杨小舟蛾MtroOBP1基因的克隆、序列分析及表达

doi:10.3969/j.issn.1000-2006.201909017

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (5): 215-221.doi: 10.3969/j.issn.1000-2006.201909017

杨小舟蛾MtroOBP1基因的克隆、序列分析及表达

张雨(), 丰凯(), 汤方^*()

南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏南京 210037

收稿日期:2019-09-09 修回日期:2020-05-01 出版日期:2020-10-30 发布日期:2020-10-30
通讯作者: 汤方
基金资助:
江苏省自然科学基金项目(BK20151517);江苏高校优势学科建设工程资助项目(PAPD)

The cloning,sequence analysis and expression of MtroOBP1 gene in Micromelalopha troglodyta(Graeser)

ZHANG Yu(), FENG Kai(), TANG Fang^*()

Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China

Received:2019-09-09 Revised:2020-05-01 Online:2020-10-30 Published:2020-10-30
Contact: TANG Fang

摘要/Abstract

摘要：

【目的】气味结合蛋白(odorant binding proteins,OBPs)是一种嗅觉相关的蛋白,该蛋白参与大多数气味分子的识别过程,并与气味分子相结合。获得杨小舟蛾[Micromelalopha troglodyta (Graeser)]OBPs以明确其特性。【方法】选择羽化后健康的杨小舟蛾触角为模板,通过反转录聚合酶链反应(RT-PCR)技术克隆杨小舟蛾MtroOBP1基因,利用生物信息学分析软件研究其基因序列和蛋白结构,运用实时荧光定量PCR(qPCR)技术研究MtroOBP1的组织表达模式。【结果】利用RT-PCR技术从杨小舟蛾触角总RNA中扩增得到MtroOBP1基因(GenBank登录号:MN056510),序列分析表明,MtroOBP1开放阅读框为777 bp,一共编码了258个氨基酸残基,且翻译的氨基酸序列仅含有4个保守的半胱氨酸位点,表明得到的OBP基因的编码蛋白不属于典型气味结合蛋白家族,而是属于Minus-C家族。蛋白质理化性质预测显示:MtroOBP1蛋白的分子量为29 664.57 u,等电点为6.23,有18个潜在的磷酸化位点,没有明显的跨膜区,疏水指数为-2.011~3.078,有一个由19个氨基酸组成的信号肽,说明为分泌型蛋白。组织表达模式表明MtroOBP1在杨小舟蛾的各部位都表达,但在触角中表达量最高。【结论】首次克隆得到杨小舟蛾MtroOBP1基因,在触角中高表达,推测其蛋白具有运输气味分子的功能,在杨小舟蛾的嗅觉识别中起着至关重要的作用。

关键词: 杨小舟蛾, MtroOBP1基因, 基因克隆, 嗅觉系统, 组织表达谱

Abstract:

【Objective】Odorant binding proteins (OBPs) are olfactory-related proteins that are involved in the recognition process of most odor molecules and combine with odor molecules. However, the research on the OBPs has not been reported at home and abroad in Micromelalopha troglodyta (Graeser) . Therefore, this study would like to obtain the OBP and clarify its characteristics in M. troglodyta. 【Method】The MtroOBP1 gene was obtained from the cDNA template of antenna using reverse transcription polymerase chain reaction (RT-PCR), and the gene sequence and protein structure were analyzed by bioinformatics analysis software, and the tissue expression pattern of MtroOBP1 was studied by real-time fluorescence quantitative PCR (qPCR) in M. troglodyta.【Result】MtroOBP1 was amplified by RT-PCR using total RNA from the antennae of M. troglodyta (GenBank accession no. MN056510). The gene harbors a 777-bp-long open reading frame and a protein-coding region with 258 amino acids. Its translated amino acid sequence contained only four conserverd cysteines, suggesting that it belonged to the Minus-C OBP . Prediction of physicochemical properties of the protein showed that the molecular weight of MtroOBP1 protein is 29 664.57 u and isoelectric point (PI) is 6.23. The protein contains 18 potential phosphorylation sites but no transmembrane region, and it is a secretory protein with an hydrophobic index of -2.011 to 3.078.There was a signal peptide consisting of 19 amino acids, indicating that it is a secreted protein. The tissue expression pattern showed that MtroOBP1 was expressed in all tissues of the M. troglodyta. but the highest expression was in the antennae.【Conclusion】In a word, the MtroOBP1 gene firstly cloned is highly expressed in the antennae. Presumably this protein has the function of transporting odor molecules, and plays an important role in olfactory recognition in M. troglodyta.

Key words: Micromelalopha troglodyta, MtroOBP1 gene, gene cloning, olfactory system, expression profiling

中图分类号:

张雨,丰凯,汤方. 杨小舟蛾MtroOBP1基因的克隆、序列分析及表达[J]. 南京林业大学学报（自然科学版）, 2020, 44(5): 215-221.

ZHANG Yu, FENG Kai, TANG Fang. The cloning,sequence analysis and expression of MtroOBP1 gene in Micromelalopha troglodyta(Graeser)[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(5): 215-221.DOI: 10.3969/j.issn.1000-2006.201909017.

图/表 4

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组分 component	用量/μL does
SYBR Premix Taq	10
引物 primer F	0.4
引物 primer R	0.4
ROX Reference DyeⅡ	0.4
cDNA	1.0
ddH₂O	7.8
总体积total Volume	20

基因 gene	引物序列(5'?3') primers sequence (5'?3')
Actin	F',GCGGCGCGACTCACCGACTAC R',GGGAAGAGAGCCTCAGGGCAAC
MtroOBP1	F',ATAAAGGCAGGCGAGGTA R',TCAAGGAAATTGGCACAG

杨小舟蛾MtroOBP1基因的克隆、序列分析及表达

The cloning,sequence analysis and expression of MtroOBP1 gene in Micromelalopha troglodyta(Graeser)

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