孟氏隐唇瓢虫气味结合蛋白ComOBP1基因的克隆和时空表达
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摘要
采用RT-PCR和RACE技术,从孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant中成功克隆出气味结合蛋白(Com OBP1)基因的全序列(Genbank登陆号:KU170686)。Com OBP1基因全长922 bp,包括5'端长为40 bp的非编码区域(UTR),及3'端长为462 bp的UTR,开放阅读框ORF长为420 bp,编码139个氨基酸,预测的分子量为15.516 k Da,等电点p I为6.57,存在AATAAA加尾信号。N-末端疏水区包含由20个氨基酸构成的信号肽,无跨膜结构,有4个保守的半胱氨酸,属于Minus-C OBP,也是在孟氏隐唇瓢虫中发现的第一个Minus-C OBP。氨基酸序列中有且仅有一个N-糖基化位点为62 NLSA,并存在2个潜在的磷酸化位点。与其它昆虫的Minus-C OBPs进行同源性比较并构建系统发育树,发现与同为鞘翅目Coleoptera昆虫的同源性较高。利用Real-time PCR、RT-PCR技术对Cmon OBP1基因在孟氏隐唇瓢虫不同发育阶段,不同组织,不同营养条件及不同食性下的表达水平进行了测定,结果显示,Cmon OBP1基因在整个发育阶段均有表达,雄性成虫期具有最高表达量,且多在成虫的头部及翅部表达。当营养条件发生变化时,表达丰度不会发生变化,当猎物由天然猎物柑橘粉蚧Planococcus citri Risso变成碗豆修尾蚜Megoura japonica Matsumura时,表达量会明显下降。该结果表明,孟氏隐唇瓢虫的不同发育阶段、不同组织及猎物种类会影响Cmon OBP1基因的表达,从而进一步影响其嗅觉行为。同时,Cmon OBP1基因可能在雄虫相关的信息素感受过程中发挥着重要作用。
The odorant binding protein gene Cmon OBP1( Genbank number: KU170686) from Cryptolaemus montrouzieri Mulsant was cloned by technology of RT-PCR and RACE. The full length was922 bp,containing a 5' UTR( untranslated region) of 40 bp and a 3' UTR of 462 bp. ORF( Open Reading Frame) was 420 bp which encoded a polypeptide of 139 amino acids with an estimated molecular weight of15. 516 k Da and p I of 6. 57. Besides,the sequence had an AATAAA tailing signal. The predicted Nterminal hydrophobic region containing 20 amino residues displayed the characteristic features of a signal peptide,and no transmembrane domain was predicted. Four conservative cysteine residues classified Cmon OBP1 as the first Minus-C OBP which was found in C. montrouzieri. The amino acid sequence had only one N-glycosylation site 62 NLSA,and two potential phosphorylation sites. Compared with other insect Minus-C OBPs,Cmon OPB1 was much identitier to the other Coleoptera. By Real-time PCR and RT-PCR methods,we determined the expression pattern of Cmon OBP1 in different developmental stages,various adult tissues,different larval food regimes and other food. Spatio-temporal expression result showed that Cmon OBP1 expressed in every stage and the male adult had the highest level. In addition,Cmon OPB1 had a higher expression level in adult head and wing than that of other adult tissues. When larval diet changed either poor or rich,the expression level wouldn' t be influenced markedly,while when the food changed from natural prey Planococcus citri Risso to Megoura japonica Matsumura,the expression level decreased significantly. The result suggested that the expression level of Cmon OBP1 gene was development,tissues and prey type dependent,indicating a distinct sensitivity in olfactory. In addition,Cmon OBP1 gene might play an important role in the process of male adult feeling related pheromone.
The odorant binding protein gene Cmon OBP1( Genbank number: KU170686) from Cryptolaemus montrouzieri Mulsant was cloned by technology of RT-PCR and RACE. The full length was922 bp,containing a 5' UTR( untranslated region) of 40 bp and a 3' UTR of 462 bp. ORF( Open Reading Frame) was 420 bp which encoded a polypeptide of 139 amino acids with an estimated molecular weight of15. 516 k Da and p I of 6. 57. Besides,the sequence had an AATAAA tailing signal. The predicted Nterminal hydrophobic region containing 20 amino residues displayed the characteristic features of a signal peptide,and no transmembrane domain was predicted. Four conservative cysteine residues classified Cmon OBP1 as the first Minus-C OBP which was found in C. montrouzieri. The amino acid sequence had only one N-glycosylation site 62 NLSA,and two potential phosphorylation sites. Compared with other insect Minus-C OBPs,Cmon OPB1 was much identitier to the other Coleoptera. By Real-time PCR and RT-PCR methods,we determined the expression pattern of Cmon OBP1 in different developmental stages,various adult tissues,different larval food regimes and other food. Spatio-temporal expression result showed that Cmon OBP1 expressed in every stage and the male adult had the highest level. In addition,Cmon OPB1 had a higher expression level in adult head and wing than that of other adult tissues. When larval diet changed either poor or rich,the expression level wouldn' t be influenced markedly,while when the food changed from natural prey Planococcus citri Risso to Megoura japonica Matsumura,the expression level decreased significantly. The result suggested that the expression level of Cmon OBP1 gene was development,tissues and prey type dependent,indicating a distinct sensitivity in olfactory. In addition,Cmon OBP1 gene might play an important role in the process of male adult feeling related pheromone.
引文
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Gu SH,Sun Y,Ren LY,et al.Cloning,expression and binding characterization analysis of odorant binding protein Alin OBP3 gene from Helicoverpa assulta[J].Chinese Science Bulletin,2010,55(27-28):2690-2701.[谷少华,孙洋,任丽燕,等.苜蓿盲蝽气味结合蛋白Alin OBP3的克隆、表达及结合特性分析[J].科学通报,2010,55(27-28):2690-2701]
Jiang RX,Zhang YH,Wu HS,et al.The color preference of Cryptolaemus montrouzieri Mulsant(Coleoptera:Coccinellidae)on substrates[J].Journal of Environmental Entomology,2011,33(2):213-218.[蒋瑞鑫,张宇宏,吴红胜,等.孟氏隐唇瓢虫对产卵基质的颜色选择行为研究[J].环境昆虫学报,2011,33(2):213-218]
Jayanthi PDK,Rajinikanth R,Ravishankar KV,et al.Influence of cG MP on feeding potential of predatory Coccinellid,Cryptolaemus montrouzieri Mulsant and isolation of partial foraging gene[J].Journal of Insect Behavior,2014,27(3):385-394.
Li J,Du X,Hosseini,et al.The research progress in protein glycosylation modification[J].Bulletin of Science and Technology,2009,25(6):773-778,783.[李军,杜鑫,Hosseini,等.蛋白质糖基化修饰研究进展[J].科技通报,2009,25(6):773-778,783]
Li HS,Jin MJ,S'lipiński A,et al.Genetic differentiation in native and introduced populations of Cryptolaemus montrouzieri(Coleoptera:Coccinellidae)and its implications for biological control programs[J].Journal of Economic Entomology,2015,108(4):743-747.
Li L,Yang WL,Guo XR,et al.Cloning,sequence analysis and spatio-temporal expression of a pheromone binding protein 2(PBP2)gene from Helicoverpa assulta(Guenee)[J].Acta Entomologica Sinica,2009,52(11):1199-1205.[李亮,杨文玲,郭线茹,等.烟夜蛾性信息素结合蛋白2(PBP2)基因的克隆、序列分析与时空表达[J].昆虫学报,2009,52(11):1199-1205]
Liang QJ,Wang PC,Bai YR.Summarization on the progress in protein phosphorylation[J].Science and Technology Review,2012,30(31):73-79.[梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):73-79]
Pang H,Li LY,Tang C.The technique for preservation of Cryptolaemus montrouzieri Mulsant adult with the artificial diet[J].Natural Enemies of Insects,1996,18(2):64-66.[庞虹,李丽英,汤才.孟氏隐唇瓢虫成虫的人工饲料保种技术[J].昆虫天敌,1996,18(2):64-66]
Pang XF,Li LY.Studies on the Cryptolaemus montrouzieri setting control of mealbug harm in Guangzhou and other places[J].Natural Enemies of Insects,1979,1(3):50.[庞雄飞,李丽英.孟氏隐唇瓢虫在广州等地定居控制石粟粉蚧的危害[J].昆虫天敌,1979,1(3):50]
Xie JQ,Wu HS,Zhang YH,et al.A way of artificial breeding,storage and reproduction of Cryptolaemus montrouzieri Mulsant[P].Chinese Patent:201410482952.1,2015-03-04.[谢佳沁,吴红胜,张宇宏,等.一种孟氏隐唇瓢虫人工饲养、保种和繁殖生产方法[P].中国专利:201410482952.1,2015-03-04]
Xie JQ,Patrick DC,Pan C,et al.Larval nutrition-induced plasticity affects reproduction and gene expression of the ladybeetle,Cryptolaemus montrouzieri[J].BMC Evolutionary Biology,2015,15(1):1-9.
Zhang F,Li S,Xiao D,et al.Progress in pest management by natural enemies in greenhouse vegetables in China[J].Scientia Agricultura Sinica,2015,48(17):3463-3476.[张帆,李姝,肖达,等.中国设施蔬菜害虫天敌昆虫应用研究进展[J].中国农业科学,2015,48(17):3463-3476]
Zhou L,Gu JX.N-glycosylation sites analysis and nonconsensus N-glycosylated sequences[J].Chinese Bulletin of Life Sciences,2011,23(6):605-611.[周蕾,顾建新.N-糖基化位点鉴定方法和非经典N-糖基化序列[J].生命科学,2011,23(6):605-611]
Zhang YH,Jiang RX,Wu HS,et al.Next-generation sequencing-based transcriptome analysis of Cryptolaemus montrouzieri under insecticide stress reveals resistance-relevant genes in ladybirds[J].Genomics,2012,100(1):35-41.
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Zhang YH,Zheng JH,Mao RQ,et al.Cloning and characterization analysis of cytochrome P450 CYP9Z401 gene from Cryptolaemus montrouzieri Mulsant[J].Journal of Environmental Entomology,2015,37(4):759-766.[张宇宏,郑基焕,毛润乾,等.孟氏隐唇瓢虫细胞色素P450基因的克隆与序列分析[J].环境昆虫学报,2015,37(4):759-766]