意大利蜜蜂AmMETTL3基因的分子克隆与表达模式

Molecular cloning and expression pattern determination of AmMETTL3 gene in Apis mellifera ligustica

关键词:  意大利蜜蜂  N⁶-甲基腺苷  甲基化转移酶3  MT-A70  分子特性  表达模式

Key words:  Apis mellifera ligustica  N⁶-methyladenosine  methyltransferase 3  MT-A70  molecular characteristics  expression pattern

摘要:

为克隆意大利蜜蜂（Apis mellifera ligustica）的AmMETTL3基因，解析AmMETTL3蛋白的分子特性，并测定AmMETTL3在工蜂不同组织及发育时期的表达谱。通过PCR扩增AmMETTL3的CDS并进行Sanger测序验证；使用相关生物信息学软件预测AmMETTL3的理化性质和分子特性，鉴定METTL3的结构域和保守基序，并构建系统进化树；采用RT-qPCR检测AmMETTL3在工蜂7个组织及5个不同发育时期的相对表达量。结果表明：成功克隆到AmMETTL3的CDS并发现AmMETTL3不含典型的跨膜结构域和信号肽；METTL3在各种昆虫中高度保守且含1个MT-A70结构域和3个相同的保守基序；AmMETTL3与东方蜜蜂METTL3的亲缘关系最近；相较于触角中的表达量，AmMETTL3的表达量在咽下腺中极显著上调（P<0.01），在中肠中极显著下调（P<0.01）；AmMETTL3在3日龄幼虫中的表达量极显著（P<0.01）低于卵中的表达量，在12日龄蛹中的表达量极显著（P<0.01）高于卵中的表达量，在2、6、12、15和18日龄工蜂体内的表达量极显著（P<0.01）低于1日龄工蜂体内的表达量。研究结果为深入探究AmMETTL3的功能及其参与的表观调控机制提供了参考和依据。

Abstract:

To clone the AmMETTL3 gene in Apis mellifera ligustica， analyze the physicochemical property and molecular characteristics of AmMETTL3 protein and determine the expression pattern of AmMETTL3 in different tissues and developmental stages of worker bees. AmMETTL3 CDS was amplified by PCR and then verified using Sanger sequencing. Related bioinformatic software were used to predict physicochemical property and molecular characteristics， identify structural domains and conserved motifs and construct phylogenetic tree of AmMETTL3. RT-qPCR was employed to detect the relative expression levels of AmMETTL3 in seven different tissues and five various developmental stages. The results indicated that the CDS of AmMETTL3 was successfully cloned， AmMETTL3 protein do not have typical transmembrane domain and signal peptide， and METTL3 is highly conserved in various insects including one structure domain MT-A70 and three same conserved motifs. AmMETTL3 and A. cerana METTL3 were clustered into one group in the phylogenetic tree. The expression level of AmMETTL3 was significantly up-regulated in hypopharyngeal gland （P<0.01）， significantly down-regulated in midgut （P<0.01） compared with that in antennae. The expression level of AmMETTL3 in 3-day-old larvae （P<0.01） was significantly lower than that in eggs. The expression level of AmMETTL3 in 12-day-old pupa （P<0.01） was significantly higher than that in eggs； the expression level of AmMETTL3 in workers at 2， 6， 12， 15 and 18 day-old （P<0.01） was significantly lower than that in workers at 1 day-old. This study provides references and a basis for further investigating the function of AmMETTL3 and itsrole in epigenetic regulatory mechanisms.