西方蜜蜂磁场感应和磁受体基因AmMagR的克隆及表达分析

【作者】 赵方媛； 李茫； 廖春华； 王子龙； 颜伟玉；

【机构】 江西农业大学蜜蜂研究所；

【摘要】 [目的]验证采集蜂磁场感应能力,以期为确定蜜蜂体内可能与磁场感应相关的基因提供基础。克隆西方蜜蜂Apis mellifera磁受体基因AmMagR的序列,并分析该基因在工蜂不同日龄、不同组织中的表达特征,以期为该基因的功能研究提供理论基础。[方法]通过磁感装置分析采集蜂对有无磁场、糖水浓度、采集位置的选择率。以西方蜜蜂工蜂为材料,提取其总RNA,通过RT-PCR技术克隆西方蜜蜂MagR基因的序列;利用多种生物信息学软件对其核酸及氨基酸序列分析;采用实时荧光定量PCR技术(qPCR)分析在西方蜜蜂工蜂不同日龄(1日龄、5日龄和21日龄)和不同组织(头部、胸部和腹部)中MagR基因的相对表达量。[结果]磁场感应结果表明,蜜蜂对有磁场一侧的选择率显著高于无磁场一侧(P<0.05);蜜蜂对训练期间放置过50%糖水一侧的选择率显著高于放置过20%糖水一侧(P<0.05);但在方向选择中,对L1、L2两侧的选择率无显著差异(P>0.05)。克隆到西方蜜蜂MagR基因,并命名为AmMagR(GenBank登录号:MH635411),其序列长度为748bp,编码区长390bp,编码130个氨基酸,其蛋白质分子量为14.142kD,理论等电点为9.06,无信号肽,无跨膜结构,且在第24-126位氨基酸之间得到一个结构域Fe-S_biosyn。系统发育树显示,西方蜜蜂AmMagR与小蜜蜂ApisfloraeAflscA (IscA别称MagR)、中华蜜蜂Apiscerana ceranaAcIscA基因聚成一支。荧光定量PCR结果表明,AmMagR基因在西方蜜蜂不同日龄的工蜂头部、胸部和腹部均有表达。5日龄工蜂的相对表达量最高,5日龄工蜂头部和胸部的AmMagR基因表达量显著高于1日龄(P<0.05)和21日龄(P<0.05),腹部AmMagR基因表达量显著高于21日龄(P<0.05),但与1日龄的比较差异不显著(P>0.05)。1日龄、5日龄和21日龄工蜂胸部的AmMagR基因表达量均显著高于头部和腹部(P<0.05);1日龄工蜂头部AmMagR基因表达量均高于腹部(P<0.05),但5日龄和21日龄工蜂AmMagR基因在头部与腹部的表达量无显著差异(P>0.05)。[结论]明确了该基因在西方蜜蜂工蜂不同日龄和不同组织中的表达模式,并推测AmMagR可能参与了西方蜜蜂的定位、归巢过程。更多还原

【Abstract】 [Aim] The aim of this study is to verify the magnetic sense of forager bees,to provide a basis for determining genes in bees that may be related to magnetic sense.And to clone the sequence of the magnetic receptor gene AmMagR in Apis mellifera,and to analyze the expression characteristics of this gene in different days and different tissues of worker bees,to provide the theoretical basis for the functional study of the gene.[Methods] The selection rate of forager bees for the presence or absence of a magnetic field, a concentration of sucrose solution,and positions was analyzed by magnetic field sensing device.Workers in Apis mellifera were collected,from which the total RNA was extracted to clone the sequence of MagR;A variety of bioinformatics software was used to analyze the nucleic acid and amino acid sequences;The relative expression level of MagR in different days(1 st day,5 th day and21 st day) and different tissues(head,thorax and abdomen) was analyzed by Real-time quantitative PCR(qPCR).[Results] The magnetic sense results showed that forager bees have a direct preference the target associated with the magnetic(P<0.05);Forager bees have a preference the target associated with the 50% sucrose during the previous discriminative training(P<0.05);There is no preference about position(P>0.05).The cDNA sequence of MagR gene of Apis mellifera was obtained,and was named as AmMagR(GenBank accession number:MH63541 1),with the length of 748 bp and a coding of 390 bp,encoding 130 amino acids.The encoded protein has the molecular weight of 14.142 kD and a theoretical isoelectric point of 9.06.The encoding protein has no signal peptide and no transmembrance structure,possesses a Fe-S_biosyn domain between 24-126 residues.The phylogenetic tree showed that AmMagR of Apis mellifera with AfIscA of Apis florae(IscA also named as MagR)and AcIscA of Apis cerana cerana gene were clustered together.The real-time PCR results showed that the AmMagR gene was expressed in different days and tissues of bees in Apis mellifera.The highest relative expression level is on 5 th day.At the same time,the relative expression level of AmMagR gene was significantly higher on 5 th day of age than(P<0.05)1 st and 21 st day of age(P<0.05) in head and thorax.And the relative expression level of AmMagR gene was significantly higher on 5 th day of age than 21 st day of age(P<0.05) in abdomen,but no significant between that on 5 th day and1 st day(P>0.05).The relative expression level of the AmMagR gene was all significantly higher in thorax than in head and abdomen on the 1 st,5 th,and 21 st day(P<0.05,And significantly higher in head than in abdomen on 1 st day of age(P<0.05),but no significant different between head and abdomen on 5 th and 21 st day(P>0.05).[Conclusion] The expression pattern of the gene in different ages and tissues mellifera was determined,and it was speculated that AmMagR may be involved in the positioning and homing process of Apis mellifera.更多还原