预扩增qPCR技术检测少量猪早期胚胎细胞基因表达的研究

doi:10.11843/j.issn.0366-6964.2024.12.021

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5567-5574.doi: 10.11843/j.issn.0366-6964.2024.12.021

预扩增qPCR技术检测少量猪早期胚胎细胞基因表达的研究

晏超¹^,²(), 刘永刚², 谢浩¹, 彭翠婷¹, 张才用¹, 赵玉兰¹^,³, 齐霖¹, 陈指龙¹^,³^,⁴^,⁵^,*(), 唐中林¹^,³^,⁴^,⁵^,*()

1. 中国农业科学院(深圳)农业基因组研究所，深圳 518000
2. 云南农业大学动物科学技术学院，昆明 650201
3. 佛山鲲鹏现代农业研究院，佛山 528225
4. 岭南现代农业科学与技术广东省实验室深圳分中心，深圳 518000
5. 农业农村部畜禽生物组学重点实验室，深圳 518000

收稿日期:2024-04-15 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 陈指龙,唐中林 E-mail:15170529593@163.com;chenzhilong@caas.cn;tangzhonglin@caas.cn
作者简介:晏超(1999-)，男，江西宜春人，硕士生，主要从事动物遗传育种与繁殖研究，E-mail: 15170529593@163.com
基金资助:
广东省基础与应用基础研究基金委员会区域联合基金—青年基金项目(2021A1515110046);深圳市可持续发展科技专项项目(KCXFZ20201221173213037);国家自然科学基金青年项目(32302747)

Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR

YAN Chao¹^,²(), LIU Yonggang², XIE Hao¹, PENG Cuiting¹, ZHANG Caiyong¹, ZHAO Yulan¹^,³, QI Lin¹, CHEN Zhilong¹^,³^,⁴^,⁵^,*(), TANG Zhonglin¹^,³^,⁴^,⁵^,*()

1. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
2. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
3. Kunpeng Institute of Modern Agriculture at Foshan, Foshan 528225, China
4. Shenzhen Branch Center of Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology, Shenzhen 518000, China
5. Key Laboratory of Livestock and Poultry Biohistology of Ministry of Agriculture and Rural Affairs, Shenzhen 518000, China

Received:2024-04-15 Online:2024-12-23 Published:2024-12-27
Contact: CHEN Zhilong, TANG Zhonglin E-mail:15170529593@163.com;chenzhilong@caas.cn;tangzhonglin@caas.cn

摘要/Abstract

摘要：

旨在比较3种qPCR方法检测少量猪早期胚胎细胞多能性和组蛋白乙酰化修饰相关基因的动态表达情况。本研究收集不同时期(1-细胞、2-细胞、4-细胞、8-细胞、桑葚胚和囊胚)猪孤雌激活胚胎，利用常规RT-qPCR、cDNA预扩增qPCR和样本直接预扩增qPCR检测微量胚胎细胞多能性和组蛋白乙酰化修饰相关基因的表达情况。结果显示，预扩增qPCR具有稳定的扩增曲线，熔解曲线呈现稳定单峰，而常规RT-qPCR检测的扩增曲线循环阈值在35以上，熔解曲线呈现多峰；胚胎细胞直接预扩增后的样品稀释20 000倍仍能检测目的基因的表达，且能稳定检测单个胚胎细胞的基因表达情况；多能性和组蛋白乙酰化修饰相关基因在猪孤雌激活早期胚胎发育过程中呈现先升高后降低的表达趋势，在基因组激活阶段的表达水平最高。综上表明，样本直接预扩增qPCR的检测灵敏性和准确性更高，且操作相对简单、成本较低，适用于微量胚胎细胞的基因表达检测，可为研究早期胚胎发育机制提供方法参考。

关键词: 预扩增, qPCR, 猪早期胚胎, 微量细胞, 基因表达

Abstract:

This study aimed to compare 3 quantitative polymerase chain reaction (qPCR) methods for detecting the dynamic expression of genes associated with pluripotency and histone acetylase modification in trace cells of porcine early embryo. Porcine parthenogenetic activated embryos at different stages (1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst) were collected, and the expression of genes associated with cell pluripotency and histone acetylase modification were detected by conventional RT-qPCR, cDNA pre-amplified qPCR and sample direct pre-amplified qPCR. The results indicated that the pre-amplified qPCR exhibited a stable amplification curve, and the melt curve displayed a consistent single peak, the conventional RT-qPCR produced cyclic thresholds above 35 and multiple peaks in the melt curve. Notably, target gene expression was successfully detected even after a 20 000-fold dilution of embryonic cells using pre-amplification, and gene expression at the single embryonic cells could be reliably assessed. The expression patterns of genes related to pluripotency and histone acetylase modification exhibited an initial increase followed by a decline across different stages of porcine parthenogenetic activated embryos, with the highest expression levels occurring at the genome activation stage. In conclusion, pre-amplified qPCR demonstrates superior sensitivity and accuracy, with a relatively simple operational protocol and lower costs, making it a suitable approach for gene expression analysis in trace cells of embryo. This methodology has the potential to advance the understanding for the mechanisms underlying early embryonic development.

Key words: preamplification, qPCR, porcine early embryo, trace cells, gene expression

中图分类号:

S828.3

晏超, 刘永刚, 谢浩, 彭翠婷, 张才用, 赵玉兰, 齐霖, 陈指龙, 唐中林. 预扩增qPCR技术检测少量猪早期胚胎细胞基因表达的研究[J]. 畜牧兽医学报, 2024, 55(12): 5567-5574.

YAN Chao, LIU Yonggang, XIE Hao, PENG Cuiting, ZHANG Caiyong, ZHAO Yulan, QI Lin, CHEN Zhilong, TANG Zhonglin. Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5567-5574.

图/表 4

表 1

图 1

图 2

图 3

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基因 Gene	引物序列(5′→3′) Primers sequence
EF1A1	F: AATGCGGTGGGATCGACAAA
	R: CACGCTCACGTTCAGCCTTT
OCT4	F: GTGTTCAGCCAAACGACCAT
	R: TTGCCTCTCACTCGGTTCTC
NANOG	F: GGTTTATGGGCCTGAAGAAA
	R: GATCCATGGAGGAAGGAAGA
SOX2	F: ATGCACAACTCGGAGATCAG
	R: TATAATCCGGGTGCTCCTTC
HADAC1	F: AGGGCACCAAGAGGAAAGTC
	R: GAGGGCGATAGATTTCCATT
HADAC2	F: AGCCAGATGTCTTCAAAAAG
	R: ACAGAGCCAAATCAGAACAG
MBD3	F: ACCTGAGCACCTTTGATTTCCG
	R: CGAAGGCGTTCAGACCACTC
HAT1	F: ACGCCTCGCTCAAGAGTAGA
	R: AGTCATCATCTGATTTCACA
CBP	F: GCGTCCTCCAAGTATTACCC
	R: GAGCAGGGTCTAAGAGAGTG
DUXC	F: CAGCCAAGTCCAGTTCTGCA
	R: AGTGCAGGTCTTCACTGGGT

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预扩增qPCR技术检测少量猪早期胚胎细胞基因表达的研究

Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR

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