西方蜜蜂发育基因AmWnt1的克隆鉴定及时空表达分析

本研究旨在克隆鉴定西方蜜蜂Apis mellifera发育相关基因AmWnt1，分析其在不同发育时期和刚出房工蜂不同组织的表达特征，为进一步研究Wnt1基因功能提供理论参考。根据NCBI中AmWnt1基因序列信息，利用Primer 6.0设计引物，RT-PCR扩增AmWnt1基因完整的CDS序列，进行生物信息学预测，用推导的氨基酸序列构建系统进化树；利用荧光定量PCR检测该基因在卵（1日龄、2日龄和3日龄）、幼虫（1日龄、3日龄和5日龄）、预蛹（1日龄和3日龄）、蛹（0日龄、2日龄、4日龄、6日龄和8日龄）、刚出房工蜂、哺育蜂和采集蜂以及刚出房工蜂8个组织中相对表达量。克隆获得西方蜜蜂的Wnt1基因CDS序列，命名为AmWnt1，上传NCBI，获得GenBank登录号MT993937。全长1 239 bp，编码412个氨基酸，预测等电点为9.48，相对分子质量为46.40313 kDa。序列比对和系统进化树结果表明：AmWnt1蛋白与其它膜翅目昆虫聚为一类，其中和东方蜜蜂Apis cerana亲缘关系最近，序列相似度为99.50%。时空表达谱结果表明：AmWnt1基因在各个时期中均有表达，且在胚胎后期表达量最高，预蛹期和蛹前期表达量相对较高，其它时期表达量相对较低；AmWnt1基因在刚出房工蜂头、胸、触角表达量高于其它组织。AmWnt1可能参与西方蜜蜂胚胎晚期的神经系统发育和化蛹过程中的四肢发育等关键历程，为进一步研究AmWnt1功能提供了理论参考。

Cloning and spatio-temporal expression of the developmental gene AmWnt1 in Apis mellifera (Hymenoptera: Apoidea)

The objective of this study is to clone and identify the AmWnt1 gene related to development from Apis mellifera, and to analyze its gene expression levels in different developmental stages and tissues in newly emerged workers, which will provide a theoretical reference for further study of the roles of AmWnt1. According to the sequence information of AmWnt1 gene in NCBI, specific primers were designed using Primer 6.0 and the complete CDS sequence of AmWnt1 gene was amplified via RT-PCR. This sequence was analysed by bioinformatics, and its deduced amino acid sequence was used to construct the phylogenetic tree. The relative expression of this gene was investigated in different development stages and 8 tissues of newly emerged workers by fluorescence quantitative PCR, including eggs (1-day-old, 2-day-old, 3-day-old), larvae (1-day-old, 3-day-old, 5-day-old), pre-pupa (1-day-old and 3-day-old), pupa (0-day-old, 2-day-old, 4-day-old, 6-day-old, 8-day-old), newly emerged workers, nurses, foragers.The Wnt1 CDS sequence of A. mellifera, which was submitted to NCBI (GenBank accession number MT993937), was cloned and named AmWnt1. The total length was 1 239 bp, encoding 412 amino acids. Predictive analysis showed that the isoelectric point and the relative molecular weight were 9.48 and 46.40313 kDa, respectively. Sequence alignment and phylogenetic tree analysis showed that AmWnt1 was clustered into one group with the sequence of other Hymenoptera insects, of which had the closest relationship with Apis cerana and the sequence similarity was 99.50%. The spatio-temporal expression profiles of AmWnt1 showed that it was expressed in all developmental stages, with the highest level in the late embryonic development and a relative higher expression in pre-pupa stage; the expression of AmWnt1 gene was higher in the head, thorax and antennae than other tissues. AmWnt1 may play a crucial role in the nervous system development during the late embryonic stages and limb development during pupation embryogenesis, providing reference for further study on the function of AmWnt1.