牦牛DRA基因克隆测序及生物信息学分析

doi:10.3969/j.issn.1004-1524.20212021

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (7): 1564-1570.DOI: 10.3969/j.issn.1004-1524.20212021

牦牛DRA基因克隆测序及生物信息学分析

张丽(), 王媛媛, 王瑞, 刘丽霞()

西北民族大学生命科学与工程学院,甘肃兰州 730030

收稿日期:2021-12-14 出版日期:2023-07-25 发布日期:2023-08-17
作者简介:张丽(1980—),女,宁夏石嘴山人,博士,副教授,主要从事现代生物技术与动物遗传育种研究。E-mail:280082343@xbmu.edu.cn
通讯作者: *刘丽霞,E-mail:skllx@xbmu.edu.cn
基金资助:
西北民族大学中央高校基本科研业务费资金资助项目(31920200074);西北民族大学中央高校基本科研业务费资金资助项目(31920210061);西北民族大学研究生科研创新项目(Yxm2021074)

Cloning sequencing and bioinformatics analysis of DRA gene of yak

ZHANG Li(), WANG Yuanyuan, WANG Rui, LIU Lixia()

College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China

Received:2021-12-14 Online:2023-07-25 Published:2023-08-17
Contact: LIU Lixia

摘要/Abstract

摘要：

为全面解析牦牛MHCⅡ类基因结构与功能,采用分子克隆测序技术对大通牦牛DRA基因编码区进行等位基因频率估算并进行生物信息学分析。结果显示:大通牦牛DRA基因编码区含有762 bp的开放阅读框,共编码253个氨基酸;共有3个SNPs(g.2376C>T、g.2851C>G、g.3016C>A),其中第2、3外显子分别存在1个C→T的同义突变和C→A的错义突变(L→M),这两个突变均降低了大通牦牛DRA基因mRNA二级结构的稳定性;g.3016C>A突变前后编码蛋白质的分子量、原子总数、脂肪系数、不稳定系数、总平均亲水性方面存在差异;生物信息学的方法分析显示,BoLA-DRA基因是MHC基因家族中高度保守的基因。该研究结果可以为大通牦牛DRA基因结构与功能的研究奠定基础。

关键词: DRA基因, 编码区克隆, 突变位点, 生物信息学

Abstract:

In order to comprehensively analyze the structure and function of yak MHCⅡ gene, molecular cloning and sequencing technology was used to estimate the coding region of DRA gene of Datong yak and conduct bioinformatics analysis. The results showed a DRA gene open reading frame in the coding region containing 762 bp, a total of 253 amino acids encoding; a total of 3 SNPs (g.2376C>T, g.2851C>G, g.3016C>A). In exons 2 and 3, there was one synonymous mutation in C→T and a missense mutation (L→M) in C→A, respectively, both mutations reduced the stability of mRNA secondary structure of DRA gene. There were differences in the molecular mass, total number of atoms, fat coefficient, instability coefficient, and total average hydrophilicity of the protein encoded by the g.3016C>A mutation. Methodological analysis by bioinformatics revealed that the BoLA-DRA gene were highly conserved genes in the MHC gene family. The results laid the foundation for the structure and function of DRA in Datong yak.

Key words: DRA gene, coding region cloning, mutation site, bioinformatics

中图分类号:

S823.85

张丽, 王媛媛, 王瑞, 刘丽霞. 牦牛DRA基因克隆测序及生物信息学分析[J]. 浙江农业学报, 2023, 35(7): 1564-1570.

ZHANG Li, WANG Yuanyuan, WANG Rui, LIU Lixia. Cloning sequencing and bioinformatics analysis of DRA gene of yak[J]. Acta Agriculturae Zhejiangensis, 2023, 35(7): 1564-1570.

图/表 9

参考文献 18

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位置 Location	引物序列 Primer sequence(5'→3')	退火温度 Annealing temperature/℃	片段长度 Fragment length/bp
外显子1 Exon1(1~93 bp) 外显子2 Exon2(263~508 bp)	F:CACCAAAGAAGTGGCC R:CCACAGTTCATTTCTCCGAAGC F:CCTGACCCCCTTTCTTGTCT R:AGGAGGACTGGAGCAGAGGT	57.3 58.5	93 246
外显子3 Exon3(938~1 219 bp)	F:GTCAGCCTCGATGGGACGAA R:CTCACCCCAGTGCTTGAGAA	63.0	282
外显子4 Exon4(1 510~1 664 bp)	F:GTGCTAAGCTGCTCCCTA R:TCCTCTGACCACAGCAGACTC	61.2	155

位置 Location	引物序列 Primer sequence(5'→3')	退火温度 Annealing temperature/℃	片段长度 Fragment length/bp
外显子1 Exon1(1~93 bp) 外显子2 Exon2(263~508 bp)	F:CACCAAAGAAGTGGCC R:CCACAGTTCATTTCTCCGAAGC F:CCTGACCCCCTTTCTTGTCT R:AGGAGGACTGGAGCAGAGGT	57.3 58.5	93 246
外显子3 Exon3(938~1 219 bp)	F:GTCAGCCTCGATGGGACGAA R:CTCACCCCAGTGCTTGAGAA	63.0	282
外显子4 Exon4(1 510~1 664 bp)	F:GTGCTAAGCTGCTCCCTA R:TCCTCTGACCACAGCAGACTC	61.2	155

蛋白质理化特性 Physicochemical properties of proteins	突变类型Mutation type
蛋白质理化特性 Physicochemical properties of proteins	突变前Before mutation	突变后After mutation
分子量Molecular weight/u	28 407.01	28 425.04
原子总数Total number of atoms	4 035	4 033
消光系数Extinction coefficients	28 085/27 960	28 085/27 960
等电点Theoretical pI	5.41	5.41
分子式Formula	C₁₃₀₅H₂₀₃₀N₃₃₀O₃₆₁S₉	C₁₃₀₄H₂₀₂₈N₃₃₀O₃₆₁S₁₀
不稳定系数The instability index (Ⅱ)	38.81	39.78
脂肪系数Aliphatic index	100.16	98.62
总平均亲水性Grand average of hydropathicity (GRAVY)	0.056	0.049
半衰期Estimated half-life/h	30	30
正电荷残基数Total number of negatively charged residues (Asp+Glu)	31	31
负电荷残基数Total number of positively charged residues (Arg+Lys)	23	23

蛋白质理化特性 Physicochemical properties of proteins	突变类型Mutation type
蛋白质理化特性 Physicochemical properties of proteins	突变前Before mutation	突变后After mutation
分子量Molecular weight/u	28 407.01	28 425.04
原子总数Total number of atoms	4 035	4 033
消光系数Extinction coefficients	28 085/27 960	28 085/27 960
等电点Theoretical pI	5.41	5.41
分子式Formula	C₁₃₀₅H₂₀₃₀N₃₃₀O₃₆₁S₉	C₁₃₀₄H₂₀₂₈N₃₃₀O₃₆₁S₁₀
不稳定系数The instability index (Ⅱ)	38.81	39.78
脂肪系数Aliphatic index	100.16	98.62
总平均亲水性Grand average of hydropathicity (GRAVY)	0.056	0.049
半衰期Estimated half-life/h	30	30
正电荷残基数Total number of negatively charged residues (Asp+Glu)	31	31
负电荷残基数Total number of positively charged residues (Arg+Lys)	23	23

突变位点 Mutant site	突变位置 Mutant location	突变类型 Mutation type	氨基酸变化 Amino acid variation	等位基因频率Allele frequency
突变位点 Mutant site	突变位置 Mutant location	突变类型 Mutation type	氨基酸变化 Amino acid variation	突变前Before mutation	突变后After mutation
g.2376C>T	Exon2	同义突变	—	0.156 2	0.843 8
		Synonymous mutation
g.2851C>G	Intron2	—	—	0.343 7	0.656 3
g.3016C>A	Exon3	错义突变	L→M	0.333 3	0.666 7
		Missense mutation

牦牛DRA基因克隆测序及生物信息学分析

Cloning sequencing and bioinformatics analysis of DRA gene of yak

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突变类型 Mutation type	α螺旋 α-Helix	β转角 β-Turn	β折叠 β-Sheet	无规则卷曲 Random coil
突变前Before mutation	18.58(47)	6.72(17)	38.58(49)	44.66(113)
突变后After mutation	18.58(47)	6.72(17)	30.04(76)	47.43(120)

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