兰州熊蜂气味受体家族鉴定及分析

doi:10.3864/j.issn.0578-1752.2017.10.015

摘要/Abstract

摘要： 【目的】了解兰州熊蜂（Bombus lantschouensis）基因组中气味受体基因（odorant receptors, Ors）情况，为进一步分析气味受体功能提供信息，从而为研究该基因家族在熊蜂觅食、交尾、通讯等行为中的重要功能打下基础。【方法】提取兰州熊蜂胸部基因组DNA，进行Illumina高通量测序，对测序所得原始序列进行质控并拼接获得基因组序列，然后使用本地blast 2.2.28+对构建兰州熊蜂基因组本地数据库，使用已知的地熊蜂（Bombus terrestris）和意大利蜜蜂（Apis mellifera ligustica）的气味受体蛋白序列作为种子序列搜索数据库获得兰州熊蜂的气味受体序列；使用EMBOSS1.5对气味受体蛋白序列进行基本理化分析，利用GSDS2.0对家族序列的内含子与外显子位置进行分析，然后使用MEME 4.11.2对氨基酸序列进行保守基序分析，最后利用ClustaW 2.1、TrimAl 1.2、PhyML3.0对兰州熊蜂、地熊蜂和意大利蜜蜂气味受体家族序列进行系统进化分析。【结果】共获得165个兰州熊蜂气味受体基因，包括1个非典型气味受体（olfactory receptor co-receptor，Orco）、5个假基因和159个气味受体。基因结构分析发现，气味受体家族外显子数目从4个到9个不等。其中Or 47—57的序列中外显子数量最少，为4个；Or 128—161中外显子的数量最多，为9个。根据基因结构的不同，将气味受体分为10大类型，每个类型中的序列都有相似的外显子长度与数量。Or 1—38、Or 69—85、Or 128—164这3大类所包含成员数较多（分别为38、17、37），其他7类包含成员个数都约10个，且每个类型中序列在染色体上都呈串联排布，其中Or 1—38中都包含一个较长的第一外显子。保守基序分析发现，在检索的10个保守基序中，除基序5为未知基序外，其他9个基序均包含在其保守结构域（7tm_6 domain）中。 Or 1—38与Or 39—46多数成员包含全部10个保守基序，基序2、3、4、9广泛存在于该家族成员序列中，这4个基序可能为该家族关键的功能区域。系统发育分析将Ors分为5个亚家族（I—V），其中亚家族II包含2种基因结构类型序列（BlOr 97—100与BlOr 69—85），亚家族V包含4种（BlOr 1—46、47—57、86—95和101—107）。BlOr 150—155与AmOr 122—139分别聚为两个分支，BlOr 47—57与AmOr 63—65也发现类似的聚类，这表明在进化中，蜜蜂与熊蜂的Ors出现了特异性的扩张与缺失。在进化树中发现Or 115较早与其他成员分离，位于树的基部，推测该序列可能更接近气味受体家族的祖先序列。【结论】探明了兰州熊蜂的Or基因数量、基因结构及其进化关系；该基因家族在进化过程中部分成员保守基序丢失，这些结果为进一步了解Or基因功能打下了基础。

关键词: 兰州熊蜂, 气味受体, 基因结构, 保守基序, 进化分析

Abstract: 【Objective】Bumblebee Bombus lantschouensis is one of the most important pollinators for wild plants and crops in North China. Odorant receptors (Ors) gene family from the genome of B. lantschouensis was identified and characterized. Result of this study will help to explore the function of this gene family in foraging, mating and other social behaviors in bumblebee.【Method】Genomic DNA of thorax of B. lantschouensis was extracted and sequenced by the high-output of next generation sequencing. The original sequence of the sequencing was quality controlled and assembled to the genomic sequence. The contigs and scaffolds were used to build local sequence database. Gene sequences of Bombus terresters and Apis mellifera ligustica were used to query the local database. The characteristics and gene structure of Ors were analyzed by EMBOSS1.5 and GSDS2.0 software, respectively. Conservative motif analysis of amino acid sequences was performed using MEME 4.11.2. The phylogenetic analysis of Ors of B. lantschouensis, B. terresters and A. mellifera was studied by ClustaW 2.1, TrimAl 1.2 and PhyML3.0.【Result】One hundred and sixty-five Ors were identified from the genome of B. lantschouensis, including an Orco, 5 pseudogenes and 159 Ors. Gene structure analysis showed that the number of exons of these Ors are varies from 4 to 9. The least number of exons found in Or 47-57 was 4 and the largest number of exons found in Or 128-161 was 9. According to gene structure, Or was classified into 10 groups. Similarity in exon lengths and numbers was identified in the sequence of each group. The numbers of subgroup members were about 10, except in Or 1-38, Or 69-85 and Or 128-164 (which has 38, 17 and 37 members, respectively). The members of each subgroup are arranged in tandem on chromosomes and Or 1-38 have a longer first exon. Conserved motif analysis revealed that 9 motifs are present in Ors conserved domains (7tm_6 domain) except for motif 5 in all 10 motifs. While Or 1-38 and Or 39-46 have all predicted motifs and motifs 2, 3, 4, and 9 are widely present in sequences, which may be the key functional regions of the family. Phylogenetic analysis showed the 5 subfamilies of Ors family. Whereas the subfamily II contains 2 groups (BlOr 97-100 and BlOr 69-85) and subfamily V contains 4 groups (BlOr 1-46, 47-57, 86-95 and 101-107). BlOr 150-155 and AmOr 122-139 were clustered into two branches, and similar phenomena were also found in BlOr 47-57 and AmOr 63-65. It indicates that Ors has species-specific loss or expansion in evolution of Apis and Bombus. Or 115 is located at the base of the tree, suggesting that the sequence may be closer to the ancestor sequence of the odor receptor family.【Conclusion】Numbers, gene structure and phylogenetic relationships of Ors from the genome of B. lantschouensis were clarified in current study. Conserved motif analysis indicated that motif has been missed during the evolution of Ors family among bees. These results would provide an important information for exploring the gene evolution and function of Ors in future.

Key words: Bombus lantschouensis, odorant receptors, gene structure, conservative motif, phylogenetic analysis

王烨，韩蕾，董捷，黄家兴，吴杰. 兰州熊蜂气味受体家族鉴定及分析[J]. 中国农业科学, 2017, 50(10): 1904-1913.

WANG Ye, HAN Lei, DONG Jie, HUANG JiaXing, WU Jie. Identification and Characteristics of Odorant Receptors in Bumblebee, Bombus lantschouensis[J]. Scientia Agricultura Sinica, 2017, 50(10): 1904-1913.

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