西方蜜蜂RBP1基因的克隆及mRNA表达分析

Cloning and mRNA expression analysis of RBP1 gene in Apis mellifera (Hymenoptera: Apidae)

【目的】RBP1是一种参与黑腹果蝇Drosophila melanogaster性别决定基因doublesex(dsx)premRNA选择性剪接的重要剪接因子。本研究旨在克隆RBP1基因在西方蜜蜂Apis mellifera中的同源基因AmRBP1,分析其结构域及其在西方蜜蜂性别决定初期胚胎和幼虫不同发育阶段中的表达模式,以期为研究该基因是否参与蜜蜂性别决定奠定基础。【方法】基于NCBI中提交的西方蜜蜂基因数据库和转录组数据库,利用黑腹果蝇rbp1对数据库进行同源检索获得RBP1的转录组序列,并设计引物进行PCR扩增克隆AmRBP1基因,利用生物信息学软件对其进行核酸及氨基酸序列分析。采用半定量RT-PCR技术分析该基因在西方蜜蜂胚胎及幼虫期的表达模式。【结果】由序列比对分析可知,西方蜜蜂AmRBP1基因的pre-mRNA含有3种不同的选择性剪切形式。通过设计不同的引物,克隆得到3种成熟的mRNA序列,分别命名为AmRBP1-R1(Gen Bank登录号:KY404154)、AmRBP1-R2(Gen Bank登录号:KY404155)和AmRBP1-R3(Gen Bank登录号:KY404156);mRNA长度分别为696,790和771 nt,编码的蛋白大小分别为130,166和162 aa;结构域分析发现AmRBP1的3种选择性剪切形式具有相同的结构域,氨基端含有一个RRM结构域,羧基端含有一个RS结构域。同源性分析表明,西方蜜蜂AmRBP1与丽蝇蛹集金小蜂Nasonia vitripennis、红火蚁Solenopsis invicta、埃及伊蚊Aedes aegypti、黑腹果蝇D.melanogaster、家蚕Bombyx mori、家蝇Musca domestica和中华按蚊Anopheles sinensis RBP1蛋白氨基酸序列一致性分别为96.97%,94.20%,84.85%,81.48%,80.28%,78.15%和69.23%。半定量RT-PCR结果表明,AmRBP1基因的3种剪切形式(AmRBP1-R1-3)在西方蜜蜂胚胎和幼虫各时期均有表达,无雌特异性或雄特异性表达的剪切形式。【结论】西方蜜蜂AmRBP1可能是一种SR家族剪切因子,其有可能参与调控基因表达的特定剪切,但其是否参与doublesex(dsx)基因pre-mRNA的性别特异性剪切需要进一步研究。

[ Aim] RBPI is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila melanogaster sex-determining gene dsx. This study aims to clone the nueleotide sequence of RBP1 gene AmRBP1 of Apis mellifera, and to analyze the domains of its amino acid sequence and itsmRNA expression profiles in different developmental stages of embryo and larva, so as to provide a basis for studying its function in honeybee sex determination. [ Methods ] Several transcriptome sequences of AmRBP1 were obtained by homology search with Drosophila melanogaster rbpl based on the predicted gene database and the transcriptome database of A. meUifera, and PCR primers were designed based on these transcriptome sequences. Then, AmRBP1 was cloned and its nucleotide and amino acid sequences were analyzed with bioinformatic software. The expression profiles of AmRBP1 in different developmental stages of embryo and larva of A. mellifera were assayed by semi-quantitative RT-PCR. [ Results ] The results showed that AmRBPI has three alternative splicing variants named AmRBP1-R1 （GenBank accession number ： KY404154）, AmRBP1-R2 （ GenBank accession number ： KY404155 ） and AmRBP1 - R3 （GenBank accession number： KY404156）, respectively, whose mRNA sequences are 696, 790 and 771 nt in length, encoding proteins of 130, 166 and 162 aa, respectively. Protein domain prediction indicated that all of the three isoforms contain the same domains, with an amino terminal RRM domain and a carboxyl terminal RS domain. Homology analysis showed that AmRBP1 has 96.97%, 94.20% , 84.85% , 81.48% , 80.28% , 78.15% and 69.23% amino acid sequence identity with RBP1 proteins of Nasonia vitripennis, Solenopsis invicta, Aedes aegypti, D. melanogaster, Bombyx mori, Musca domestica and Anopheles sinensis, respectively. The semi-quantitative RT-PCR results showed that AmRBP1-R1 - 3 were expressed in various developmental stages of embryo and larva, with no obvious sex difference. [ Conclusion] AmRBP1 may be a member of SR family splicing factors with no obvious sex difference, and whether it is involved in the sex-specific splicing of doublesex （dsx） pre-mRNA needs further research.