牦牛FHL2基因的克隆、组织表达与蛋白定位分析

doi:10.11843/j.issn.0366-6964.2023.01.014

Abstract

Abstract: The objectives of the study were to obtain the sequence of yak FHL2 gene and to elucidate its sequence, protein structure and properties, mRNA expression in tissues, protein localization in the reproductive organs and granulosa cells of female yaks (Bos grunniens). Tissue samples of heart, liver, spleen, lung, kidney, ovaries, uterus and oviducts of 5 healthy non-pregnant adult female yaks and ovaries, uterus and oviducts of 5 pregnant female yaks were used as study materials. The FHL2 gene was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and its bioinformatics properties were analyzed using online softwares. mRNA expression levels in tissues were detected by a real-time quantitative PCR (RT-qPCR). The distribution of FHL2 protein in female reproductive organs and localization in granulosa cells was detected by immunohistochemistry (IHC) and immunofluorescence (IF) techniques. The results showed that the CDS region of FHL2 gene was 840 bp (ON456866) in length and encoded 279 amino acids. The FHL2 protein was a weakly basic hydrophilic unstable protein with no transmembrane structure. Based on amino acid sequence comparison and evolutionary tree results among multiple species, the coding region of the FHL2 gene was relatively conserved during evolution. RT-qPCR results showed that the FHL2 gene was expressed in all detected tissues, and the expression in the heart was significantly higher than in other tissues(P<0.01). Its expression levels in lung, ovary, oviducts and uterus were significantly higher than in liver, spleen and kidney (P<0.01). Its expression in the uterus during pregnancy was significantly higher than that in the non-pregnant period (P<0.01), but in the ovary its expression was significantly lower during pregnancy than in the non-pregnant period (P<0.01). IHC result showed that FHL2 protein was mainly expressed in follicular granulosa cells, endometrium and oviductal mucosa. IF result showed that FLH2 protein was mainly localized in nuclear of granulosa cells. In conclusion, the sequence of yak FHL2 gene is relatively conserved during animal evolution. It is highly expressed in heart, lung, ovaries, oviducts and uterus, indicating that it may be involved in the regulation of physiological functions such as hypoxia adaptation, follicular development and pregnancy maintenance in yak.

Key words: Bos grunniens, FHL2 gene, expression characteristics, real-time quantitative PCR, immunohistochemistry

CLC Number:

S823.85

GONG Sanni, JIANG Xudong, MA Yao, LU Jianyuan, ZI Xiangdong. Cloning, Tissue Expression and Protein Localization of FHL2 Gene in Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 146-156.

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Cloning, Tissue Expression and Protein Localization of FHL2 Gene in Yaks

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