发情周期对香猪卵巢生物钟相关基因表达的影响

doi:10.13304/j.nykjdb.2023.0132

摘要/Abstract

摘要：

生物钟在生物体（如猪）的生殖系统中起着重要作用。为研究发情周期对香猪卵巢生物钟相关基因表达的影响，本研究分析了香猪卵巢在发情期和未发情期生物钟相关基因的表达和可变剪接。结果在香猪卵巢中共检测到90个生物钟相关基因的表达，其中有33个生物钟相关基因在发情期和未发情期差异表达。从34个生物钟相关基因的转录本中鉴定出44个差异可变剪接事件。此外，还发现包括核心时钟成分 arntl和 cry1在内的20个基因仅在可变剪接水平上被差异调节，包括 per1和 clock在内的14个基因同时在基因表达和可变剪接水平被差异调节。通过RT-qPCR 实验，证实了核心时钟基因 per1、cry1、 clock和 arntl以及时钟相关基因 ppp1cb和 ntrk1在香猪卵巢中的表达具有节律性。结果表明，香猪卵巢生物钟可能通过转录和转录后调控水平在调节卵巢生理功能中发挥着重要作用。

关键词: 昼夜节律, 香猪, 卵巢, 发情周期, 可变剪接

Abstract:

The biological clock plays a critical role in the reproductive system of various living organisms like swine. To examine the effects of estrus cycle on the expression of ovarian biological clock-related gene in Xiang pig， this study analyzed the expression and alternative splicing of biological clock-related genes in ovary during estrus and diestrus periods. In total， 90 clock-related genes expressed in the ovaries of the Xiang pig were detected and 33 clock-related genes differentially expressed between estrous and diestrous stages. 44 differential splicing events were identified from the transcripts of 34 biological clock-related genes. Furthermore， it was found 20 genes including the core clock components， arntl and cry1 were differentially regulated only at AS level and 14 genes， including per1 and clock， were differentially regulated at both expression and AS levels. We also proved that the core clock genes per1， cry1， clock and arntl and the clock-related genes， ppp1cb and ntrk1 were rhythmically expressed in Xiang pig ovaries by RT-qPCR experiments. The results demonstrated that the biological clock in the ovaries of Xiang pig might play an important role in regulating the ovarian physiological functions by the transcriptional and post-transcriptional regulation.

Key words: circadian rhythm, Xiang pig, ovary, estrus cycle, alternative splicing

中图分类号:

S828

唐靓婷, 黄世会, 牛熙, 李升, 王嘉福, 冉雪琴. 发情周期对香猪卵巢生物钟相关基因表达的影响[J]. 中国农业科技导报, 2023, 25(12): 67-84.

Liangting TANG, Shihui HUANG, Xi NIU, Sheng LI, Jiafu WANG, Xueqin RAN. Effects of Estrus Cycle on Expression of Ovarian Biological Clock-related Genes of Xiang Pig[J]. Journal of Agricultural Science and Technology, 2023, 25(12): 67-84.

图/表 12

参考文献 67

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qPCR detection			RT-PCR detection
Primer name	Primer sequence （5’-3’）	Size/bp	Primer name	Primer sequence （5’-3’）	Size/bp
ppp1cb-F	AAGAGGAAACCATGAGTGTGCTAG	112	ppp1cb-MXE-1F	GGAAGACCTTCACTGATTGT	131
ppp1cb-R	GCAGTTGAAACAATCAGTGAAGGTC	112	ppp1cb-MXE-1R	CCAACCTTGCACATCCTTA	131
atf4-F	TTGGTCAGTGCCTCAGACAACAG	95	ppp1cb- MXE-2F	GCAGTCTATGGAGCAGATT	156
atf4-R	GCATCGAAGTCGAACTCCTTCAGAT	95	ppp1cb-MXE-2R	TCATTCCACCAGCATTATCA	156
csnk1d-F	GCGGGTCCTTCGGAGACATCTAT	102	thrap3- MXE- 1F	CCTGACTGTAAATTGGAGAAG	112
csnk1d-F	GCGGGTCCTTCGGAGACATCTAT	102	thrap3- MXE- 1R	GAACGAGACCTGGAACTTAT	112
per1-F	CATCGCTTACAGCCTCCTGAGTG	105	thrap3- MXE- 2F	AGTCTGGATCTCGCTCTT	143
per1-R	ATGAGTTCTTTCTGGGTCCTTGCC	105	thrap3- MXE- 2R	ATTCTCTGTCTTCTGCTCAT	143
klf9-F	CCATTACAGAGTGCATACAGGTGAA	139	top2a- MXE- 1F	TCTGCTAGTCCTCGATACATCT	112
klf9-R	ACACAGCGGACAGCGGAACT	139	top2a- MXE- 1R	GCCACTTCACCACTAATCACA	112
nampt-F	AGATCCAAGAAGCCAAAGAGGTGTA	141	top2a- MXE- 2F	CCCAACTTTGATGTTCGTGAAG	125
namptR	GAATGACAGAGCCCTCAGGAACAG	141	top2a- MXE- 2R	GGTGTCTTCTCAGTGCCATTC	125
bhlhe40-F	ACTGGAGCCTGGAGACCTACCA	91	clock- SE-F	GGTGGTCAGTTTCAGTTCT	111
bhlhe40-R	GCTGTCCTCGCTCCGCTTTATTC	91	clock- SE-R	TTCTTCTTGTTGCCGATGA	111

qPCR detection			RT-PCR detection
Primer name	Primer sequence （5’-3’）	Size/bp	Primer name	Primer sequence （5’-3’）	Size/bp
ppp1cb-F	AAGAGGAAACCATGAGTGTGCTAG	112	ppp1cb-MXE-1F	GGAAGACCTTCACTGATTGT	131
ppp1cb-R	GCAGTTGAAACAATCAGTGAAGGTC	112	ppp1cb-MXE-1R	CCAACCTTGCACATCCTTA	131
atf4-F	TTGGTCAGTGCCTCAGACAACAG	95	ppp1cb- MXE-2F	GCAGTCTATGGAGCAGATT	156
atf4-R	GCATCGAAGTCGAACTCCTTCAGAT	95	ppp1cb-MXE-2R	TCATTCCACCAGCATTATCA	156
csnk1d-F	GCGGGTCCTTCGGAGACATCTAT	102	thrap3- MXE- 1F	CCTGACTGTAAATTGGAGAAG	112
csnk1d-F	GCGGGTCCTTCGGAGACATCTAT	102	thrap3- MXE- 1R	GAACGAGACCTGGAACTTAT	112
per1-F	CATCGCTTACAGCCTCCTGAGTG	105	thrap3- MXE- 2F	AGTCTGGATCTCGCTCTT	143
per1-R	ATGAGTTCTTTCTGGGTCCTTGCC	105	thrap3- MXE- 2R	ATTCTCTGTCTTCTGCTCAT	143
klf9-F	CCATTACAGAGTGCATACAGGTGAA	139	top2a- MXE- 1F	TCTGCTAGTCCTCGATACATCT	112
klf9-R	ACACAGCGGACAGCGGAACT	139	top2a- MXE- 1R	GCCACTTCACCACTAATCACA	112
nampt-F	AGATCCAAGAAGCCAAAGAGGTGTA	141	top2a- MXE- 2F	CCCAACTTTGATGTTCGTGAAG	125
namptR	GAATGACAGAGCCCTCAGGAACAG	141	top2a- MXE- 2R	GGTGTCTTCTCAGTGCCATTC	125
bhlhe40-F	ACTGGAGCCTGGAGACCTACCA	91	clock- SE-F	GGTGGTCAGTTTCAGTTCT	111
bhlhe40-R	GCTGTCCTCGCTCCGCTTTATTC	91	clock- SE-R	TTCTTCTTGTTGCCGATGA	111

qPCR detection			RT-PCR detection
Primer name	Primer sequence （5’-3’）	Size/bp	Primer name	Primer sequence （5’-3’）	Size/bp
id4-F	CAGTGCGATATGAACGACTGCTA	89	cipc- SE-F	GCAGAGCCAATAACCCTAA	236
id4-R	AGGATCTCCACTTTGCTGACTTTCT	89	cipc- SE-R	CGAGTTGACGAGACACTT	236
ass1-F	F：CTGCATCCTCGTGTGGCTGAA	95	mapk9- SE-F	CGTTACCAGCAGTTGAAACCA	157
ass1-R	R：TCTTCCTGGCTTCCTCAAAGTCTTC	95	mapk9- SE-R	AGAGGACAAGTTCACGATAAGC	157
ntrk1-F	TACATCGAGAACCAGCAGCACCT	146	cry- RI-F	AGCAAGGTAAGAATGTAGCA	147
ntrk1-R	AGATGACTGAGCCGAGGAGTGAA	146	cry- RI-R	CGTGTCCTCTTCCTGACT	147
			per1- RI-F	CTGCTGTAGTCTCCTGTCT	189
			per1- RI-R	GGAAGGTCACCTGTGGAT	189
			ppp1cb- RI-F	GCGTGACTTGTAGGTGAG	105
			ppp1cb- RI-R	CCATCTTGTCGGCACTAG	105

qPCR detection			RT-PCR detection
Primer name	Primer sequence （5’-3’）	Size/bp	Primer name	Primer sequence （5’-3’）	Size/bp
id4-F	CAGTGCGATATGAACGACTGCTA	89	cipc- SE-F	GCAGAGCCAATAACCCTAA	236
id4-R	AGGATCTCCACTTTGCTGACTTTCT	89	cipc- SE-R	CGAGTTGACGAGACACTT	236
ass1-F	F：CTGCATCCTCGTGTGGCTGAA	95	mapk9- SE-F	CGTTACCAGCAGTTGAAACCA	157
ass1-R	R：TCTTCCTGGCTTCCTCAAAGTCTTC	95	mapk9- SE-R	AGAGGACAAGTTCACGATAAGC	157
ntrk1-F	TACATCGAGAACCAGCAGCACCT	146	cry- RI-F	AGCAAGGTAAGAATGTAGCA	147
ntrk1-R	AGATGACTGAGCCGAGGAGTGAA	146	cry- RI-R	CGTGTCCTCTTCCTGACT	147
			per1- RI-F	CTGCTGTAGTCTCCTGTCT	189
			per1- RI-R	GGAAGGTCACCTGTGGAT	189
			ppp1cb- RI-F	GCGTGACTTGTAGGTGAG	105
			ppp1cb- RI-R	CCATCTTGTCGGCACTAG	105

Period	Sample	Input read pairs	Both surviving	Forward only surviving	Reverse only surviving	Dropped	Percentage of mapped reads/%
Diestrus	D1	77 089 626	74 382 355 （96.49%）	2 471 254 （3.21%）	164 408 （0.21%）	69 380 （0.09%）	96.01
	D2	77 040 960	74 260 107 （96.39%）	2 550 481 （3.31%）	160 145 （0.21%）	70 227 （0.09%）	95.88
	D3	76 170 080	73 520 243 （96.52%）	2 408 511 （3.16%）	167 409 （0.22%）	73 917 （0.10%）	95.93
	D4	77 100 634	74 147 772 （96.17%）	2 713 372 （3.52%）	165 857 （0.22%）	73 633 （0.10%）	96.26
	D5	76 860 177	74 164 234 （96.49%）	2 449 821 （3.19%）	177 911 （0.23%）	68 211 （0.09%）	96.06
	D6	76 903 234	74 486 606 （96.86%）	2 179 432 （2.83%）	173 980 （0.23%）	63 216 （0.08%）	96.11
	D7	77 054 008	74 411 266 （96.57%）	2 408 339 （3.13%）	163 511 （0.21%）	70 892 （0.09%）	95.95
Estrus	E1	75 005 968	74 960 964 （99.94%）	45 004 （0.06%）	0 （0.00%）	0 （0.00%）	95.98
	E2	74 990 854	74 960 858 （99.96%）	29 996 （0.04%）	0 （0.00%）	0 （0.00%）	95.90
	E3	75 206 715	75 176 632 （99.96%）	30 083 （0.04%）	0 （0.00%）	0 （0.00%）	95.93
	E4	75 187 643	75 135 012 （99.93%）	52 631 （0.07%）	31 （0.00%）	0 （0.00%）	96.24
	E5	75 184 086	75 154 012 （99.96%）	30 074 （0.04%）	31 （0.00%）	1 （0.00%）	96.15
	E6	76 109 159	74 099 877 （97.36%）	1 682 012 （2.21%）	243 549 （0.32%）	83 720 （0.11%）	94.13
	E7	75 002 160	73 149 607 （97.53%）	1 545 044 （2.06%）	225 006 （0.30%）	82 502 （0.11%）	93.57