桔小实蝇14-3-3基因的克隆和表达谱分析  

Cloning and expression pattern analysis of 14-3-3 in Bactrocera dorsalis(Hendel)

作　　者：申建梅[1] 胡黎明[1] 宾淑英[1] 林进添[1] 

机构地区：[1]仲恺农业工程学院外来有害生物预警与控制研究所,广东广州510225

出　　处：《西北农林科技大学学报（自然科学版）》2011年第7期97-104,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30971923);仲恺农业工程学院校级科研基金项目(G3100003)

摘　　要：【目的】克隆桔小实蝇14-3-3蛋白的基因(Bactrocera dorsalis14-3-3,Bdor14-3-3),研究Bdor14-3-3mRNA在不同组织和不同发育时期的表达情况,探索其是否参与了桔小实蝇的发育过程。【方法】采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆Bdor14-3-3;采用实时荧光定量PCR(real-time PCR)方法,研究Bdor14-3-3 mRNA在桔小实蝇不同组织及不同发育时期的相对表达量。【结果】克隆获得了Bdor14-3-3基因,其编码区长度为747 bp,编码248个氨基酸残基。氨基酸序列一致性分析表明,在昆虫纲中,桔小实蝇与刺舌蝇14-3-3蛋白的序列一致性最高,为98.8%,与豌豆蚜的序列一致性最低,为85.4%。实时荧光定量PCR分析表明,Bdor14-3-3 mRNA在桔小实蝇不同组织和发育时期都有表达;在雌虫头(去除触角)和翅中的相对表达量均较高,分别是触角的1.39和1.44倍;在雄虫胸和后足中的相对表达量均较高,分别是前足的1.28和1.23倍。在蛹的早期发育过程中Bdor14-3-3 mRNA表达量逐渐升高,到7 d蛹期相对表达量达到最高峰,是10 d蛹的4.91倍。【结论】克隆获得了Bdor14-3-3基因,其表达的14-3-3蛋白可能参与了桔小实蝇的变态发育过程,尤其在蛹的发育过程中Bdor14-3-3可能发挥着重要作用。【Objective】 To determine whether Bdor14-3-3 was involved in its development,cDNA cloning and the expression levels of Bdor14-3-3 were studied.【Method】 Rapid amplification cDNA ends（RACE） method and reverse transcription PCR（RT-PCR） method were used to clone 14-3-3 gene.The temporal and spatial expression levels of Bdor14-3-3 mRNA were investigated by real-time quantitative RT-PCR method.【Result】 The sequencing results showed that the full-length of open reading frame in Bdor14-3-3 is 747 bp in length,encoding 248 amino acid residues.The putative amino acid shared the highest similarity（98.8%）with 14-3-3 from Glossina morsitans,and the lowest sequence identity（85.4%） with 14-3-3 from Acyrthosiphon pisum in insecta.The result of real-time PCR indicated that Bdor14-3-3 expressed in all tissues and the whole developmental stages.The relative expression level of Bdor14-3-3 in female head and wings was about 1.39 and 1.44 folds of that in female antenna,respectively.And the relative expression level of Bdor14-3-3 in male thorax and hind legs was 1.28 and 1.23 folds of that in male front legs,respectively.The relative expression level of Bdor14-3-3 was gradually increased from 1d-old pupa to 7 d-old pupa.Bdor14-3-3 expression level in 7 d-old pupa was about 4.91 times of that in 10 d-old pupa.【Conclusion】 Bdor14-3-3 gene was cloned.Bdor14-3-3 may be involved in the metamorphosis development and played an important role in the development of pupae.

关 键 词：桔小实蝇 14-3-3蛋白 基因克隆 实时定量PCR 

分 类 号：S436.661.2[农业科学—农业昆虫与害虫防治] Q965.9[农业科学—植物保护]