中华豆芫菁β-actin基因cDNA的克隆、序列分析及表达量检测  

Molecular cloning,sequence analysis and expression detection of β-actin gene in the blister beetle Epicauta chinensis Laporte

作　　者：霍棠[1] 姜鸣[1] 吕淑敏[1] 张雅林[1] 

机构地区：[1]西北农林科技大学植保资源与病虫害防治教育部重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2012年第11期97-102,111,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：公益性行业(农业)科研专项(200903052);中国博士后科学基金项目(20090451405)

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cD-NA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 673bp,其中5′非编码区88bp,3′非编码区455bp,开放阅读框为1 120bp,编码376个氨基酸,推测其编码蛋白分子质量约为93.6ku,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。【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（P0.05）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.

关 键 词：中华豆芫菁 Β-ACTIN基因 反转录PCR RACE技术 实时定量PCR 

分 类 号：Q78[生物学—分子生物学]