| 摘要: |
| 【目的】克隆桔小实蝇14-3-3蛋白的基因（Bactrocera dorsalis 14-3-3,Bdor 14-3-3），研究Bdor 14-3-3 mRNA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Bdor 14-3-3；采用实时荧光定量PCR（real-time PCR）方法，研究Bdor 14-3-3 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Bdor 14-3-3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，Bdor 14-3-3 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Bdor 14-3-3 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Bdor 14-3-3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Bdor 14-3-3可能发挥着重要作用。 |
| 关键词: 桔小实蝇 14-3-3蛋白 基因克隆 实时定量PCR |
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| 基金项目: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) |
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| Cloning and expression pattern analysis of 14-3-3 in Bactrocera dorsalis（Hendel） |
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| Abstract: |
| 【Objective】To determine whether Bdor 14-3-3 was involved in its development,cDNA cloning and the expression levels of Bdor 14-3-3 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 Bdor 14-3-3 mRNA were investigated by real-time quantitative RT-PCR method.【Result】The sequencing results showed that the full-length of open reading frame in Bdor 14-3-3 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 Bdor 14-3-3 expressed in all tissues and the whole developmental stages.The relative expression level of Bdor 14-3-3 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 Bdor 14-3-3 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 Bdor 14-3-3 was gradually increased from 1d-old pupa to 7 d-old pupa.Bdor 14-3-3 expression level in 7 d-old pupa was about 4.91 times of that in 10 d-old pupa.【Conclusion】Bdor 14-3-3 gene was cloned.Bdor 14-3-3 may be involved in the metamorphosis development and played an important role in the development of pupae. |
| Key words: Bactrocera dorsalis (Hendel) 14-3-3 protein gene cloning real-time PCR |
