中华蜜蜂<i>Smoothened</i>基因的鉴定、表达及生物信息学分析

doi:10.7668/hbnxb.20194415

摘要/Abstract

摘要：

旨在明确中华蜜蜂Smoothened基因(AcerSmo)的序列特征及表达特性,为进一步研究其在蜜蜂体内的功能奠定理论基础,对中华蜜蜂AcerSmo基因DNA序列进行扩增鉴定,运用多种生物信息学软件对其进行结构和功能分析,并采用RT-qRCR对中华蜜蜂不同组织和发育时期的表达特性进行检测。序列分析结果表明,基因cDNA全长3 495 bp(GenBank登录号:OP616035),其中开放阅读框(ORF)区域2 952 bp,共编码936个氨基酸。该蛋白理论分子质量为111.15 ku,理论等电点为8.82,属于不稳定的两性蛋白。该蛋白N-末端有一段含21个氨基酸的信号肽,有6个跨膜结构域,亚细胞定位主要在内质网和细胞核。该蛋白二级结构和三级结构以无规则卷曲(50.76%)为主,其次为α-螺旋(25.33%),从昆虫到哺乳动物,AcerSmo蛋白序列高度保守;荧光定量PCR结果表明,AcerSmo在30日龄中华蜜蜂不同组织中均存在表达,而在触角与头的表达量显著高于其他组织,在中华蜜蜂触角与头组织中,5日龄时AcerSmo的mRNA表达量最高。AcerSmo蛋白具有Smoothened蛋白的保守结构,且在触角中呈高丰度表达,推测AcerSmo基因可能在蜜蜂的嗅觉识别过程发挥着重要作用。

关键词: 中华蜜蜂, Smoothened, 序列特征, 生物信息学, 表达特性

Abstract:

The aim of this study was to clarify the sequence characteristics and expression properties of the Smoothened gene in Apis cerana cerana.The DNA sequence of AcerSmo was amplified and identified,its structure and function were analysed using various bioinformatics software,and the expression characteristics were examined by RT-qPCR for different tissues and developmental periods of Apis cerana cerana.Sequence analysis showed that the AcerSmo was 3 495 bp(GenBank accession number:OP616035),the open reading frame(ORF)region was 2 952 bp,encoding 936 amino acids.The molecular weight of this protein was 111.15 ku and the predicted isoelectric point was 8.82,which had a signal peptide containing 21 amino acids at the N-terminus,and there were 6 transmembrane structures domains.Subcellular localization mostly takes place in the nucleus and endoplasmic reticulum.The secondary and tertiary structures of this protein were dominated by irregular coiled(50.76%),followed by α-helix(25.33%).The sequence of AcerSmo protein sequence was highly conserved from insects to mammals.The results of RT-qPCR showed that AcerSmo was expressed in different tissues of Apis cerana cerana at the age of 30 days and the expression level of AcerSmo in the antennae and head were significantly higher than in other tissues,and in the tissues of the antennae and head of the Apis cerana cerana,the mRNA expression of AcerSmo was the highest at 5 days of age.AcerSmo has the conserved structure of Smoothened protein and was highly expressed in high abundance in antennae,which may play an important role in the olfactory recognition process of honey bees.

Key words: Apis cerana cerana, Smoothened, Sequence characterization, Bioinformatics analysis, Expression characteristics

于点点, 申红英, 王珏, 郭媛, 郭丽娜. 中华蜜蜂Smoothened基因的鉴定、表达及生物信息学分析[J]. 华北农学报, 2024, 39(3): 230-238. doi: 10.7668/hbnxb.20194415.

YU Diandian, SHEN Hongying, WANG Jue, GUO Yuan, GUO Lina. Identification,Expression and Bioinformatics Analysis of Smoothened Gene of Apis cerana cerana[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 230-238. doi: 10.7668/hbnxb.20194415.

http://www.hbnxb.net/CN/Y2024/V39/I3/230

图/表 11

表1 引物序列信息

Tab.1 Primer sequence formation

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
P1	F: TAATATTTGTTCATTATTTCTAAAATTTCC	序列扩增
	R: AGCCCAAGCAATAAAAGAATGAAT
P2	F: TAATATTTGTTCATTATTTCTAAAATTTCC
	R: AGCCCAAGCAATAAAAGAATGAAT
P3	F: ATTTCTAGCAAATGCTTAAAGCCT
	R: GTAAAGCAGCTGCCCATGTTG
P4	F: CATTCGCAAAACGGAAAACATT
	R:GAAAAATTAGTGTTACGTAGTAATATTTTTG
qAcerSmo	F: TGGCAGCTATGGTGTGGTTT	荧光定量
	R: GACAGTGAGGACAAGTGGCA
qArp1	F: TGCTGCACTCGTAGTTGACAATGG
	R: ACCCTGGTGGCGTGGTCTTC

图1 PCR产物电泳 M.3000 Marker;1—3.PCR产物。

Fig.1 Electrophoresis of the PCR products of AcerSmo M.3000 Marker;1—3.PCR product.

图2 AcerSmo ORF区及推导的氨基酸序列

Fig.2 AcerSmo ORF region and deduced amino acid sequence

图3 AcerSmo蛋白信号肽与跨膜区结构预测 A.信号肽结构预测;B.蛋白跨膜区预测。

Fig.3 Predicted signal peptide and transmembrane of AcerSmo A.Predicted signal peptide;B.Transmembrane prediction.

图4 AcerSmo蛋白的二、三级结构预测结果 A.二级结构预测;B.三级结构预测;C.三级结构模型的拉曼图分析。

Fig.4 The secondary and tertiary structure prediction of AcerSmo A.Secondary structure prediction;B.The tertiary structure prediction;C.Ramachandran map analysis of the tertiary structural model.

图5 AcerSmo蛋白模体结构与保守结构域预测 A.模体结构预测;B.保守结构域预测。

Fig.5 Motif and conserved domain prediction of AcerSmo A.Motif prediction;B.The prediction of conserved domain.

图6 AcerSmo多重序列比对

Fig.6 Multiple alignment of AcerSmo

图7 系统发育树树枝上方数字为>50%的自展值(1 000次重复)。

Fig.7 Phylogenetic tree Data in the upper branches are bootstrap of>50% from 1 000 repeats.

图8 AcerSmo在30日龄中华蜜蜂各组织中的相对表达量 AN.触角;HE.头(不含触角);T.胸部;AB.腹部;L.足;W.翅膀。不同小写字母表示不同组织间差异显著(P<0.05)。

Fig.8 Relative expression of AcerSmo in different tissues of 30-day-old Apis cerana cerana AN.Antennae;HE.Head(without antenna);T.Thorax;AB.Abodem;L.Legs;W.Wing.Different lowercase letters indicate significant differences in different tissues(P<0.05).

图9 AcerSmo在不同日龄中华蜜蜂触角中的相对表达量不同小写字母表示不同日龄间差异显著(P<0.05)。图10同。

Fig.9 Relative expression of AcerSmo in the antennae of Apis cerana cerana at different days Different lowercase letters indicated significant differences in different days(P<0.05).The same as Fig.10.

图10 AcerSmo在不同日龄中华蜜蜂头部中表达量

Fig.10 Relative expression of AcerSmo in the antennae of Apis cerana cerana at different days

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中华蜜蜂Smoothened基因的鉴定、表达及生物信息学分析

Identification,Expression and Bioinformatics Analysis of Smoothened Gene of Apis cerana cerana

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