转录因子ZBTB6激活猪卵巢颗粒细胞中lncRNA NORHA转录和功能

doi:10.3864/j.issn.0578-1752.2026.13.016

摘要/Abstract

摘要：

【目的】了解猪lncRNA NORHA基因远端核心启动子特征，探索转录因子ZBTB6激活其转录和功能的机制，为解析NORHA的转录调控网络提供依据。【方法】利用PCR扩增和测序技术获得大白猪NORHA基因远端核心启动子序列，利用生物信息学方法预测转录因子结合位点、分析转录因子ZBTB6编码基因的序列特征。利用酶切技术构建猪ZBTB6基因过表达载体，Western blot等技术验证其在猪卵巢颗粒细胞（sow granulosa cells，sGCs）中的过表达效率。利用荧光素酶活性分析和qPCR技术分析ZBTB6对sGCs中NORHA基因远端核心启动子活性和转录的影响。利用ChIP和DNA测序技术检测ZBTB6与sGCs中NORHA基因远端核心启动子ZBTB6结合位点（ZBS）的结合情况。利用ELISA技术检测卵泡液中孕酮（progesterone，P4）和雌二醇（estradiol，E2）浓度。利用qPCR技术检测卵泡中ZBTB6和NORHA表达水平、分析ZBTB6对sGCs中BCL2和BAX表达的影响。【结果】在526bp大白猪NORHA基因远端核心启动子上预测到包括ZBTB6在内的255个转录因子的结合位点。构建的过表达载体在sGCs中成功实现ZBTB6的过表达。过表达ZBTB6后，sGCs中NORHA的转录水平极显著上调。相关性分析发现猪卵泡中ZBTB6与NORHA表达水平呈显著正相关。在NORHA基因远端核心启动子-2276/-2264 nt处发现一个ZBS位点，荧光素酶活性分析发现ZBTB6可通过这个位点激活远端核心启动子活性。ChIP和测序显示，ZBTB6可与sGCs中NORHA基因远端核心启动子ZBS位点直接结合。猪与哺乳动物其他物种ZBTB6基因在进化上保守，均含有保守的DNA结合域。ZBTB6在猪卵泡闭锁过程中极显著上调，与卵泡闭锁标志指标P4/E2比值呈极显著正相关。共转试验显示，过表达ZBTB6可显著下调抗凋亡基因BCL2的表达，上调促凋亡基因BAX的表达，以及下调凋亡标志指标BCL2/BAX比值，而敲减NORHA则可使之逆转。【结论】ZBTB6是sGCs中NORHA基因的转录激活因子，通过直接激活NORHA基因的转录，促进sGCs凋亡和卵泡闭锁。

关键词: 猪, NORHA, ZBTB6, 远端核心启动子, 卵巢颗粒细胞凋亡

Abstract:

【Objective】 This study was aimed to characterize the distal core promoter of the porcine lncRNA NORHA gene, and explore the mechanism by which the transcription factor (TF) ZBTB6 activated its transcription and function, so as to provide a basis for analyzing the transcriptional regulatory network of NORHA. 【Method】 The sequence of the distal core promoter of the NORHA gene in Yorkshire pigs was obtained by PCR amplification and sequencing. Bioinformatic method was used to predict the TF-binding sites (TFBSs) and to characterize the ZBTB6 encoding sequence. A ZBTB6 overexpression vector was constructed by restriction enzymatic digestion, and its overexpression efficiency in sow granulosa cells (sGCs) was verified by qPCR and western blot. The effects of ZBTB6 on the activity of the distal core promoter and transcription of NORHA in sGCs were determined by luciferase assay and qPCR. The binding of ZBTB6 to the ZBTB6-binding site (ZBS) in the distal core promoter in sGCs was detected by chromatin immunoprecipitation (ChIP) and DNA sequencing. The concentrations of progesterone (P4) and estradiol (E2) in follicular fluid were measured by ELISA. ZBTB6 and NORHA levels in follicles, and BCL2 and BAX levels in ZBTB6-overexpressing sGCs were detected by qPCR. 【Result】 TFBSs of 255 TFs, including ZBTB6, were predicted in 526-bp of the distal core promoter of Yorkshire NORHA gene. The overexpression vector successfully achieved the overexpression of ZBTB6 levels in sGCs. The transcriptional levels of NORHA in sGCs were markedly upregulated in ZBTB6-overexpressing sGCs. Correlation analysis revealed that ZBTB6 and NORHA levels in follicles were markedly positively correlated. A ZBS motif was discovered at -2276/-2264 nt in the distal core promoter. Luciferase assay revealed that ZBTB6 activates the activity of the distal core promoter via the ZBS motif. ChIP and sequencing confirmed that ZBTB6 directly bound to the ZBS motif in the distal core promoter of the NORHA gene in sGCs. The ZBTB6 gene in pigs and other mammalian species was evolutionally conserved and all contained a conserved DNA-binding domain. ZBTB6 was markedly upregulated during sow follicular atresia and showed a marked positive correlation with the P4/E2 ratio, a marker for follicular atresia. Co-transfection experiments showed that overexpression of ZBTB6 markedly downregulated the anti-apoptotic gene BCL2 levels, upregulated the pro-apoptotic gene BAX levels, and downregulated the BCL2/BAX ratio, an apoptotic marker, whereas knockdown of NORHA reversed this. 【Conclusion】 ZBTB6 was a transcription activator of the NORHA gene in sGCs, which accelerated sGC apoptosis and follicular atresia by directly activating NORHA transcription.

Key words: pig, NORHA, ZBTB6, the distal core promoter, sGC apoptosis

孙振宇, 崔伊歌, 李玉琦, 王思琪, 杜星, 李齐发. 转录因子ZBTB6激活猪卵巢颗粒细胞中lncRNA NORHA转录和功能[J]. 中国农业科学, 2026, 59(13): 2962-2974.

SUN ZhenYu, CUI YiGe, LI YuQi, WANG SiQi, DU Xing, LI QiFa. Transcription Factor ZBTB6 Activates the Transcription and Function of NORHA in Sow Granulosa Cells[J]. Scientia Agricultura Sinica, 2026, 59(13): 2962-2974.

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引物名称Primer name	引物序列Sequence (5'-3')	退火温度Tm (℃)	大小Size (bp)	用途Usage
NORHA-promoter	F:TTGGCATTGGAGAGTGGC	58.0	606	PCR扩增 PCR amplification
NORHA-promoter	R:GTGCGTTTCCCAAAGTGC	58.0	606	PCR扩增 PCR amplification
ZBTB6-CDS	F:GTTCTGAGTCGATTGTGACCATG	58.3	1331	载体构建 Vector construction
ZBTB6-CDS	R:ACGGTCTAAGGCTTACATAGCA	58.3	1331	载体构建 Vector construction
NORHA-RT	F:TCATCCTGCAGCCCTCATTATAC	60.0	288	qPCR
NORHA-RT	R:CCTTCTCAACCCAACTCGCTATT	60.0	288
ZBTB6-RT	F:GTTTGAACAGCAAGGAGATGTAGTC	60.0	107
ZBTB6-RT	R:GCCCTTGGAACTCAGTGTCG	60.0	107
RPLP0-RT	F:CGCTAAGGTGCTCGGTTCT	60.0	119
RPLP0-RT	R:CAGATGAGGCTCCCGATG	60.0	119
BCL2-RT	F:TTCTTTGAGTTCGGTGGGG	60.0	108
BCL2-RT	R:CCAGGAGAAATCAAATAGAGGC	60.0	108
BAX-RT	F:TTTCTGACGGCAACTTCAACTGGG	60.0	165
BAX-RT	R:TGTCCAGCCCATGATGGTTCTGAT	60.0	165
ZBS-CHIP	F:GCTCTCTGCTTTTCTGTATTGATG	58.0	249	ZBS-ChIP
ZBS-CHIP	R:CAGCCTTTGTAATTTCTTGCC	58.0	249	ZBS-ChIP