双向启动子调控鸡胚性腺PRLR和SPEF2的转录表达

doi:10.11843/j.issn.0366-6964.2025.02.018

摘要/Abstract

摘要：

旨在揭示鸡催乳素受体基因(PRLR)和精子鞭毛蛋白2基因(SPEF2)在鸡胚性腺的双向转录调控特征。采集4个胚龄(E12.5、E16.5、E18.5、E21.5)180只大午金凤鸡胚左右侧性腺，每3个样品混池组成1个重复，除E21.5母鸡右侧卵巢完全退化，其他同胚龄同性别同侧性腺共14分组，利用RT-qPCR检测PRLR和SPEF2的表达变化。利用cDNA末端快速扩增技术(5'RACE)和双荧光素酶报告基因系统(DLRA)分别鉴定PRLR和SPEF2的转录起始位点(TSS)及其核心启动子区，利用亚硫酸氢盐测序技术(BSP)检测启动子区甲基化水平。结果发现，PRLR在E12.5~E21.5睾丸中的表达显著高于卵巢(P<0.05)，而SPEF2在E18.5~E21.5卵巢中的表达显著高于睾丸(P<0.05)。PRLR的10个TSS中5个具有启动子活性，SPEF2的3个TSS全部具有启动子活性。PRLR的PA1启动子和SPEF2的SC启动子活性最高(P<0.05)，进一步检测发现二者的最高活性区域分别是长565 bp和478 bp的反向互补双向启动子区。478 bp的双向启动活性显著高于565 bp(P<0.05)，且二者对PRLR的启动活性均显著高于SPEF2(P<0.05)，这与E12.5~E21.5鸡胚性腺中PRLR的转录表达显著高于SPEF2(P<0.05)一致。E21.5卵巢双向启动子区443 bp的CpG岛甲基化水平显著高于睾丸(P<0.05)，与睾丸PRLR表达显著高于卵巢一致；位于SPEF2第1内含子区159 bp的CpG岛甲基化水平睾丸显著高于卵巢(P<0.05)，与睾丸SPEF2的表达显著低于卵巢一致。综上，478 bp的双向核心启动子区调控鸡胚性腺中PRLR和SPEF2的转录表达，并且启动PRLR的转录活性高于SPEF2，PRLR的转录表达水平高于SPEF2；甲基化参与双向启动子调控性腺PRLR的转录表达，E21.5卵巢甲基化水平高，PRLR在卵巢表达低于睾丸。这些研究结果为揭示PRLR和SPEF2在鸡胚性腺发育中的转录调控机制研究提供理论依据。

关键词: 鸡, PRLR, SPEF2, 双向启动子, 甲基化

Abstract:

This study aimed to reveal the bidirectional transcriptional regulation character of prolactin receptor gene (PRLR) and sperm flagellar protein 2 (SPEF2) in chicken embryonic gonads. The left and right gonads of 180 Dawu Jinfeng chicken embryos at 4 embryonic ages were collected, and each 3 samples mixing pools formed a repeat. Except for the complete degeneration of the right ovary of E21.5 hens, the other ipsilateral gonads of the same embryonic age and sex were divided into 14 groups. The expression changes of PRLR and SPEF2 in gonads of Dawujinfeng chickens at 4 embryonic ages (E12.5, E16.5, E18.5 and E21.5) were detected using real-time quantitative PCR (RT-qPCR). The transcription start sites (TSS) of the two genes were identified using rapid amplification of cDNA ends (5'RACE), their core promoter region was identified using dual luciferase reporter assay (DLRA). The methylation level in promoter region was detected using bisulfite genomic sequencing PCR (BSP). Results were as follows: The expression of PRLR in testis from E12.5 to E21.5 was higher than in ovary (P<0.05), while the expression of SPEF2 in ovary from E18.5 to E21.5 was higher than in testis (P<0.05). Among the 10 TSSs of PRLR, 5 had promoter activity, and all the 3 TSSs of SPEF2 had promoter activity. PA1 promoter region of PRLR and SC of SPEF2 exhibited the highest promoter activity (P<0.05), respectively. Further detection found that 565 bp of PA1 and 478 bp of SC in reverse complement had the highest promoter activity, respectively. The bidirectional promoter activity of 478 bp region was significantly higher than 565 bp (P<0.05), and both had significantly higher promoter activity for PRLR than for SPEF2 (P<0.05), which was in conformity with that the expression of PRLR was significantly higher than SPEF2 in chicken embryonic gonads at E12.5-E21.5 (P<0.05). That the methylation level of 443 bp CpG island in the bidirectional promoter region was significantly higher in E21.5 ovary than in testis (P<0.05), was in accordance with the higher expression of PRLR in testis. The methylation level of 159 bp CpG island in the first intron of SPEF2 was significantly higher in testis than in ovary (P<0.05), correlated with the higher expression of SPEF2 in ovary. In summary, the transcriptional expression of PRLR and SPEF2 in chicken embryonic gonads was regulated by bidirectional promoter with 478 bp core region, which had higher promoter activity for PRLR than SPEF2, and the expression of PRLR in gonad was higher than SPEF2. The methylation of the CpG island in BDP participated in regulating the transcriptional expression of PRLR, with ovary at E21.5 had higher methylation level, and PRLR was expressed lower in ovary. These results provided a theoretical basis for revealing transcriptional regulation mechanism of PRLR and SPEF2 in chicken gonadal development.

Key words: chicken, PRLR, SPEF2, bidirectional promoter, methylation

中图分类号:

S831.2

王涛, 王麒, 董娇娇, 王德贺, 李兰会. 双向启动子调控鸡胚性腺PRLR和SPEF2的转录表达[J]. 畜牧兽医学报, 2025, 56(2): 666-678.

WANG Tao, WANG Qi, DONG Jiaojiao, WANG Dehe, LI Lanhui. Bidirectional Promoter Regulate Transcriptional Expression of PRLR and SPEF2 in Chicken Embryonic Gonads[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 666-678.

图/表 12

表 1

试验所用引物"

引物名称 Primer	引物序列(5′→3′) Primer sequence	用途 Usage
PRLR-5′RACE-1	gattaccgccaagcttCCAGGGTCTAACGTACAGCGGACCTGAA	RACE
PRLR-5′RACE-2	gattaccgccaagcttTTATCTTTGGTCCTGGAACTGGCGGTAG	RACE
SPEF2-5′RACE-1	gattaccgccaagcttACAGCAATCCTCCGCTCCTGCTGAGA	RACE
SPEF2-5′RACE-2	gattaccgccaagcttCAGTTGGGCACCATTCCCCTGTCATA	RACE
pGL3-PA1	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$TGTGACATGAGTGTGTCAGT	DLRA
pGL3-PA1	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CTGGGGAACGATGTAGACCT	DLRA
pGL3-PA2	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CACACATCAAATCGCACAACGG	DLRA
pGL3-PA2	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$AGTGCAGAACACAGACATCTTTC	DLRA
pGL3-PC1	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CCAGTACTTCCTTGATTCGCCT	DLRA
pGL3-PC1	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$TGGTATTGAGAAGTATGTCGACCC	DLRA
pGL3-PB5	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CCTTCCGTGTGTTTACCTGACT	DLRA
pGL3-PB5	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CAAGGTCCATGCCAAATGAGAG	DLRA
pGL3-PC2	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$GGTAGAGGGTTAAGCTGAGGC	DLRA
pGL3-PC2	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$TCAATCAGACCTTCAGGAACCC	DLRA
pGL3-PA10	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$GTAAGGGTTCCTGAAGGTCTGATT	DLRA
pGL3-PA10	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$GTGTTGTGGCTTCTCCTCTGTC	DLRA
pGL3-PA4	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$AAGCCCATCCTATGAGCAGC	DLRA
pGL3-PA4	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCAGCACATTTTACACAGAAACAC	DLRA
pGL3-PB2	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$ATGGAAGAACTCAGGCGTCA	DLRA
pGL3-PB2	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$ACCATTCTTGTTCAGGCAGTATCT	DLRA
pGL3-SDA	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CTGGGGAACGATGTAGACCT	DLRA
pGL3-SDA	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$TGTGACATGAGTGTGTCAGT	DLRA
pGL3-SA	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$AGTGCAGAACACAGACATCTTTC	DLRA
pGL3-SA	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CACACATCAAATCGCACAACGG	DLRA
pGL3-SB	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$ACACGAACGCCTTACACAAGA	DLRA
pGL3-SB	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$GGAACCACTTCCCCATTCCA	DLRA
pGL3-SC	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CCTAAGAGTCCCCTGCGGTG	DLRA
pGL3-SC	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
PRLR-P1-865	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CTGTGGAAAGTCGCTCACACG	DLRA
PRLR-P1-865	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$ACAGCCACCTGTAGTGAGGA	DLRA
PRLR-P2-713	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$TACCAAGAGCGACGGAGCCT	DLRA
PRLR-P2-713	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$ACAGCCACCTGTAGTGAGGA	DLRA
PRLR-P3-565	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CTCAGACTCGCCGCATCCT	DLRA
PRLR-P3-565	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$ACAGCCACCTGTAGTGAGGA	DLRA
PRLR-P4-345	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$GCATGCGCAAAGGAGAGGA	DLRA
PRLR-P4-345	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$ACAGCCACCTGTAGTGAGGA	DLRA
SPEF2-P1-2178	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$AGTGCAGAACACAGACATCTTTC	DLRA
SPEF2-P1-2178	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P2-1852	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$TCAAGGTCAGAGGTTATACGGAA	DLRA
SPEF2-P2-1852	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P3-1684	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$ACACTCAGATGGCAGACACTC	DLRA
SPEF2-P3-1684	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P4-1428	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$GGCAACTCAATAACTCGCCC	DLRA
SPEF2-P4-1428	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P5-1142	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CTGGGGAACGATGTAGACCT	DLRA
SPEF2-P5-1142	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P6-931	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$TTCTGGCACACGAACGCCTT	DLRA
SPEF2-P6-931	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P7-703	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CCTAAGAGTCCCCTGCGGTG	DLRA
SPEF2-P7-703	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P8-478	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$ATGACAGCCACCTGTAGTGAGG	DLRA
SPEF2-P8-478	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
SPEF2-P9-205	cagaacatttctctatcgata$\underline{{\rm{GGTACC}}}$CATGCGCGGCCCTCTC	DLRA
SPEF2-P9-205	aagcttacttagatcgcagat$\underline{{\rm{CTCGAG}}}$CCCACGCTCCGGGAAAGTT	DLRA
PRLR	TGGTGGAAGATGAAGAAGAGCAT	RT-qPCR
PRLR	AGACCTGTTTTGTTTGACCTGTG	RT-qPCR
SPEF2	GCTGCCTGTGCTGTAGTTTC	RT-qPCR
SPEF2	AAGAGAAGCGGAGGACACTT	RT-qPCR
β-actin	CTGTGCCCATCTATGAAGGCTA	RT-qPCR
β-actin	ATTTCTCTCTCGGCTGTGGTG	RT-qPCR
CpG-PRLR	TTTTGTTATGTGTTGTAGGTTTTTAAGT	BSP
CpG-PRLR	TCCAAAAATTAAAAAAATTTTAATAAATTT	BSP
CpG-PRLR-1	TGAGGAAAATTAATTTTATTTTTGAAAG	BSP
CpG-PRLR-1	AACAACCACCTATAATAAAAACCCC	BSP
CpG-PRLR-2	TTTTTGTTATTTTTTGAGGTAG	BSP
CpG-PRLR-2	CCACCTATAATAAAAACCCCC	BSP
CpG-SPEF2	GGTTTTTTAGTTTAAGAGTTTTTTG	BSP
CpG-SPEF2	ACTTACTACTCCCTCTATCAACCTC	BSP

表 1

图 1

表 2

图 2

图 3

图 4

图 5

图 6

图 7

图 8

图 9

图 10

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组织 Tissue	左Left				右Right
组织 Tissue	E12.5	E16.5	E18.5	E21.5	E12.5	E16.5	E18.5	E21.5
睾丸Testis	-0.333	-0.340	0.132	0.638^*	-0.206	-0.500	0.980^**	0.265
卵巢Ovary	0.407	0.859^*	0.786^*	0.762^*	0.900^*	0.004	—	—

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