Acta Veterinaria et Zootechnica Sinica ›› 2021, Vol. 52 ›› Issue (9): 2394-2405.doi: 10.11843/j.issn.0366-6964.2021.09.003

• ANIMAL GENETICS AND BREEDING • Previous Articles     Next Articles

Mechanism of ZBED6 Gene on Spleen Development of Bama Xiang Pig

WANG Shengnan1, WANG Dandan1, TIAN Wenjie1, PU Yabin1, PAN Dengke1, XING Xiangyang3, MA Yuehui1, JIANG Lin1*   

  1. 1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
    3. Chengdu Clonorgan Biotechnology Co. Ltd., Chengdu 610000, China
  • Received:2021-03-11 Online:2021-09-23 Published:2021-09-26

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Abstract: As a transcription factor, ZBED6 gene can bind to IGF2 to regulate muscle growth and development. But its role in the growth and development of the spleen is unclear. RNA-seq sequencing technology was used to compare the spleen tissue transcription of ZBED6 gene knockout Bama Xiang pigs (ZBED6-KO) and wild-type Bama Xiang pigs (WT) at the same age, so as to explore the effects of ZBED6 gene on spleen tissue development of Bama Xiang pigs ZBED6 knockout. The t-test was used to analyze the phenotypic differences of spleen tissue, and the gene expression of IGF2 that a target gene directly regulated by ZBED6 transcription factors between ZBED6-KO pigs and WT pigs. The total RNA from the spleen tissues of ZBED6 knockout group and wild-type group of Bama Xiang pigs was extracted, and RNA-seq analysis was performed on the Illumina Hiseq 2500 platform. Using Sus scrofa11.1 as reference genome, the differentially expressed genes in spleen tissues of ZBED6-KO and WT Bama Xiang pigs were screened by the standard process of transcriptome analysis. Using DAVID online website to analyze GO and KEGG enrichment analysis of differentially expressed genes. Seven differentially expressed genes were randomly selected to verify the accuracy and reliability of the sequencing results by the RT-qPCR technique. Compared with WT Bama Xiang pigs, the spleen weight and IGF2 gene expression of ZBED6-KO pigs were significantly increased (P<0.05), indicating that ZBED6 gene knockout could promote the growth and development of spleen tissue of Bama Xiang pigs. The sequencing results showed that at least 4G of data was obtained from each sample, and the ratio of Clean Ratio to Q30 of each sample was more than 90%, and more than 83.94% of the reads could be mapped with the pig reference genome, which indicating that the sequencing data were true and reliable. A total of 161 differentially expressed genes were screened by transcriptome analysis, including 90 up-regulated genes and 71 down-regulated genes. Hierarchical cluster analysis of differentially expressed genes showed that the expression patterns of 3 individuals (spleen1, spleen3, spleen6) of ZBED6-KO pigs were similar, and the expression patterns of 3 individuals (spleen2, spleen4, spleen5) of WT were similar, which further proved that the sequencing data were accurate. In GO and KEGG enrichment analysis, 10 significant GO terms and 5 significant pathways were enriched, in which 2 pathways were related to muscle development. The expression patterns of 7 differentially expressed genes were randomly detected by real-time fluorescent quantative PCR experiments consistent with the results of RNA-seq analysis, which confirmed the reliability of sequencing data. Using the Bama Xiang pig as a model, the RNA-seq technology was used to study the effect of ZBED6 gene knockout on the spleen development of domestic pigs, which provided a basis for mining more functions of the ZBED6 gene.

Key words: ZBED6, CRISPR/Cas9, RNA-seq, growth and development, spleen

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