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

doi:10.7668/hbnxb.20193874

Abstract

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

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.

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Figures/Tables 6

References 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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