| 摘要: |
| 【目的】克隆中华豆芫菁（Epicauta chinensis Laporte）β-actin基因全长cDNA序列，并检测其在相对实时定量研究中作为内参基因的可靠性。【方法】采用RT PCR和RACE技术扩增中华豆芫菁β-actin基因全长cDNA序列，利用生物信息学软件对其进行序列分析；并用实时定量PCR方法检测在中华豆芫菁雄性成虫脑、中肠、精巢和脂肪体4种不同组织中及注射刺激和非刺激条件下β-actin基因的表达量。【结果】中华豆芫菁β-actin基因全长1 673 bp，其中5′非编码区88 bp，3′非编码区455 bp，开放阅读框为1 120 bp，编码376个氨基酸，推测其编码蛋白分子质量约为93.6 ku，理论等电点为5.09，富含6类特定功能位点，其氨基酸序列与其他昆虫的β-actin序列一致性可达97%～99%；实时定量PCR结果显示,该基因在中华豆芫菁不同组织、注射刺激与非刺激情况下表达量无显著差异(P>0.05)。【结论】中华豆芫菁β-actin基因可作为基因表达定量研究中的可靠内参。该基因的cDNA序列已递交GenBank,获得登录号为JQ764814。 |
| 关键词: 中华豆芫菁 β-actin基因 反转录PCR RACE技术 实时定量PCR |
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| 基金项目:公益性行业（农业）科研专项（200903052)；中国博士后科学基金项目（20090451405） |
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| Molecular cloning,sequence analysis and expression detection of β-actin gene in the blister beetle Epicauta chinensis Laporte |
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| Abstract: |
| 【Objective】This research cloned the full-length cDNA of β-actin from Epicauta chinensis Laporte to prove the reliability of this gene as an internal control gene in gene quantitative analysis.【Method】β-actin gene was cloned using RT-PCR and RACE technique and the sequence was analyzed by bioinformatics software.Moreover,the real-time PCR was performed to detect the expression of β-actin in four different tissues of E.chinensis with and without being injected.【Result】The results showed that the cDNA had 1 673 base pairs (bp) in length and contained a 5′-untranslated region (5′-UTR) with 88 bp and a 3′-UTR with 455 bp.The open reading frame (ORF) of 1 120 bp encoded 376 amino acid residues with a predicted molecular weight of 93.6 ku and an isoeletric point value of 5.09.The predicted protein contained 6 kinds of functional sites and was 97%-99% idential with β-actin from other insects.Quantitative assays indicated that there was no significant difference(P>0.05)between either the 4 different tissues or the injected and uninjected bodies.【Conclusion】β-actin is a reliable internal control gene in gene quantitative analysis of E.chinensis.The β-actin cDNA has been submitted to GenBank and the accession number is JQ764814. |
| Key words: Epicauta chinensis Laporte β-actin reverse transcription PCR RACE technology RT-PCR |
