鸡 BMP15 基因克隆、表达模式及启动子活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1224

摘要/Abstract

摘要：

为了解鸡骨形态生成蛋白15（bone morphogenetic protein 15, BMP15）基因的功能、结构及启动子活性区域，探究该基因的转录调控机制。通过RACE（rapid amplification of cDNA ends）技术克隆鸡 BMP15 基因cDNA全长序列，生物信息学分析其编码蛋白的性质及结构。实时荧光定量PCR（RT-qPCR）检测鸡 BMP15 基因在不同组织中的表达情况，双荧光素酶报告系统确定该基因的核心启动子区。结果表明，鸡 BMP15 基因存在一个长度为1 865 bp的转录本，编码350个氨基酸，其遗传距离与火鸡最近。该蛋白为水溶性蛋白，其结构主要由无规则卷曲构成；存在信号肽区域，无跨膜结构域。构建的组织表达谱结果表明，BMP15 基因在鸡的卵巢组织和颗粒细胞中表达极显著（P<0.01）。双荧光报告结果显示，BMP15 基因启动子-153 - -1 bp为核心区域。为进一步确定 BMP15 基因功能及探究其转录调控机制奠定基础。

关键词: 太行鸡, BMP15 基因, 生物信息学, 启动子活性

Abstract:

The purpose of this study is to investigate the transcriptional regulation mechanism of the bone morphogenetic protein 15（BMP15）gene in chickens, as well as its function, structure, and promoter active region. RACE（rapid amplification of cDNA ends）was used to clone the full-length cDNA of the chicken BMP15gene, and bioinformatics was to analyze the properties and structures of its encoding protein. Quantitative real-time PCR was plied to detect the expressions of chicken BMP15 gene in different tissues. Dual luciferase was used to identify the primary promoter region of the gene. According to the results, the chicken BMP15 gene has a transcript of 1 865 bp in length, encoding 350 amino acids, and its hereditary correlation is the closest to that of turkey. The protein structure primarily consists of disordered coils, there is a signal peptide region and no transmembrane domain. Additionally, BMP15 gene was significantly expressed in both ovaries and granulosa cells of the chicken（P<0.01）according to the constructed tissue-expression profile. Dual-luciferase assay revealed that the core promoter region of chicken BMP15 gene was positioned at -1 bp to -153 bp. This sets the foundations for further research into the BMP15 gene's function and its transcriptional regulatory mechanism.

Key words: Taihang chicken, BMP15 gene, bioinformatics, promoter activity

杨岚, 张晨曦, 樊学伟, 王阳光, 王春秀, 李文婷. 鸡 BMP15 基因克隆、表达模式及启动子活性分析[J]. 生物技术通报, 2023, 39(4): 304-312.

YANG Lan, ZHANG Chen-xi, FAN Xue-wei, WANG Yang-guang, WANG Chun-xiu, LI Wen-ting. Gene Cloning, Expression Pattern, and Promoter Activity Analysis of Chicken BMP15[J]. Biotechnology Bulletin, 2023, 39(4): 304-312.

图/表 13

参考文献 30

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引物名称Name	引物序列 Primer sequence（5'-3'）	用途 Purpose
BMP15- F	ATGGCTTTGCTCCGGCCCTT	BMP15CDS扩增
BMP15-R	GCGGCAGGTGCAGGACTCTGCAAT	BMP15CDS扩增
5R-R1	GCTCGGCCGGATCGCACGCAGATGTGC	5'RACE一轮巢式
5R-R2	GGCAGCGGCAGGCTCTGAGGGTAGGC	5'RACE二轮巢式
5R-R3	CACGGTGTTGGTGCTCAGACTGCGGC	5'RACE三轮巢式
3R-F1	CCCTGCGGACCCCTTCCTCCTCCTCTTC	3'RACE一轮巢式
3R-F2	CTGCGCTCCTTCCCCGTCAGCTTCGCCC	3'RACE二轮巢式
3R-F3	TGCCCACGGCTCCTCCGGGACGGCTACC	3'RACE三轮巢式
NEST	AAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM	CATTTACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
BMP15-q-F	CTGTCACCTCACCCCTTGTC	RT-PCR
BMP15-q-R	CGTTGGTACAGCTCCAGCAT
β-actin-q-F	GAGAGAAGATGACACAGAC
β-actin-q-R	GTCCATCACAATACCAGTGG
pGL4-F1	TTCTCTTTGTGCCGAGCTGA	启动子截短体扩增
pGL4-F2	CCCCACTGCACCCCTTCCCCA
pGL4-F3	CTCTCCTCCAGCCTGACCCTT
pGL4-F4	CAGCTCAGGTACCACCTGGC
pGL4-F5	AGTTTGGCTTGGCACGGCTC
pGL4-F6	TGGGAAGCAGGCAAAGGGAG

引物名称Name	引物序列 Primer sequence（5'-3'）	用途 Purpose
BMP15- F	ATGGCTTTGCTCCGGCCCTT	BMP15CDS扩增
BMP15-R	GCGGCAGGTGCAGGACTCTGCAAT	BMP15CDS扩增
5R-R1	GCTCGGCCGGATCGCACGCAGATGTGC	5'RACE一轮巢式
5R-R2	GGCAGCGGCAGGCTCTGAGGGTAGGC	5'RACE二轮巢式
5R-R3	CACGGTGTTGGTGCTCAGACTGCGGC	5'RACE三轮巢式
3R-F1	CCCTGCGGACCCCTTCCTCCTCCTCTTC	3'RACE一轮巢式
3R-F2	CTGCGCTCCTTCCCCGTCAGCTTCGCCC	3'RACE二轮巢式
3R-F3	TGCCCACGGCTCCTCCGGGACGGCTACC	3'RACE三轮巢式
NEST	AAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM	CATTTACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
BMP15-q-F	CTGTCACCTCACCCCTTGTC	RT-PCR
BMP15-q-R	CGTTGGTACAGCTCCAGCAT
β-actin-q-F	GAGAGAAGATGACACAGAC
β-actin-q-R	GTCCATCACAATACCAGTGG
pGL4-F1	TTCTCTTTGTGCCGAGCTGA	启动子截短体扩增
pGL4-F2	CCCCACTGCACCCCTTCCCCA
pGL4-F3	CTCTCCTCCAGCCTGACCCTT
pGL4-F4	CAGCTCAGGTACCACCTGGC
pGL4-F5	AGTTTGGCTTGGCACGGCTC
pGL4-F6	TGGGAAGCAGGCAAAGGGAG

项目 Item	反应温度 Reaction temperature/℃	时间 Time	循环次数 Cycles
一轮巢式 One round of the nest	95	5 min	1
	95	30 s	20
	89	30 s
	72	1 min
	95	30 s	20
	79	30 s
	72	1 min
	12	∞
二/三轮巢式 Two/Three round of the nest	95	5 min	1
	95	30 s	20
	68	30 s
	72	1 min
	72	10 min	1
	12	∞

项目 Item	反应温度 Reaction temperature/℃	时间 Time	循环次数 Cycles
一轮巢式 One round of the nest	95	5 min	1
	95	30 s	20
	89	30 s
	72	1 min
	95	30 s	20
	79	30 s
	72	1 min
	12	∞
二/三轮巢式 Two/Three round of the nest	95	5 min	1
	95	30 s	20
	68	30 s
	72	1 min
	72	10 min	1
	12	∞

软件名称Software name	网址Website	用途Purpose
BDGP	https://www.fruitfly.org/seq_tools/promoter.html	启动子预测
ExPASy	https://web.expasy.org/	理化性质分析
TMHMM	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0	跨膜域预测
SignalP5.0	https://services.healthtech.dtu.dk/service.php?SignalP-5.0	信号肽预测
NetSurfP-2.0	https://services.healthtech.dtu.dk/service.php?NetSurfP-2.0	蛋白质二级结构预测
SWISS-MODEL	https://swissmodel.expasy.org/interactive	蛋白质三级结构预测
MEGA 7.0软件		NJ法构建系统进化树比
UCSC	http://genome.ucsc.edu/ENCODE/	保守性分析
NCBI	http://www.ncbi.nlm.nih.gov/blast	同源性分析
AliBaba2.1	http://gene-regulation.com/pub/programs/alibaba2/index.html	转录因子结合位点预测
MethPrimer	http://www.urogene.org/methprimer2/	CpG岛预测