小峰熊蜂可溶型海藻糖酶基因的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2015.02.17

摘要/Abstract

摘要： 【目的】克隆小峰熊蜂（Bombus hypocrita）可溶型海藻糖酶基因（soluble trehalase，Tre-1）全长cDNA序列，预测该基因及其编码蛋白的理化性质，明确该基因在小峰熊蜂不同组织及不同发育时期的表达特性，为研究该基因在小峰熊蜂生长发育中的生物学功能奠定基础。【方法】根据地熊蜂（B. terrestris）和B. impatiens可溶型海藻糖酶基因Tre-1编码蛋白的保守区序列，利用Primer Premier 5.0设计简并引物扩增得到小峰熊蜂保守区片段；随后根据该片段设计基因特异性引物，用RACE方法获得5'端和3'端片段。在此基础上，根据RACE扩增所得片段和保守序列，预测开放阅读框（ORF）序列，分别在起始密码子近5'端和终止密码子的近3'端设计ORF区特异性引物扩增得到ORF，最后采用BioEdit软件比对、拼接获得小峰熊蜂Tre-1 cDNA全长序列。运用ExPASy、SignalP 4.1、NetOGlyc 1.0 sever、ClustalW和MEGA 5.0等软件对该基因进行生物信息学分析，并利用实时荧光定量PCR技术，采用2^-^ΔΔCt方法检测不同组织及不同发育时期可溶型海藻糖酶基因的相对表达量。【结果】克隆所得基因cDNA全长为3 129 bp，命名为BhTre-1（GenBank登录号：KJ025078），其中包含5'端441 bp非编码区和3'端945 bp非编码区，ORF为1 743 bp，共编码580个氨基酸。其编码的蛋白预测分子质量为67.16 kD，等电点为5.95；有1个信号肽结构（1—21位），1个甘氨酸富集区（GGGGEY），2个特色“标签序列”（PGGRFKEFYYWDSY和QWDFPNAWPP），6个Asn-Xaa-Ser/Thr（N-X-S/T）序列，无跨膜区。序列分析发现BhTre-1氨基酸序列与地熊蜂BtTre-1和B. impatiens BiTre-1的一致性很高，分别为99%和98%，与西方蜜蜂（Apis mellifera）和云南小蜜蜂（A. florea）的Tre-1一致性也达到78%；系统发育分析也表明BhTre-1与BtTre-1、BiTre-1的亲缘关系最近，与AmTre-1、AfTre-1之间的亲缘关系次之。基因定量分析结果显示BhTre-1在成虫被检测的各组织中均有表达，在中肠的表达量最高，其次是马氏管，其他各组织表达量较低。成年工蜂的BhTre-1表达量高于幼虫和蛹，工蜂出房后随着龄期的增加表达量呈现先升高后降低的规律，在第15天表达量最高。【结论】克隆得到了BhTre-1全长cDNA序列，其分子生物学特性与其他昆虫Tre-1相似，该基因在小峰熊蜂中肠表达量最高，此外，幼虫和蛹的表达量低于成年工蜂，工蜂出房后表达量呈现先升高后降低的趋势，为进一步研究该基因在小峰熊蜂体内的生物学功能奠定了基础。

关键词: 小峰熊蜂, 可溶型海藻糖酶基因, 克隆, 序列分析, 基因表达

Abstract: 【Objective】The objective of this study is to clone full-length cDNA sequence of the soluble trehalase gene (Tre-1) in Bombus hypocrita, predict the physicochemical properties, clarify its expression in different tissues and developmental stages, and to understand the function of this gene in the development of B. hypocrita.【Method】 Degenerate primers were designed by using Primer Premier 5.0 software according to the conservative sequences of Tre-1 in B. terrestris and B. impatiens to get the corresponding conservative fragment in B. hypocrita. Gene-specific primers were designed according to the conserved fragment. The rapid-amplification of cDNA ends (RACE) method was used to clone B. hypocrita 5′ and 3′ sequences, then the open reading frame (ORF) was amplified by the specific primers based on the initiation codon and termination codon near 5′ and 3′, respectively. Different fragments were spliced by BioEdit to obtain the full-length cDNA. The full-length cDNA was analyzed by a variety of bioinformatics softwares such as ExPASy, SignalP 4.1, NetOGlyc 1.0 sever, ClustalW and MEGA 5.0. Finally, the relative expression of Tre-1 in different tissues and developmental stages in B. hypocrita were analyzed by real-time PCR and 2^-^ΔΔCt method.【Result】The full-length cDNA of B. hypocrita Tre-1 is 3 129 bp, and designated as BhTre-1 (GenBank number: KJ025078). The bioinformatics analysis suggested that BhTre-1 has an ORF of 1 743 bp, a 5′ (untranslated region) UTR of 441 bp and a 3′ UTR of 945 bp. The ORF of BhTre-1 encodes a polypeptide of 580 amino acids with a predicted molecular weight of 67.16 kD, and an isoeletric point value of 5.95. Thededuced amino acid sequence has six predicted sequences of Asn-Xaa-Ser/Thr, a signature peptide, a highly conserved glycine-rich region (GGGGEY), and two conserved signature motifs (PGGRFKEFYYWDSY and QWDFPNAWPP), but no transmembrane domain. Homology comparison found that BhTre-1 has the greatest similarity to B【Conclusion】The cDNA sequence of BhTre-1 was successfully cloned from B. hypocrita, and the properties were similar with Tre-1 of other insects. BhTre-1 hasthe highest expression in midgut. In addition, the expression of BhTre-1 of adult worker was higher than at larva and pupa stages,and the expression of BhTre-1 increased first and then decreased. This study indicated that the expression patterns in different tissues and developmental stages of B. hypocrita, which will lay a foundation for biological functional research of BhTre-1.. terrestris BtTre-1 (99%)and B. impatiens BiTre-1 (98%), it also has greater similarity to Tre-1 of Apis mellifera and A. florea which have 78% sequence homology. The phylogenetic tree analysis showed that BhTre-1 was first clustered with BtTre-1, BiTre-1, AmTre-1 and AfTre-1. The results agreed with the sequence homology analysis. Tissue-specific expression results indicated that BhTre-1was expressed in all the major tissues, it had the highest expression in midgut, then Malpighian tubules, and lower expression in other tissues. The expression of BhTre-1 in adult worker was higher than at larva and pupa stages, and it increased from the 1st day after emergence, and had the maximum expression in the 15-day-old adults, then, it decreased with the age.

Key words: Bombus hypocrita, soluble trehalase gene, clone, sequence analysis, gene expression

秦加敏，罗术东，廖秀丽，黄家兴，和绍禹，吴杰. 小峰熊蜂可溶型海藻糖酶基因的克隆及表达分析[J]. 中国农业科学, 2015, 48(2): 370-380.

QIN Jia-min, LUO Shu-dong, LIAO Xiu-li, HUANG Jia-xing, HE Shao-yu, WU Jie. Molecular Cloning and Expression Analysis of a Soluble Trehalase Gene Tre-1 in Bombus hypocrita[J]. Scientia Agricultura Sinica, 2015, 48(2): 370-380.

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