Ser Arg Ser Arg Tyr Lys
355 360 365
Tyr Arg Asp Arg Gly Tyr Asn Tyr Ser Arg Arg Ser Pro Pro His Ala
370 375 380
Ser Ala Ile Thr Ser Val Glu Thr Lys Ser Gly Leu Ser Phe Asn Asn
385 390 395 400
Glu Ser Ile Ala Ser Phe Ile Asn Glu Asn Lys Glu Gln Asn Met Asp
405 410 415
Gln Arg Val Val Leu Gln Leu Thr Lys Leu Pro Ser Val Thr Ser Thr
420 425 430
Val Val Ser Ser Thr Pro Leu Leu Ile Thr Val Ser Asp Glu Ser Asn
435 440 445
Glu Thr Ser Gly Pro Ile Gln Thr Thr Thr Glu Phe Thr Lys Asn Phe
450 455 460
Val Ser Asp Ser Pro Ile Ile Leu Tyr Pro Thr Thr Leu Arg Val His
465 470 475 480
Ser Thr Asn Ala Thr Met Lys Thr Phe Val Arg Asn Thr Thr Val Pro
485 490 495
Asn Asp Ser Ile Ile Lys Ile Thr Ser Ser Ala Phe Arg Lys Leu Ser
500 505 510
Pro Val Ser Pro Thr Val Arg Pro Arg Thr Val Asp Glu Glu Gly Ile
515 520 525
Arg Asn Pro Asp Phe Leu Asn Gln Lys Val Thr Gln Glu Ile Asn Ser
530 535 540
His Glu Thr Val Lys Pro Thr Thr Asn Asn Thr Asn Ser Arg Ala Ile
545 550 555 560
Val Lys Glu Asp Ile Glu Lys Leu Arg Ala Leu Gly Pro Ser Leu Thr
565 570 575
Gln Glu Ile Lys Asn Asp Glu Asn Ile Val Ile Phe Leu Asn Gln Ser
580 585 590
Ser Pro Asp Arg Ser Asn Gly Glu Ile Glu Ile Ile Asp Lys Pro Gly
595 600 605
Ala Ile Leu His Pro Thr Leu Lys Ser Arg His Ser Tyr Glu Asn Pro
610 615 620
Ile Thr Glu Thr Pro Ser Val Pro Leu Glu Ser Ser Ser Pro Asn Ser
625 630 635 640
Asn Glu Asp Ile Glu Pro Ser Leu Val Gly Val Ile Glu Thr Ala Pro
645 650 655
Ile Lys Lys Gln Thr Gln Leu Glu Gln Thr Asn Ile Asn Arg Gly Asp
660 665 670
Ile Lys Tyr Thr Phe Val Ala Asn Asp Arg Glu Val Ser Leu Asp Met
675 680 685
Arg Arg Val Asn Ile Ala Thr Met Val Leu Ala Gly Ile Gly Ile Ile
690 695 700
Pro Leu Cys Ala Leu Thr Leu Tyr Leu Val Arg Ser Tyr Ile Phe Arg
705 710 715 720
Arg Thr Val Lys Ile Glu Glu Asp Phe Asp Val Cys Ile Gly Asp Gln
725 730 735
Gln Pro Ile Ser Pro Val Lys Lys Leu Asp Ser Lys Phe Gln Asn Lys
740 745 750
Gly Glu Lys Glu Asn Tyr Glu Ile Asp His His Arg Ser Asp His Pro
755 760 765
Lys Ala Ile Asn Lys Val Thr Ser Arg Lys Thr Asn Asp Gly Met Arg
770 775 780
Tyr Asp Glu Gln Cys Ser Val Thr Ser Glu Gln Glu Phe Asp Arg Ser
785 790 795 800
Asn Ile Arg Leu Lys Ser Leu Leu Gly Glu Gly Asn Phe Gly Gln Val
805 810 815
Trp Lys Ala Glu Ala Asp Asn Leu Ser Gly His Phe Gly Ala Thr Arg
820 825 830
Ile Val Ala Val Lys Thr Ile Arg Lys Phe Ser Pro Gln Ser Ser Leu
835 840 845
Lys Glu Glu Ala Asp Ile Met Arg Lys Leu Gly Ser His Gln Asn Val
850 855 860
Val Thr Leu Leu Gly Val Cys Leu Glu Ile Glu Pro His Met Leu Ile
865 870 875 880
Met Glu Tyr Ala Met Arg Gly Arg Leu Leu Ser Leu Leu Arg Ala Ala
885 890 895
Arg Ser Ala Val Asn Ile Leu Pro Ala Ser Val Pro Gly Gly Arg Ser
900 905 910
Ser Thr Pro Leu Ser Pro Arg Thr Leu Gly Gly Phe Ala Leu Asp Ile
915 920 925
Ala Cys Gly Met Glu Tyr Ile Ala Glu Lys Arg Ile Val His Arg Asp
930 935 940
Leu Ala Ala Arg Asn Val Leu Leu Asp His Asn Gly Val Cys Lys Ile
945 950 955 960
Cys Asp Phe Gly Met Ser Ile Asp Leu Glu Gly Glu Cys Lys Gln Gln
965 970 975
Glu Val Asp Leu Lys Ser Ala Asn Lys Ile Ile Ser Ser Asn Thr Lys
980 985 990
Arg Lys Leu Asp Phe Gly Ser Arg Phe Ile Ile Asn His Trp Asn Asn
995 1000 1005
Asn Phe Asn Thr Asn Gln Ile Pro Thr Lys Asp Ala Ile Glu Lys Arg
1010 1015 1020
Thr His Ser His Asp His Ser His Gly His Ala His Asp Ser Ile Asn
1025 1030 1035 1040
Arg Arg Pro Ala Leu Pro Ile Arg Trp Met Ala Pro Glu Ala Leu Gln
1045 1050 1055
Tyr His Ile Phe Thr His Glu Thr Asp Val Trp Ala Phe Gly Ile Val
1060 1065 1070
Leu Trp Glu Ile Ser Thr Leu Gly Ser Thr Pro Tyr Ala Asn Leu Thr
1075 1080 1085
Gly Arg Glu Val Ile Arg Arg Val Pro Asn Gly Leu Arg Pro Glu Leu
1090 1095 1100
Pro Lys Glu Ser Arg Leu Glu Phe Tyr Asn Leu Met Thr Arg Cys Trp
1105 1110 1115 1120
His Lys Asp Ser His Leu Arg Pro Ser Phe Ser Tyr Ala Arg Gln Glu
1125 1130 1135
Ile Ser Arg Ser Leu His Lys Trp Asp Glu Asp Asp Ser Ala Glu Ser
1140 1145 1150
Asp Tyr Met Asp Val Ser Gly Phe Ser Glu Asp Leu Glu His Gly Met
1155 1160 1165
Val Tyr Phe Asn Gln Arg Ile Ser Glu Phe Glu Cys Glu Ile
1170 1175 1180
<210> 3
<211> 330
<212> DNA
<213> Artificial Sequence
<400> 3
ctgcttggcg tctgcttaga aattgagcca cacatgctaa ttatggagta tgcaatgcgt 60
ggacgattgt tgtcattact gcgcgctgcg cgtagtgctg taaatatttt accagcctca 120
gtgcctggtg gtcgcagttc cacacctctg tcgccacgga cgctaggtgg atttgcattg 180
gacattgctt gtggaatgga gtacattgct gaaaagagga ttgtacatcg tgatcttgcc 240
gcacgcaacg tccttctgga tcataatggt gtttgtaaaa tttgtgattt tggaatgtca 300
attgatttag aaggtgaatg taagcagcaa 330
Claims (4)
1.一种抑制桔小实蝇雌虫生殖发育的方法,其特征在于,靶向沉默桔小实蝇生殖发育相关基因UCP,基因UCP的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法,其特征在于,靶向沉默桔小实蝇生殖发育相关基因UCP的dsRNA由序列如SEQ ID NO.3所述的核苷酸和与其反向互补的核苷酸组成。
3.dsRNA,其特征在于,所述的dsRNA由序列如SEQ ID NO.3所述的核苷酸和与其反向互补的核苷酸组成。
4.权利要求3所述的dsRNA在桔小实蝇防治中的应用。
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