CN111378670B - 一种分离的中黑盲蝽Taiman基因及其编码的蛋白 - Google Patents
一种分离的中黑盲蝽Taiman基因及其编码的蛋白 Download PDFInfo
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Abstract
本发明涉及昆虫基因工程技术领域。具体涉及一种分离的中黑盲蝽Taiman基因及其编码的蛋白。所述基因的核苷酸序列如SEQ ID No:1所示。将本发明的Taiman基因的dsRNA通过显微注射至中黑盲蝽初羽化雌虫体内,检测发现Taiman基因表达量在整个生育期均被极显著抑制,抑制Taiman基因的表达显著减少了中黑盲蝽卵巢内卵的数量和中黑盲蝽的终生产卵量,最终影响了中黑盲蝽的生殖能力和种群的发展。Taiman基因可作为抗中黑盲蝽转基因植物培育的备选基因。
Description
技术领域
本发明涉及昆虫基因工程技术领域。具体涉及一种分离的中黑盲蝽Taiman基因及其编码的蛋白。
背景技术
中黑盲蝽(Adelphocorissuturalis)属于半翅目盲蝽科苜蓿盲蝽属,是一种高度杂食性害虫,广泛的分布于日本、中国、朝鲜以及前苏联,中黑盲蝽的寄主范围非常广泛,其主要危害棉花,大豆,苜蓿等,1997年转基因Bt棉花商业化种植以来,很好的控制了靶标鳞翅目害虫的危害,但对于非靶标害虫中黑盲蝽逐渐由次要害虫上升为主要害虫,并呈进一步蔓延和大面积灾变的发展趋势。目前对于盲蝽科害虫的防治主要是以化学防治为主,由于中黑盲蝽危害较为隐蔽,活动迅速敏捷,且容易转移寄主危害,严重影响化学药剂的防治效果。由于长期大量使用化学农药带来的环境压力以及抗药性等问题,开发和利用符合环保、健康、持续发展理念的无公害防治措施成为了当前的防治热点。
科学研究发现,保幼激素在昆虫***和生殖方面起到关键作用,保幼激素能够促进昆虫卵黄发生和卵子的生成。保幼激素的分子作用依赖于由两种bHLH-PAS转录因子组成的细胞内受体复合物(Methoprenetolerant,Met)和Taiman(Tai)。Tai/Met异源二聚体在保幼激素信号通路中起到关键调控作用。越来越多的化学农药的使用导致害虫抗药性的增强,寻找科学环保的杀虫策略迫在眉睫。随着科学技术的发展,更多的通过转基因植物来达到防治害虫的手段受到人们的关注,也是科学环保杀虫的重要战略。Taiman基因对昆虫的生殖调控可以应用到转基因植物对中黑盲蝽防治中来,促进绿色环保、科学治虫理念的发展。
发明内容
本发明的目的是提供的中黑盲蝽Taiman基因。
本发明的再一目的是提供上述中黑盲蝽Taiman基因所编码的蛋白。
本发明的再一目的是提供含有上述中黑盲蝽Taiman基因的重组质粒。
本发明的再一目的是提供含有上述中黑盲蝽Taiman基因的重组细胞。
本发明的再一目的是提供抗中黑盲蝽的转基因植物。
本发明的再一目的是提供提高植物中黑盲蝽抗性的方法。
根据本发明的中黑盲蝽Taiman基因,其核苷酸序列如SEQIDNo:1所示。
SEQIDNO:1ATGTCGACGCTTCTCGCCGAAAATGCAGGGTTGGTCCGATGTGAGCTGCCATCAGACCCCCTGTGGGCTAAGATGAGCTCTACCGGAGCCAGCTCTAAGAAGCGAAAGAAACCCGACACAAAACCCCAGTCTCAACTCGCCAAAAGCCTTAATGAAAAACGTAGGCGTGAGCTTGAAAACGTGTTCATCGAAGAGCTGGCTGAACTCATATCGGCGAGCTTTGCTGATATGAGCTCGCTCGCTGTCAAGCCCGACAAGTGCGCTATACTTCAAGAAACTGTAAACCAGATCAAGCACATAAAAGAGCAAGAGGCCGCAGCGACGATAGACGCAGTTCAACAGGGAGAAGTTTCCTCGTCGAAACCTACCATCCTCACTAACGACTTTTTTGGTCCACTTCTGTTAGAGGCTCTCGAAGGTTTCCTTTTCGTAGTCAACCAAGACGGCAAAGTAGAGTACGTGACTGATAACATCGCCACGTTCATCAAATTCTCGAAAGACGAGGTTTTGGGAAAGCCCGTCTATAACATCCTTCACCACGGGGACCATGGCCGTTTCAACTCCGCCCTCCTGCCTACGGTTTGGTCCACTCTTGAAGGCTCCTCCTCGTCTGTCGTAGCCTCAGGAGGTGGTTCTTCCTCCAATTCGTCGAACCCTCCGCGGAATCGAACTTTCAACTGCCGATTCCTCATCAAACCTCCTGACGACTCGAACCAAACCGTCGAGGAGAAACAGCAGAGGGTATCGAAGTATGAGAATATGCAAGTTGTCCAGATCAATTCGACGCAAATCGCTCAATTGACATCAGGCGGCGACAAGGACGAAGAGGGCAACGATATCGGTCCGTGCCTCATGTGCGTGGCTCGGCGGATTATGCCGAACGAGAAACATCACGCGGGGACGTCCATGGAACAGTTTTCAATGAAAATGGACCCCCAAGGGAAGATCATCGGAATAGACACGACGGGGTTGTCGCCAGGGTTATCCCAATTCATCGTCAAGAAAGATCTCCTTGGAAGAAATCTCGACGACATAGTTCATGCTGGCGATTTTAGCAAAGTATCGAATCATGTGAAAGACGTTCAATCCCATTCGCATGGAACTTCATCTATCTACAGGATACGGGTGACTTCACCGAACTCCGGTGACAAATACATTCACGTGCAGACTAAGTCCAAGCTTTTCAAATCAATGTCTGAGCCGGACTTCATAATGTCTACCCACTCCATCGTTACCGCGGGTGGAGAAGAGCAGTCATTCAATATGGTGGAAGCGAATTCGCCTGCGTCTATAAGCGGGCAGAGCACATCCAGCTCTATGGGAAACCAAATGATGTTGTCTGGAACTGTCAACGGTGCTCCTATTAGAAGTGAAAACACCCCTAGTAGCGTGAGTTCTTCAGAGCTGGCAAACACCATCAACTCACTCAGCAATACCTCCAATTTTCAAACTGTGTCTCTGAACCAGGGTCTGGTTTCCAACGAACTTAGTCACGATATCACTTTCGCAGACCTGTTTCCTTCGACTACTTGGCCTGACATTGGTGAACGGATCGGAACGGGGTTGGCCGAACGAGGAAGTTGGGAAAGATCCGAATCCCGTGCTTCCATGACATCAGCATCTAGTCCATTAGCGCCTGCCCCCGCCACACCATCCTTCTCGAACTCCAATTTCCCCTTCTCGCCTCCCGTCATGGACGAAAAGGAGTCGAATGAAGCGCAAGGAGGCGGCAATCCCTGCCGGCTAAGGAACCTCCTGACTGAAGGGCTTGGAGCGATAAAGAGAGAATCGACAGACTCAGAGTCTGGAGCCGGTGGCGGGGGCAATAGCTCTCAGGAACATCCTGGTGAGAAAAACAAGTACAGCCTCCTTACCAAAATCCTGAATGAAAAGGAAGAAGACCAGCAGGGGTCGGGACACCATTCAGAGCAGTCATCTCACAACAAGCCTAAGAACAACATGTTACTGCAGCTGTTGAACAAAAACGACGATGACGATATGGAGCAGAAGGGAGGAGCGAGTGATGATCTTCTGCTCAATAATTTGGGGCTTGGCTCCCTGACCTCAACGCAGGCTGAGACCATCAGACGAGGCACGAAGCGCCCAAGCTCTGAAGACAACCAGGGAATGGGGAGTGGTGGAAACACTCCACAGGGTGGTGATGATGGTCCGAACTCGGCCAAACGACCTGCCAACTCCCTTTTGGACACTGTACCTCCGTCTGCGCCAGTTTCATCTTCTTCAGTCAACTCGAAGCTGTGGGAGAAAAACCGGATGTTGGCCTCGTTACTGGCAAACCCTCCATCCACTCCCACCACAATCCCTCCTGTACCTGCCAGTGTCATTTCCGCCACGCCTCAGGACAAGTTACCACGAGTTATTAAAAATAATGCCCAAAATTCTTGGTCTGGCTGTAACGTTCAACAAGGAGGCGGGATTCCTCAATCTCCTCAGGGAGCTGGAGGTCAGGCTATTGGGCGACAGGGAACTGGGCAAACCCTTCATTCCGTTTCTTCACCTGACTTCCGGTGGGCCGGTGAAGACTCATCGGACCCTTACCTCTCTCATCTGTTAGATCAAGTGATAGAAATAGCTCCCGAAGATTCCCCTGCTCTAGCTAACATAATAAATTCTATTGAAGCAAACTCCCAAGGGGTTTTGGGTGGAAGCCCTTTCCAGCAAGATCAACTCACCGCTGAAAAGATCAACGAACGGATGGCAATTAATGCCATTCAAAAGTCGCTTATGCAATGCGAGACGTTGCAAAAAACACCTGTTTATACTGTTTCTCAGCTATCCTCCGTTCAAGGACAGCAACAATTTCCTCCTCCACCTGTTTATCAACAGAATAGGCAACGATTGACCTTATCTGCTGGTCTGAGGCAAGCACAGTACAACCTTGGACCTCAACAACAGCAACTGTTGATACAGCAGCAGCAACAGCGGACTAAACTGTTGCAACAACAAACTAAGCAGCGGTTGTTACAGTTGCAACAGCAACAACAGCTGCTAATTCCTTCTAATGCTGCTGCCACATCGGGGGATCCTTCCGGAATCCATAATATTGACTCTCTTATGAATAACACGGTTGCGCCCAACGTTTCTCTTCAGAGGTCATCTAGCGTTCCTGATTCCCAGCTGTCGCCAGTCGGAAGCTATAGTGGCTCTGGTGCCAGCACGCCCAACATCATCGGATCTGGGAGCTCACAGATATCACCTGGCCAGAGGCAACCCTTCTCTCCGCAACCTTTCTCTCCTGTAAATGGTGGTATGAATAGTTTCCAAGGAAGTGGAGGGAGTGGAGGTTCAACTACTCCTAACCAGAGTGGGCAAGTTGTAACCTCACAAGGAGCGCAGCAGGCTCGTATGAGTCCTAGTTCGCTTCAGAATTTCCAGCAGGCTCAACTGTCCCCTCGGCTTTCACAGGGACAAACGGCAGGTTATGTTCAGACCCAGAGCACTTCATGGTCCCAACAACAAGCCAATAGGATCTCAATTCAACAACAACAAAACCCTATGCTCAATGCCCAGCTCACTGGCGGCAGTTATAACTCCAGTGGTGCTAGAAACTACAACCCTGGAACCCCTCCTGGTGCTGGGGGTGGAACGCCTCCCACCTCTGCAGCAGGCAGTGGCAGTCGTACAGGTCTGCCACCCGTCCGGTCTCTAACCAGTCCAGGATCACGGCAGAGTCCGTTCCCTCCCGAACTGTCACCAACGGCAGCAGGGAGTGCCACCAACTACCAGTTTAGAATCCAAAGGTCACTTTCAGGACCAACCCACCAGTCAGTCGTTCCTCAAGCAACCACTCATCTTCCAGGAGGTCCAATAGGAGGAGGTGGATGCAGCCGGATGTACAAGGAGCAGGTCCCTTACCACCACCACTACCATCCCCCCCATGTGACCCATCAACACCCCCAACATCCTCTCATTTACCAACCACACCCCACGGACCAGGGTTATCCTGGCCAGGGATACCCTGATGGCTACAGACAACACCCGACGGGTAAGTTCCCTGTCCTCCAGGTACACGGACACAAACACGACCCCCACCTTCCGACACCATAG
根据本发明的上述中黑盲蝽Taiman基因所编码的蛋白,其氨基酸序列如SEQIDNo:2所示。
MSTLLAENAGLVRCELPSDPLWAKMSSTGASSKKRKKPDTKPQSQLAKSLNEKRRRELENVFIEELAELISASFADMSSLAVKPDKCAILQETVNQIKHIKEQEAAATIDAVQQGEVSSSKPTILTNDFFGPLLLEALEGFLFVVNQDGKVEYVTDNIATFIKFSKDEVLGKPVYNILHHGDHGRFNSALLPTVWSTLEGSSSSVVASGGGSSSNSSNPPRNRTFNCRFLIKPPDDSNQTVEEKQQRVSKYENMQVVQINSTQIAQLTSGGDKDEEGNDIGPCLMCVARRIMPNEKHHAGTSMEQFSMKMDPQGKIIGIDTTGLSPGLSQFIVKKDLLGRNLDDIVHAGDFSKVSNHVKDVQSHSHGTSSIYRIRVTSPNSGDKYIHVQTKSKLFKSMSEPDFIMSTHSIVTAGGEEQSFNMVEANSPASISGQSTSSSMGNQMMLSGTVNGAPIRSENTPSSVSSSELANTINSLSNTSNFQTVSLNQGLVSNELSHDITFADLFPSTTWPDIGERIGTGLAERGSWERSESRASMTSASSPLAPAPATPSFSNSNFPFSPPVMDEKESNEAQGGGNPCRLRNLLTEGLGAIKRESTDSESGAGGGGNSSQEHPGEKNKYSLLTKILNEKEEDQQGSGHHSEQSSHNKPKNNMLLQLLNKNDDDDMEQKGGASDDLLLNNLGLGSLTSTQAETIRRGTKRPSSEDNQGMGSGGNTPQGGDDGPNSAKRPANSLLDTVPPSAPVSSSSVNSKLWEKNRMLASLLANPPSTPTTIPPVPASVISATPQDKLPRVIKNNAQNSWSGCNVQQGGGIPQSPQGAGGQAIGRQGTGQTLHSVSSPDFRWAGEDSSDPYLSHLLDQVIEIAPEDSPALANIINSIEANSQGVLGGSPFQQDQLTAEKINERMAINAIQKSLMQCETLQKTPVYTVSQLSSVQGQQQFPPPPVYQQNRQRLTLSAGLRQAQYNLGPQQQQLLIQQQQQRTKLLQQQTKQRLLQLQQQQQLLIPSNAAATSGDPSGIHNIDSLMNNTVAPNVSLQRSSSVPDSQLSPVGSYSGSGASTPNIIGSGSSQISPGQRQPFSPQPFSPVNGGMNSFQGSGGSGGSTTPNQSGQVVTSQGAQQARMSPSSLQNFQQAQLSPRLSQGQTAGYVQTQSTSWSQQQANRISIQQQQNPMLNAQLTGGSYNSSGARNYNPGTPPGAGGGTPPTSAAGSGSRTGLPPVRSLTSPGSRQSPFPPELSPTAAGSATNYQFRIQRSLSGPTHQSVVPQATTHLPGGPIGGGGCSRMYKEQVPYHHHYHPPHVTHQHPQHPLIYQPHPTDQGYPGQGYPDGYRQHPTGKFPVLQVHGHKHDPHLPTP
本发明还提供了包含上述中黑盲蝽Taiman基因的重组质粒。
本发明还提供了包含上述中黑盲蝽Taiman基因的重组细胞,例如含有上述中黑盲蝽Taiman基因的重组植物细胞,昆虫细胞,真菌等。
本发明还提供了抗中黑盲蝽的转基因植物,所述转基因植物含有黑盲蝽Taiman基因的干扰序列。
根据本发明的具体实施方式,所述黑盲蝽Taiman基因的干扰序列通过使用以下特异性引物扩增而得:
上游引物序列dsTAI-F:5’GCACAGTACAACCTTGGACC3’,
下游引物序列dsTAI-R:5’GTTGAACCTCCACTCCCTCC3’。
根据本发明的提高植物中黑盲蝽抗性的方法,包括向植物中导入黑盲蝽Taiman基因的干扰序列的步骤。
将本发明的Taiman基因的dsRNA通过显微注射至中黑盲蝽初羽化雌虫体内,检测发现Taiman基因表达量在整个生育期均被极显著抑制,抑制Taiman基因的表达显著减少了中黑盲蝽卵巢内卵的数量和中黑盲蝽的终生产卵量,最终影响了中黑盲蝽的生殖能力和种群的发展。Taiman基因可作为抗中黑盲蝽转基因植物培育的备选基因。
附图说明
图1显示注射Taiman基因干扰序列的dsRNA后,Taiman基因的沉默效率。其中“**”表示p<0.01。图中英文缩写含义:“dsTAI”表示注射Taiman基因dsRNA的处理组;“dsGFP”表示注射GFP基因dsRNA的对照组.
图2显示注射Taiman基因干扰序列的dsRNA对中黑盲蝽生殖能力的影响,A图:注射Taiman基因干扰序列的dsRNA对中黑盲蝽卵巢内卵数量的影响;B图:注射Taiman基因干扰序列的dsRNA对中黑盲蝽终生产卵量的影响。“**”表示差异极显著性:“**”P<0.01。图中英文缩写含义:“dsTAI”表示注射Taiman基因干扰序列dsRNA的处理组;“dsGFP”表示注射GFP基因dsRNA的对照组。
具体实施方式
实施方式中所使用的技术,包括TRIzol法提取RNA、cDNA克隆、PCR扩增与检测和合成dsRNA等分子生物学技术,均为本领域内技术人员已知的常规技术。所使用的仪器设备、试剂等均为本领域技术人员可通过公关途径或商业途径获得。
实施例1:中黑盲蝽Taiman基因的克隆与分析
TRIzol法提取中黑盲蝽RNA,使用RTMasterMix(perfectrealtime)试剂盒将提取的总RNA合成cDNA模板(具体步骤按照试剂盒说明书)。设计引物序列如下。
上游引物序列TAI-F:5'-CGCTTATGCAATGCGAGACG-3',
下游引物序列TAI-R:5'-GCCAGTGAGCTGGGCATTGA-3'。
以中黑盲蝽cDNA为模板,利用上述引物TAI-F和TAI-R,进行PCR扩增。回收的PCR产物与pEASY-T1载体进行连接,重组载体转化入感受态细胞T1,氨苄抗性LB培养基过夜培养。培养过夜后,挑取8个阳性克隆进行PCR验证,将菌落PCR扩增为阳性的克隆取新鲜菌液送测序。
Taiman基因开放阅读框架全长4068bp,编码1355个氨基酸残基(SEQIDNO:2),预测分子质量为144.9kDa,理论等电点为4.73。
实施例2:合成dsRNA
根据实施例1中得到的Taiman基因序列,设计特异性扩增引物(5'-端加上T7
启动子序列),用于Taiman基因的dsRNA片段的扩增,设计的特异性引物如下:
上游引物序列dsTAI-F:GCACAGTACAACCTTGGACC,
下游引物序列dsTAI-R:GTTGAACCTCCACTCCCTCC。
以中黑盲蝽cDNA为模板,利用上述引物dsTAI-F和dsTAI-R进行PCR扩增,将PCR产物连接到pEASY-T1载体。挑取菌落PCR检测为阳性的单菌落,送测序,检测基因序列的正确性,对测序正确的单菌落,6mlLB+AMP培养基过夜摇菌培养。
提取含有目的片段的质粒,以该质粒为模板,使用上述特异性引物dsTAI-F和dsTAI-R进行第二次PCR扩增,PCR体系和反应程序同上。
将第二次PCR的产物利用酚氯仿抽提法纯化,合成dsRNA,将dsRNA浓度稀释为10μg/μl备用。
实施例3注射Taiman基因dsRNA后基因的沉默效率及其对雌虫卵巢内卵的数量和生殖力变化情况
以绿色荧光蛋白基因(GFP)双链dsRNA作为对照,将Taiman基因dsRNA通过显微注射法从中黑盲蝽后胸与腹部节间膜的最外侧注射进入新羽化雌虫体内。
如图1所示,沉默效率检测:分别收集注射处理后4天、6天、8天、10天、12天、14天、16天和18天的中黑盲蝽全虫,提取RNA并反转录为cDNA后,使用日本Takara公司的PremixExTaqTMII和Bio-RadDetectioniQ2System检测Taiman基因的沉默效应。
卵巢内卵数量统计:注射dsRNA11天后,利用体式显微镜解剖雌虫卵巢,每个处理解剖20头未交配雌虫,3个生物学重复,观察卵巢形态,统计并分析中黑盲蝽卵巢内卵的数量。
生殖力:雌虫注射dsRNA后,与新羽化的雄虫一一配对,放入一次性塑料杯内(5cm×7cm)交配,若发现雄虫死亡,马上补充新的性成熟的雄虫,以40对虫子为一个处理组,3个生物学重复,统计终生产卵量,以此评价注射Taiman基因dsRNA对中黑盲蝽产卵量的影响。
试验结果显示:
(1)Taiman沉默效率
与对照组相比,注射Taiman基因的dsRNA后,Taiman基因表达量在整个生育期显著下降,说明该中黑盲蝽Taiman基因的干扰序列可显著抑制Taiman基因的表达。
(2)注射Taiman基因dsRNA对中黑盲蝽雌虫卵巢内卵数量的影响
注射处理11天后,解剖雌虫卵巢,统计卵巢内卵的数量并观察卵巢形态,与对照组相比,注射Taiman基因dsRNA处理组的中黑盲蝽卵巢内卵的数量减少了44.34%(见图2中的A图)。说明注射Taiman基因的dsRNA可显著减少了中黑盲蝽雌虫卵巢内卵的数量。
(3)注射Taiman基因dsRNA对中黑盲蝽生殖能力的影响
新羽化雌虫注射dsRNA处理后与雄虫交配,统计终生产卵量,与对照组相比,处理组的中黑盲蝽产卵量下降了75.5%(见图2中的B图)。可见将Taiman基因干扰后,显著抑制了中黑盲蝽卵巢内卵的数量与雌虫生殖力,对中黑盲蝽的生殖能力和种群的发展产生了不利影响。因此本发明提供的RNA干扰序列可应用于转基因抗中黑盲蝽植物的开发。进一步开发其蛋白可应用于中黑盲蝽的生物防治。
序列表
<110> 华中农业大学
<120> 一种分离的中黑盲蝽Taiman基因及其编码的蛋白
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4068
<212> DNA
<213> 中黑盲蝽(Adelphocoris suturalis)
<400> 1
atgtcgacgc ttctcgccga aaatgcaggg ttggtccgat gtgagctgcc atcagacccc 60
ctgtgggcta agatgagctc taccggagcc agctctaaga agcgaaagaa acccgacaca 120
aaaccccagt ctcaactcgc caaaagcctt aatgaaaaac gtaggcgtga gcttgaaaac 180
gtgttcatcg aagagctggc tgaactcata tcggcgagct ttgctgatat gagctcgctc 240
gctgtcaagc ccgacaagtg cgctatactt caagaaactg taaaccagat caagcacata 300
aaagagcaag aggccgcagc gacgatagac gcagttcaac agggagaagt ttcctcgtcg 360
aaacctacca tcctcactaa cgactttttt ggtccacttc tgttagaggc tctcgaaggt 420
ttccttttcg tagtcaacca agacggcaaa gtagagtacg tgactgataa catcgccacg 480
ttcatcaaat tctcgaaaga cgaggttttg ggaaagcccg tctataacat ccttcaccac 540
ggggaccatg gccgtttcaa ctccgccctc ctgcctacgg tttggtccac tcttgaaggc 600
tcctcctcgt ctgtcgtagc ctcaggaggt ggttcttcct ccaattcgtc gaaccctccg 660
cggaatcgaa ctttcaactg ccgattcctc atcaaacctc ctgacgactc gaaccaaacc 720
gtcgaggaga aacagcagag ggtatcgaag tatgagaata tgcaagttgt ccagatcaat 780
tcgacgcaaa tcgctcaatt gacatcaggc ggcgacaagg acgaagaggg caacgatatc 840
ggtccgtgcc tcatgtgcgt ggctcggcgg attatgccga acgagaaaca tcacgcgggg 900
acgtccatgg aacagttttc aatgaaaatg gacccccaag ggaagatcat cggaatagac 960
acgacggggt tgtcgccagg gttatcccaa ttcatcgtca agaaagatct ccttggaaga 1020
aatctcgacg acatagttca tgctggcgat tttagcaaag tatcgaatca tgtgaaagac 1080
gttcaatccc attcgcatgg aacttcatct atctacagga tacgggtgac ttcaccgaac 1140
tccggtgaca aatacattca cgtgcagact aagtccaagc ttttcaaatc aatgtctgag 1200
ccggacttca taatgtctac ccactccatc gttaccgcgg gtggagaaga gcagtcattc 1260
aatatggtgg aagcgaattc gcctgcgtct ataagcgggc agagcacatc cagctctatg 1320
ggaaaccaaa tgatgttgtc tggaactgtc aacggtgctc ctattagaag tgaaaacacc 1380
cctagtagcg tgagttcttc agagctggca aacaccatca actcactcag caatacctcc 1440
aattttcaaa ctgtgtctct gaaccagggt ctggtttcca acgaacttag tcacgatatc 1500
actttcgcag acctgtttcc ttcgactact tggcctgaca ttggtgaacg gatcggaacg 1560
gggttggccg aacgaggaag ttgggaaaga tccgaatccc gtgcttccat gacatcagca 1620
tctagtccat tagcgcctgc ccccgccaca ccatccttct cgaactccaa tttccccttc 1680
tcgcctcccg tcatggacga aaaggagtcg aatgaagcgc aaggaggcgg caatccctgc 1740
cggctaagga acctcctgac tgaagggctt ggagcgataa agagagaatc gacagactca 1800
gagtctggag ccggtggcgg gggcaatagc tctcaggaac atcctggtga gaaaaacaag 1860
tacagcctcc ttaccaaaat cctgaatgaa aaggaagaag accagcaggg gtcgggacac 1920
cattcagagc agtcatctca caacaagcct aagaacaaca tgttactgca gctgttgaac 1980
aaaaacgacg atgacgatat ggagcagaag ggaggagcga gtgatgatct tctgctcaat 2040
aatttggggc ttggctccct gacctcaacg caggctgaga ccatcagacg aggcacgaag 2100
cgcccaagct ctgaagacaa ccagggaatg gggagtggtg gaaacactcc acagggtggt 2160
gatgatggtc cgaactcggc caaacgacct gccaactccc ttttggacac tgtacctccg 2220
tctgcgccag tttcatcttc ttcagtcaac tcgaagctgt gggagaaaaa ccggatgttg 2280
gcctcgttac tggcaaaccc tccatccact cccaccacaa tccctcctgt acctgccagt 2340
gtcatttccg ccacgcctca ggacaagtta ccacgagtta ttaaaaataa tgcccaaaat 2400
tcttggtctg gctgtaacgt tcaacaagga ggcgggattc ctcaatctcc tcagggagct 2460
ggaggtcagg ctattgggcg acagggaact gggcaaaccc ttcattccgt ttcttcacct 2520
gacttccggt gggccggtga agactcatcg gacccttacc tctctcatct gttagatcaa 2580
gtgatagaaa tagctcccga agattcccct gctctagcta acataataaa ttctattgaa 2640
gcaaactccc aaggggtttt gggtggaagc cctttccagc aagatcaact caccgctgaa 2700
aagatcaacg aacggatggc aattaatgcc attcaaaagt cgcttatgca atgcgagacg 2760
ttgcaaaaaa cacctgttta tactgtttct cagctatcct ccgttcaagg acagcaacaa 2820
tttcctcctc cacctgttta tcaacagaat aggcaacgat tgaccttatc tgctggtctg 2880
aggcaagcac agtacaacct tggacctcaa caacagcaac tgttgataca gcagcagcaa 2940
cagcggacta aactgttgca acaacaaact aagcagcggt tgttacagtt gcaacagcaa 3000
caacagctgc taattccttc taatgctgct gccacatcgg gggatccttc cggaatccat 3060
aatattgact ctcttatgaa taacacggtt gcgcccaacg tttctcttca gaggtcatct 3120
agcgttcctg attcccagct gtcgccagtc ggaagctata gtggctctgg tgccagcacg 3180
cccaacatca tcggatctgg gagctcacag atatcacctg gccagaggca acccttctct 3240
ccgcaacctt tctctcctgt aaatggtggt atgaatagtt tccaaggaag tggagggagt 3300
ggaggttcaa ctactcctaa ccagagtggg caagttgtaa cctcacaagg agcgcagcag 3360
gctcgtatga gtcctagttc gcttcagaat ttccagcagg ctcaactgtc ccctcggctt 3420
tcacagggac aaacggcagg ttatgttcag acccagagca cttcatggtc ccaacaacaa 3480
gccaatagga tctcaattca acaacaacaa aaccctatgc tcaatgccca gctcactggc 3540
ggcagttata actccagtgg tgctagaaac tacaaccctg gaacccctcc tggtgctggg 3600
ggtggaacgc ctcccacctc tgcagcaggc agtggcagtc gtacaggtct gccacccgtc 3660
cggtctctaa ccagtccagg atcacggcag agtccgttcc ctcccgaact gtcaccaacg 3720
gcagcaggga gtgccaccaa ctaccagttt agaatccaaa ggtcactttc aggaccaacc 3780
caccagtcag tcgttcctca agcaaccact catcttccag gaggtccaat aggaggaggt 3840
ggatgcagcc ggatgtacaa ggagcaggtc ccttaccacc accactacca tcccccccat 3900
gtgacccatc aacaccccca acatcctctc atttaccaac cacaccccac ggaccagggt 3960
tatcctggcc agggataccc tgatggctac agacaacacc cgacgggtaa gttccctgtc 4020
ctccaggtac acggacacaa acacgacccc caccttccga caccatag 4068
<210> 2
<211> 1355
<212> PRT
<213> 中黑盲蝽(Adelphocoris suturalis)
<400> 2
Met Ser Thr Leu Leu Ala Glu Asn Ala Gly Leu Val Arg Cys Glu Leu
1 5 10 15
Pro Ser Asp Pro Leu Trp Ala Lys Met Ser Ser Thr Gly Ala Ser Ser
20 25 30
Lys Lys Arg Lys Lys Pro Asp Thr Lys Pro Gln Ser Gln Leu Ala Lys
35 40 45
Ser Leu Asn Glu Lys Arg Arg Arg Glu Leu Glu Asn Val Phe Ile Glu
50 55 60
Glu Leu Ala Glu Leu Ile Ser Ala Ser Phe Ala Asp Met Ser Ser Leu
65 70 75 80
Ala Val Lys Pro Asp Lys Cys Ala Ile Leu Gln Glu Thr Val Asn Gln
85 90 95
Ile Lys His Ile Lys Glu Gln Glu Ala Ala Ala Thr Ile Asp Ala Val
100 105 110
Gln Gln Gly Glu Val Ser Ser Ser Lys Pro Thr Ile Leu Thr Asn Asp
115 120 125
Phe Phe Gly Pro Leu Leu Leu Glu Ala Leu Glu Gly Phe Leu Phe Val
130 135 140
Val Asn Gln Asp Gly Lys Val Glu Tyr Val Thr Asp Asn Ile Ala Thr
145 150 155 160
Phe Ile Lys Phe Ser Lys Asp Glu Val Leu Gly Lys Pro Val Tyr Asn
165 170 175
Ile Leu His His Gly Asp His Gly Arg Phe Asn Ser Ala Leu Leu Pro
180 185 190
Thr Val Trp Ser Thr Leu Glu Gly Ser Ser Ser Ser Val Val Ala Ser
195 200 205
Gly Gly Gly Ser Ser Ser Asn Ser Ser Asn Pro Pro Arg Asn Arg Thr
210 215 220
Phe Asn Cys Arg Phe Leu Ile Lys Pro Pro Asp Asp Ser Asn Gln Thr
225 230 235 240
Val Glu Glu Lys Gln Gln Arg Val Ser Lys Tyr Glu Asn Met Gln Val
245 250 255
Val Gln Ile Asn Ser Thr Gln Ile Ala Gln Leu Thr Ser Gly Gly Asp
260 265 270
Lys Asp Glu Glu Gly Asn Asp Ile Gly Pro Cys Leu Met Cys Val Ala
275 280 285
Arg Arg Ile Met Pro Asn Glu Lys His His Ala Gly Thr Ser Met Glu
290 295 300
Gln Phe Ser Met Lys Met Asp Pro Gln Gly Lys Ile Ile Gly Ile Asp
305 310 315 320
Thr Thr Gly Leu Ser Pro Gly Leu Ser Gln Phe Ile Val Lys Lys Asp
325 330 335
Leu Leu Gly Arg Asn Leu Asp Asp Ile Val His Ala Gly Asp Phe Ser
340 345 350
Lys Val Ser Asn His Val Lys Asp Val Gln Ser His Ser His Gly Thr
355 360 365
Ser Ser Ile Tyr Arg Ile Arg Val Thr Ser Pro Asn Ser Gly Asp Lys
370 375 380
Tyr Ile His Val Gln Thr Lys Ser Lys Leu Phe Lys Ser Met Ser Glu
385 390 395 400
Pro Asp Phe Ile Met Ser Thr His Ser Ile Val Thr Ala Gly Gly Glu
405 410 415
Glu Gln Ser Phe Asn Met Val Glu Ala Asn Ser Pro Ala Ser Ile Ser
420 425 430
Gly Gln Ser Thr Ser Ser Ser Met Gly Asn Gln Met Met Leu Ser Gly
435 440 445
Thr Val Asn Gly Ala Pro Ile Arg Ser Glu Asn Thr Pro Ser Ser Val
450 455 460
Ser Ser Ser Glu Leu Ala Asn Thr Ile Asn Ser Leu Ser Asn Thr Ser
465 470 475 480
Asn Phe Gln Thr Val Ser Leu Asn Gln Gly Leu Val Ser Asn Glu Leu
485 490 495
Ser His Asp Ile Thr Phe Ala Asp Leu Phe Pro Ser Thr Thr Trp Pro
500 505 510
Asp Ile Gly Glu Arg Ile Gly Thr Gly Leu Ala Glu Arg Gly Ser Trp
515 520 525
Glu Arg Ser Glu Ser Arg Ala Ser Met Thr Ser Ala Ser Ser Pro Leu
530 535 540
Ala Pro Ala Pro Ala Thr Pro Ser Phe Ser Asn Ser Asn Phe Pro Phe
545 550 555 560
Ser Pro Pro Val Met Asp Glu Lys Glu Ser Asn Glu Ala Gln Gly Gly
565 570 575
Gly Asn Pro Cys Arg Leu Arg Asn Leu Leu Thr Glu Gly Leu Gly Ala
580 585 590
Ile Lys Arg Glu Ser Thr Asp Ser Glu Ser Gly Ala Gly Gly Gly Gly
595 600 605
Asn Ser Ser Gln Glu His Pro Gly Glu Lys Asn Lys Tyr Ser Leu Leu
610 615 620
Thr Lys Ile Leu Asn Glu Lys Glu Glu Asp Gln Gln Gly Ser Gly His
625 630 635 640
His Ser Glu Gln Ser Ser His Asn Lys Pro Lys Asn Asn Met Leu Leu
645 650 655
Gln Leu Leu Asn Lys Asn Asp Asp Asp Asp Met Glu Gln Lys Gly Gly
660 665 670
Ala Ser Asp Asp Leu Leu Leu Asn Asn Leu Gly Leu Gly Ser Leu Thr
675 680 685
Ser Thr Gln Ala Glu Thr Ile Arg Arg Gly Thr Lys Arg Pro Ser Ser
690 695 700
Glu Asp Asn Gln Gly Met Gly Ser Gly Gly Asn Thr Pro Gln Gly Gly
705 710 715 720
Asp Asp Gly Pro Asn Ser Ala Lys Arg Pro Ala Asn Ser Leu Leu Asp
725 730 735
Thr Val Pro Pro Ser Ala Pro Val Ser Ser Ser Ser Val Asn Ser Lys
740 745 750
Leu Trp Glu Lys Asn Arg Met Leu Ala Ser Leu Leu Ala Asn Pro Pro
755 760 765
Ser Thr Pro Thr Thr Ile Pro Pro Val Pro Ala Ser Val Ile Ser Ala
770 775 780
Thr Pro Gln Asp Lys Leu Pro Arg Val Ile Lys Asn Asn Ala Gln Asn
785 790 795 800
Ser Trp Ser Gly Cys Asn Val Gln Gln Gly Gly Gly Ile Pro Gln Ser
805 810 815
Pro Gln Gly Ala Gly Gly Gln Ala Ile Gly Arg Gln Gly Thr Gly Gln
820 825 830
Thr Leu His Ser Val Ser Ser Pro Asp Phe Arg Trp Ala Gly Glu Asp
835 840 845
Ser Ser Asp Pro Tyr Leu Ser His Leu Leu Asp Gln Val Ile Glu Ile
850 855 860
Ala Pro Glu Asp Ser Pro Ala Leu Ala Asn Ile Ile Asn Ser Ile Glu
865 870 875 880
Ala Asn Ser Gln Gly Val Leu Gly Gly Ser Pro Phe Gln Gln Asp Gln
885 890 895
Leu Thr Ala Glu Lys Ile Asn Glu Arg Met Ala Ile Asn Ala Ile Gln
900 905 910
Lys Ser Leu Met Gln Cys Glu Thr Leu Gln Lys Thr Pro Val Tyr Thr
915 920 925
Val Ser Gln Leu Ser Ser Val Gln Gly Gln Gln Gln Phe Pro Pro Pro
930 935 940
Pro Val Tyr Gln Gln Asn Arg Gln Arg Leu Thr Leu Ser Ala Gly Leu
945 950 955 960
Arg Gln Ala Gln Tyr Asn Leu Gly Pro Gln Gln Gln Gln Leu Leu Ile
965 970 975
Gln Gln Gln Gln Gln Arg Thr Lys Leu Leu Gln Gln Gln Thr Lys Gln
980 985 990
Arg Leu Leu Gln Leu Gln Gln Gln Gln Gln Leu Leu Ile Pro Ser Asn
995 1000 1005
Ala Ala Ala Thr Ser Gly Asp Pro Ser Gly Ile His Asn Ile Asp Ser
1010 1015 1020
Leu Met Asn Asn Thr Val Ala Pro Asn Val Ser Leu Gln Arg Ser Ser
1025 1030 1035 1040
Ser Val Pro Asp Ser Gln Leu Ser Pro Val Gly Ser Tyr Ser Gly Ser
1045 1050 1055
Gly Ala Ser Thr Pro Asn Ile Ile Gly Ser Gly Ser Ser Gln Ile Ser
1060 1065 1070
Pro Gly Gln Arg Gln Pro Phe Ser Pro Gln Pro Phe Ser Pro Val Asn
1075 1080 1085
Gly Gly Met Asn Ser Phe Gln Gly Ser Gly Gly Ser Gly Gly Ser Thr
1090 1095 1100
Thr Pro Asn Gln Ser Gly Gln Val Val Thr Ser Gln Gly Ala Gln Gln
1105 1110 1115 1120
Ala Arg Met Ser Pro Ser Ser Leu Gln Asn Phe Gln Gln Ala Gln Leu
1125 1130 1135
Ser Pro Arg Leu Ser Gln Gly Gln Thr Ala Gly Tyr Val Gln Thr Gln
1140 1145 1150
Ser Thr Ser Trp Ser Gln Gln Gln Ala Asn Arg Ile Ser Ile Gln Gln
1155 1160 1165
Gln Gln Asn Pro Met Leu Asn Ala Gln Leu Thr Gly Gly Ser Tyr Asn
1170 1175 1180
Ser Ser Gly Ala Arg Asn Tyr Asn Pro Gly Thr Pro Pro Gly Ala Gly
1185 1190 1195 1200
Gly Gly Thr Pro Pro Thr Ser Ala Ala Gly Ser Gly Ser Arg Thr Gly
1205 1210 1215
Leu Pro Pro Val Arg Ser Leu Thr Ser Pro Gly Ser Arg Gln Ser Pro
1220 1225 1230
Phe Pro Pro Glu Leu Ser Pro Thr Ala Ala Gly Ser Ala Thr Asn Tyr
1235 1240 1245
Gln Phe Arg Ile Gln Arg Ser Leu Ser Gly Pro Thr His Gln Ser Val
1250 1255 1260
Val Pro Gln Ala Thr Thr His Leu Pro Gly Gly Pro Ile Gly Gly Gly
1265 1270 1275 1280
Gly Cys Ser Arg Met Tyr Lys Glu Gln Val Pro Tyr His His His Tyr
1285 1290 1295
His Pro Pro His Val Thr His Gln His Pro Gln His Pro Leu Ile Tyr
1300 1305 1310
Gln Pro His Pro Thr Asp Gln Gly Tyr Pro Gly Gln Gly Tyr Pro Asp
1315 1320 1325
Gly Tyr Arg Gln His Pro Thr Gly Lys Phe Pro Val Leu Gln Val His
1330 1335 1340
Gly His Lys His Asp Pro His Leu Pro Thr Pro
1345 1350 1355
Claims (4)
1.中黑盲蝽Taiman基因,其特征在于,所述基因的核苷酸序列如SEQ ID No:1所示。
2.权利要求1所述的中黑盲蝽Taiman基因所编码的蛋白。
3.包含权利要求1所述的中黑盲蝽Taiman基因的重组质粒。
4.权利要求1所述中黑盲蝽Taiman基因的干扰序列用于抑制中黑盲蝽Taiman基因表达的应用,其特征在于,所述中黑盲蝽Taiman基因的干扰序列通过使用以下特异性引物扩增而得:
上游引物序列dsTAI-F:5’GCACAGTACAACCTTGGACC3’,
下游引物序列dsTAI-R:5’GTTGAACCTCCACTCCCTCC3’。
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CN110483631A (zh) * | 2019-08-07 | 2019-11-22 | 华中农业大学 | 中黑盲蝽生殖相关蛋白PG及其编码基因、dsRNA干扰序列和应用 |
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PVF1, a PDGF/VEGF homolog, is sufficient to guide border cells and interacts genetically with Taiman;Jocelyn A. McDonald 等;《Development》;20030831;第130卷(第15期);第3469-3478页 * |
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