猪<i>CRYBB2</i>基因启动子的克隆、活性及转录调控元件分析

doi:10.7668/hbnxb.20193874

摘要/Abstract

摘要：

旨在初步分析猪CRYBB2的启动子活性以及转录调控元件。利用生物信息学分析、PCR扩增、基因克隆、细胞转染、双荧光素酶活性分析等方法,获得了CRYBB2启动子区域的序列特征,构建了不同片段长度的CRYBB2启动子区的双荧光素酶报告基因载体并分析了其荧光素酶活性,进而确定了CRYBB2的核心启动子区域以及关键的调控区域,最后还预测了关键调控区域的转录因子及其结合位点。结果表明,CRYBB2候选启动子区可能包含4个核心启动子以及1个CpG岛,-52~-3 bp可能为CRYBB2基因的核心启动子区域,-505~-19 bp为CRYBB2基因启动子的关键调控区域,且发挥正向调节作用,而-2 060~-505 bp不存在任何对CRYBB2基因启动子活性有影响的调控元件;CRYBB2启动子的关键调控区域包含多个潜在的转录因子结合位点,如TBP、NFIA、FOXP1、NKX2-8、KLF4、Tcf3、Crx、SNAI3、Rfx1和CREB1等。为进一步研究猪CRYBB2的表达机制奠定了基础。

关键词: 猪, CRYBB2, 启动子, 转录活性, 转录因子

Abstract:

The purpose of this study was to preliminarily analyze the promoter activity and transcription regulatory elements of porcine CRYBB2. Using bioinformatics analysis,PCR amplification,gene cloning,cell transfection,double luciferase activity analysis and other methods,we obtained the sequence characteristics of CRYBB2 promoter region,constructed double luciferase reporter gene vectors of CRYBB2 promoter region with different fragment lengths,analyzed its luciferase activity,and then determined the core promoter region and key regulatory region of CRYBB2.Finally,the transcription factors and their binding sites in key regulatory regions were predicted. The results showed that the candidate promoter region of CRYBB2 might contain four core promoters and one CpG island.-52—-3 bp might be the core promoter region of CRYBB2 gene,and-505—-19 bp might be the key regulatory region of CRYBB2 gene promoter,which played a positive regulatory role,while-2 060—-505 bp didn't have any regulatory elements that affected the activity of CRYBB2 gene promoter.The key regulatory region of CRYBB2 promoter contained multiple potential transcription factor binding sites,such as TBP,NFIA,FOXP1,NKX2-8,KLF4,Tcf3,Crx,SNAI3,Rfx1 and CREB1. This study laid a foundation for further study on the expression mechanism of porcine CRYBB2.

Key words: Pig, CRYBB2, Promoter, Transcriptional activity, Transcription factor

龙熙, 袁晓, 陈力, 吴平先, 张亮, 潘红梅, 郭宗义, 柴捷. 猪CRYBB2基因启动子的克隆、活性及转录调控元件分析[J]. 华北农学报, 2023, 38(S1): 400-407. doi: 10.7668/hbnxb.20193874.

LONG Xi, YUAN Xiao, CHEN Li, WU Pingxian, ZHANG Liang, PAN Hongmei, GUO Zongyi, CHAI Jie. Cloning,Transcriptional Activity and Regulatory Elements Analysis of Porcine CRYBB2 Gene Promoter[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(S1): 400-407. doi: 10.7668/hbnxb.20193874.

http://www.hbnxb.net/CN/Y2023/V38/IS1/400

图/表 6

参考文献 60

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引物名称 Primer name	引物序列(5'—3') Sequence (5'—3')	扩增长度/bp Length
pGL4.17-2060	F:CCGctcgagTTTCCCTAGATGGTTGCAGACT	2 311
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-1507	F:CCGctcgagATGAACGGGCTCTCGTAAAATA	1 757
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-505	F:CCGctcgagGAAGCTGCACCATTTTATACCC	754
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-19	F:CCGctcgagCCCGCCCAGGAGTAAAGAT	268
	R:CCCaagcttGCTTTCTGTCCCATCACGTC

引物名称 Primer name	引物序列(5'—3') Sequence (5'—3')	扩增长度/bp Length
pGL4.17-2060	F:CCGctcgagTTTCCCTAGATGGTTGCAGACT	2 311
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-1507	F:CCGctcgagATGAACGGGCTCTCGTAAAATA	1 757
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-505	F:CCGctcgagGAAGCTGCACCATTTTATACCC	754
	R:CCCaagcttGCTTTCTGTCCCATCACGTC
pGL4.17-19	F:CCGctcgagCCCGCCCAGGAGTAAAGAT	268
	R:CCCaagcttGCTTTCTGTCCCATCACGTC

起始位点/bp Start	结束位点/bp End	启动子序列(5'—3') Promoter sequence(5'—3')	分数 Score(0-1)
-1 944	-1 895	GGTATCAGCATATAAATACCCCAGCACCCTCCCCTTGGTGGGAACTTGA	0.99
-1 056	-1 007	CCTTCTGTCTCTAAATGCAGCTGCACTAAGAACCTCATACAAATGCAGTC	0.92
-52	-3	GTCAGTTCCCCTATAAAAGGCAGCCTCCTTCTCCCCGCCCAGGAGTAAAG	0.98
-19	30	CCCGCCCAGGAGTAAAGATGGCGGCTGTGTCCTGCTCTTTGTGGGCTGCA	0.87

起始位点/bp Start	结束位点/bp End	启动子序列(5'—3') Promoter sequence(5'—3')	分数 Score(0-1)
-1 944	-1 895	GGTATCAGCATATAAATACCCCAGCACCCTCCCCTTGGTGGGAACTTGA	0.99
-1 056	-1 007	CCTTCTGTCTCTAAATGCAGCTGCACTAAGAACCTCATACAAATGCAGTC	0.92
-52	-3	GTCAGTTCCCCTATAAAAGGCAGCCTCCTTCTCCCCGCCCAGGAGTAAAG	0.98
-19	30	CCCGCCCAGGAGTAAAGATGGCGGCTGTGTCCTGCTCTTTGTGGGCTGCA	0.87

名称 Name	得分 Score	相对得分 Relative score	起点/bp Start	终点/bp End	链 Strand	预测序列 Predicted sequence
TBP	11.086 1	0.906 820	6	20	-	GTATAAAATGGTGCA
NFIA	11.603 6	0.970 820	63	72	-	ACTGCCAAAA
FOXP1	15.954 0	0.932 665	73	87	+	CCTAAGAAAACAAAA
NKX2-8	12.380 3	0.994 405	145	153	+	GCACTTGAA
KLF4	11.314 4	0.902 393	266	277	+	TACTCCACCCAC
Tcf3	13.729 4	0.967 873	307	317	+	CCCAGCTGGTG
SNAI3	13.804 8	0.959 655	346	355	-	AGCAGGTGGA
Rfx1	15.536 7	0.919 571	423	436	+	GTTCCCCTGGTAAC

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猪CRYBB2基因启动子的克隆、活性及转录调控元件分析

Cloning,Transcriptional Activity and Regulatory Elements Analysis of Porcine CRYBB2 Gene Promoter

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