牛FMR1基因5'UTR克隆及生物信息学分析

doi:10.16178/j.issn.0528-9017.20230843

摘要/Abstract

摘要：

为了探讨西门塔尔牛脆性X智力低下基因1(fragile X mental retardation 1,FMR1)的生物学功能,并为研究FMR1在西门塔尔牛屡配不孕中发挥的作用提供数据支持。实验以西门塔尔牛血液DNA为模板,通过PCR方法克隆FMR1基因完整的5'UTR序列;与NCBI提供的牛FMR1基因mRNA全序列进行比对;利用在线软件进行遗传分析并构建系统进化树,分析FMR1基因5'UTR端脆性;分析该基因编码蛋白的理化性质、亲疏水性、磷酸化位点、跨膜结构、信号肽、二级结构、三级结构和互作蛋白。结果表明:西门塔尔牛FMR1基因5'UTR序列与NCBI提供的牛FMR1基因序列基本一致,生物信息学分析得到牛FMR1基因最长开放阅读框1 710 bp,基因共编码569个氨基酸,分子量64 221.90 u,其编码蛋白是不稳定的亲水蛋白,没有明显的信号肽切割位点,并且不包括跨膜螺旋,二级结构预测表示其主要以α螺旋和无规卷曲形式为主。说明西门塔尔牛FMR1基因编码序列长度为40 254 bp,编码569个氨基酸,编码的蛋白质为亲水不稳定蛋白,与绵羊遗传距离最近。

关键词: 西门塔尔牛, FMR1基因, 克隆, 生物信息学分析, 系统进化树

Abstract:

To explore the biological function of fragile X mental retardation 1 (FMR1) in Simmental cattle and to provide data support for the study of the role of FMR1 in the infertility of Simmental cattle, in this study, the complete 5'UTR sequence of FMR1 gene was cloned by PCR using Simmental cattle blood DNA as a template. It was compared with the full mRNA sequence of bovine FMR1 gene provided by NCBI. Online software was used for genetic analysis and phylogenetic tree construction to analyze the fragility of the 5'UTR end of FMR1 gene. The physicochemical properties, hydrophilicity, phosphorylation sites, transmembrane structure, signal peptide, secondary structure, tertiary structure and interacting proteins of the protein encoded by this gene were analyzed. The results showed that the 5'UTR sequence of Simmental cattle FMR1 gene was basically consistent with the sequence of cattle FMR1 gene provided by NCBI, and the longest open reading frame of bovine FMR1 gene was 1 710 bp, the gene encoded 569 amino acids, and the molecular weight was 64 221.90 u, and the encoded protein was an unstable hydrophilic protein, with no obvious signal peptide cleavage site, and did not include the transmembrane helix, and the secondary structure prediction indicated that it was mainly in α-helix and random coil form. The results indicated that the coding sequence of Simmental cattle FMR1 gene was 40 254 bp and encoded 569 amino acids, and the encoded protein was a hydrophilic unstable protein, which showed the closest genetic distance from sheep.

Key words: Simmental cattle, FMR1 gene, cloning, bioinformatics analysis, phylogenetic tree

中图分类号:

S823

王选艺, 孙亚伟, 王俪颖, 龙雨薇, 叶彤, 周瑜新, 马雪连, 李娜, 赵红琼, 姚刚. 牛FMR1基因5'UTR克隆及生物信息学分析[J]. 浙江农业科学, 2024, 65(9): 2190-2197.

WANG Xuanyi, SUN Yawei, WANG Liying, LONG Yuwei, YE Tong, ZHOU Yuxin, MA Xuelian, LI Na, ZHAO Hongqiong, YAO Gang. Cloning and bioinformatics analysis of 5'UTR of bovine FMR1 gene[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(9): 2190-2197.

图/表 17

表1 12个不同物种FMR1核酸及蛋白登录信息

Table 1 Login information of FMR1 nucleic acid and protein in 12 different species

物种	GenBank核酸登录号	GenBank蛋白登录号
猪	NC_010461.5	XP_005674011.1
小鼠	NC_000086.8	XP_006527876.1
人	NG_007529.2	NP_001172004.1
牛	NC_037357.1	NP_001179079.1
猫	NC_058386.1	XP_019679976.1
马	NC_009175.3	XP_023489885.1
鸡	NC_052535.1	XP_025005602.1
恒河猴	NC_041774.1	XP_014983855.1
大鼠	NC_051356.1	XP_038955419.1
狗	NC_051843.1	XP_038306968.1
斑马鱼	NC_007125.7	NP_694495.1
羊	NC_056080.1	XP_027819346.1

图1 牛FMR1基因5'UTR端PCR扩增产物 M—DL-500 Marker;1~4分别为不同西门塔尔牛FMR1的5'UTR扩增产物;阴—阴性对照。

Fig.1 PCR amplification products at 5'UTR of bovine FMR1 gene

图2 FMR1基因测序结果与NCBI上牛FMR1基因的序列比对原始序列为NCBI提供的印度牛FMR1基因序列;正向测序结果为新疆西门塔尔牛FMR1基因正向引物所得;反向测序结果为新疆西门塔尔牛FMR1基因反向引物所得。

Fig.2 Sequence alignment of bovine FMR1 gene with FMR1 gene in NCBI

表2 FMR1氨基酸序列遗传距离分析表

Table 2 Genetic distance analysis of FMR1 amino acid sequence

物种	1	2	3	4	5	6	7	8	9	10	11
2	0.622
3	0.101	0.090
4	0.100	0.083	0.092
5	0.383	0.417	0.425	0.431
6	0.063	0.053	0.077	0.067	0.423
7	0.235	0.233	0.238	0.246	0.497	0.225
8	0.076	0.065	0.075	0.031	0.427	0.050	0.230
9	0.102	0.090	0.026	0.091	0.430	0.078	0.238	0.075
10	0.062	0.052	0.078	0.069	0.427	0.040	0.218	0.052	0.076
11	0.357	0.368	0.370	0.393	0.457	0.360	0.366	0.370	0.370	0.363
12	0.010	0.056	0.093	0.087	0.425	0.058	0.236	0.068	0.093	0.055	0.365

图3 FMR1氨基酸序列系统进化树

Fig.3 Phylogenetic tree of FMR1 amino acid sequence

图4 牛FMR1编码的蛋白质各氨基酸含量

Fig.4 The amino acid content of the protein encoded by bovine FMR1

图5 牛FMR1基因5'UTR端编码的蛋白质各类氨基酸含量

Fig.5 Amino acids of the protein encoded at the 5'UTR of bovine FMR1 gene

图6 牛FMR1蛋白疏水性分析

Fig.6 Hydrophobicity analysis of bovine FMR1 protein

图7 牛FMR1基因5'UTR端蛋白疏水性分析

Fig.7 Hydrophobicity analysis of 5'UTR terminal protein of bovine FMR1 gene

图8 N-糖基化位点预测

Fig.8 N-Glycodylation point prediction

图9 西门塔尔牛FMR1蛋白5'UTR端蛋白信号肽预测

Fig.9 Prediction of 5'UTR terminal protein signal peptide of Simmental cattle FMR1 protein

图10 牛FMR1 CDSs区蛋白信号肽预测

Fig.10 Protein signal peptide prediction of Simmental cattle FMR1 CDSs region

图11 西门塔尔牛FMR1的5'UTR端蛋白跨膜结构预测

Fig.11 Prediction of the transmembrane structure of 5'UTR-terminal protein of Simmental cattle FMR1

图12 西门塔尔牛FMR1蛋白跨膜结构预测

Fig.12 Prediction of transmembrane structure of Simmental cattle FMR1 protein

图13 FMR1蛋白二级结构预测蓝色为α螺旋;绿色为β折叠;黄色为无规卷曲;红色为延伸链。

Fig.13 Prediction of secondary structure of FMR1 protein

图14 牛FMR1 CDSs区编码蛋白质三级结构预测

Fig.14 Prediction of tertiary structure of protein encoding protein in bovine FMR1 CDSs

图15 FMR1蛋白互作网络

Fig.15 Protein-protein interaction network encoded by FMR1

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