亚洲玉米螟幼虫体内保幼激素二醇激酶cDNA片段克隆与序列分析
Cloning and sequence analysis of cDNA fragment of juvenile hormonediol kinase from the larvae of Asian corn borer,Ostrinia furnacalis
冯从经**， 翟会峰
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作者单位：扬州大学园艺与植物保护学院植物保护系 扬州 225009
中文关键词：亚洲玉米螟，保幼激素二醇激酶，基因克隆，序列分析，半定量PCR
英文关键词：Ostrinia furnacalis，juvenile hormone diol kinase， gene cloning， sequence analysis，semi-quantitative PCR
中文摘要：
为研究亚洲玉米螟Ostrinia furnacalis (Guenée)幼虫体内保幼激素二醇激酶（JHDK）表达调控的分子机理，根据不同昆虫保幼激素二醇激酶基因序列的保守区域，设计合成简并引物，采用RT-PCR技术从亚洲玉米螟5龄幼虫中扩增出一段cDNA片段，大小为189 bp，编码63个氨基酸，预测分子量为6.78 ku，理论等电点pI值为4.57。该基因序列中含有保守的GTP结合蛋白特征指纹基序∑3和∑1。BlastP分析结果表明：该片段氨基酸序列与烟草天蛾JHDK氨基酸序列的一致性最高，为69%；与家蚕和小菜蛾JHDK氨基酸序列的一致性分别为55%和52%。构建系统发育树分析了3种鳞翅目昆虫JHDK进化关系，结果显示：亚洲玉米螟cDNA片段氨基酸序列与家蚕JHDK的亲缘关系最近，与小菜蛾JHDK的亲缘关系最远。半定量PCR结果表明：JHDK基因在中肠中表达量最高，随着5龄幼虫的发育，JHDK基因在血细胞、脂肪体和体壁组织中表达量有下降趋势，但在中肠组织中表达量明显增强。
英文摘要： In order to explore the molecular mechanism controlling the expression and regulation of the juvenile hormone diol kinase (JHDK) in Ostrinia furnacalis (Guenée) larvae,sequences of conserved regions of the JHDK gene from different individuals were used to design degenerate primers and a cDNA fragment of 189 bp,encoding 63 amino acids,was amplified from 5th instar larvae by RT-PCR.This fragment had a predicted molecular weight of 6.78 ku and a pI value of 4.57.Two conserved fingerprint motifs of the GTP-binding proteins ∑3 and ∑1 in the JHDK gene were found.BlastP showed that the amino acid sequence of O.furnacalis JHDK (Of-JHDK) was most similar to that of Manduca sexta JHDK (69%) and its relative similarity to the Bombyx mori and Plutella xylostella JHDK was 55% and 52%.Phylogenetic analysis showed that Of-JHDK was most closely related to B.mori JHDK and least related to P.xylostella JHDK.Semi-quantitative PCR showed that the expression of JHDK in 5th instar larvae was highest in the midgut.JHDK expression increased in the midgut while decreasing in the hemocytes,fat body and integument tissue as larvae developed.