中华蜜蜂CREB基因的克隆及表达

doi:10.3864/j.issn.0578-1752.2016.08.017

摘要/Abstract

摘要： 【目的】克隆中华蜜蜂（Apis cerana cerana）的cAMP反应原件结合蛋白（cAMP response element binding protein，CREB)基因全长cDNA序列,预测该基因及其编码蛋白的理化性质，并解析中华蜜蜂CREB在大脑中的表达特征，为研究该基因在中华蜜蜂学习记忆过程中的生物学功能提供基础。【方法】以中华蜜蜂为试验材料，解剖其大脑获得脑组织，提取大脑组织的总RNA。在此基础上，利用RT-PCR技术克隆中华蜜蜂大脑的CREB基因(AcCREB)，然后用DNAstar软件中的Seqman程序将CREB正反向测序后的序列拼接获得CREB基因的cDNA序列，其氨基酸序列通过Bioedit软件按照六框翻译而成；用BLASTn和BLASTp进行序列比对分析；用ClustalX进行多重序列比对和同源性比较；用MEGA软件的邻接法（neighbor-joining）构建系统进化树，明确其系统进化关系。并通过SMART数据库分析AcCREB的结构域。另外，将解剖好的蜜蜂大脑制成石蜡切片，并采用地高辛原位杂交技术分析AcCREB在中华蜜蜂大脑中的分布表达情况。【结果】中华蜜蜂CREB基因的cDNA全序列全长890 bp，编码240个氨基酸残基，序列提交到GenBank（登录号为KC814690）。其编码的蛋白预测分子质量为25.691 kD，等电点为5.82。结构域分析结果显示，该蛋白具有AA 89—131的PKID和AA 176—237的BRLA两个保守结构域。氨基酸序列比对结果显示，中华蜜蜂与西方蜜蜂、熊蜂、切叶蜂、大黄蜂、印度跳蚁、家蚕、埃及伊蚊、疟原虫、人类、小鼠10个物种的同源蛋白相似度分别为98.76%、98.35%、97.11%、94.09%、72.22%、58.97%、44.69%、41.69%、36.89%、36.89%。系统发育树显示该基因编码的蛋白质与西方蜜蜂的CREB亲缘关系最近，与熊蜂、大黄蜂之间的亲缘关系次之。另外，经过地高辛原位杂交技术处理后，在中蜂大脑蘑菇体的凯尼恩细胞、触角叶周围的细胞和大脑的髓质与小叶之间的细胞均有阳性着色，而且AcCREB在中华蜜蜂工蜂左右大脑的阳性着色不对称。【结论】AcCREB的氨基酸序列与西方蜜蜂、熊蜂、切叶蜂、大黄蜂高度同源，且基因在大脑中的分布暗示其可能参与了中华蜜蜂学习记忆过程。

关键词: 中华蜜蜂, CREB基因, 克隆, 表达分析

Abstract: 【Objective】 The objective of this study is to clone the full-length cDNA sequence of the cAMP response element binding protein gene of Apis cerana cerana (AcCREB), predict the gene and its physicochemical properties of the corresponding encoded protein, and then clarify its expression in the brain tissues, which will provide a research basis of the physiological function of AcCREB in the learning and memory. 【Method】Total RNA was extracted from the dissected brain tissues of A. cerana cerana (A.c.c.) as the main experimental materials. On this basis, the AcCREB was cloned using RT-PCR technique. The forward and reverse sequences were then spliced by Bioedit to obtain the full-length cDNA using the Seqman of DNAstar software, and its amino acid sequences were translated using Bioedit software according to the six-box translation. The sequences was analyzed using BLASTn and BLASTp. Multiple sequence alignment and homology were analyzed using ClustalX. A phylogenetic tree was constructed using neighbor-joining method of software MEGA. The conserved structural domains were deduced by analyzing the SMART database. In addition, the paraffin sections of brain were made based on the dissected brain tissues, and the distribution of its expression in the brain was investigated by in situ hybridization histochemistry. 【Result】The full length of AcCREB cDNA is 890 bp, which encodes 240 amino acids. The sequence was submitted to GenBank (Accession number KC814690). The molecular weight and an isoelectric point of AcCREB protein were 25.691 kD and 5.82, respectively. Structural domain analysis indicated that the encoded protein contained two highly conserved structures of AA 89-131 PKID and AA 176-237 BRLA. Homology comparison illustrated that AcCREB has a similarity with CREB of Apis mellifera (AmCREB), Bombus impatiens (BiCREB), Megachile rotundata (MrCREB), Bombus terrestris (BtCREB), Harpegnathos saltator (HsCREB), Bombyx mori (BmCREB), Aedes aegypt (AaCREB),Anopheles gambiae (AgCREB), Homo sapiens (HsCREB)and Mus musculus (MmCREB), with the similarity of 98.76%, 98.35%, 97.11%, 94.09%, 72.22%, 58.97%, 44.69%, 41.69%, 36.89%, and 36.89%, respectively. Meanwhile, the phylogenetic tree analysis showed that AcCREB was first clustered with AmCREB, BiCREB, BtCREB. Moreover, in situ hybridization showed that AcCREB staining was mainly localized to the Kenyon cells of the mushroom bodies, cells around the antennal lobes, cells between medulla and lobula, and display asymmetrical expression between the right and left brain of worker bees in A.c.c..【Conclusion】The amino sequences of AcCREB own high homology with AmCREB, BiCREB, MrCREB, BtCREB, and the distribution of the gene in the brains of honeybee suggested that AcCREB may be involved in the process of learning and memory of the A. c. c. which provides useful information for future studies of CREB in the leaning-memory of honeybees.

Key words: Apis cerana cerana, cAMP response element binding protein gene, molecular cloning, expression analysis

张丽珍，王子龙，曾志将. 中华蜜蜂CREB基因的克隆及表达[J]. 中国农业科学, 2016, 49(8): 1599-1606.

ZHANG Li-zhen, WANG Zi-long, ZENG Zhi-jiang. Cloning and Expression of cAMP Response Element Binding Protein (CREB) Gene of Apis cerana cerana[J]. Scientia Agricultura Sinica, 2016, 49(8): 1599-1606.

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