藏羊HSP27基因过表达和沉默载体构建及对卵泡发育的功能初步分析

doi:10.11843/j.issn.0366-6964.2021.012.009

Abstract

Abstract: In order to explore the role of HSP27 gene in the follicular development of Tibetan sheep, in this study, 6 healthy (3-4 years old) Tibetan ewes were selected and slaughtered to obtain ovarian tissue for cloning Tibetan sheep HSP27 gene. The CDS region sequence of HSP27 gene was analyzed using bioinformatics methods, HSP27 gene overexpression and silencing vectors were constructed, Tibetan sheep ovarian granulosa cells were isolated and cultured, and cell transfection experiment was conducted with the constructed vectors in different groups, namely blank group, overexpression vector group, silent vector group, negative control group, each group had 3 multiple wells. Microscopic observation of the cell morphology changes and cell counts of each transfection group at 0, 24, 48, 72 h incubation time was performed. The expression of of HSP27, GDF9, BMPR-1B, Erβ genes mRNA were detected by RT-PCR. The results showed that the the Tibetan sheep HSP27 gene was successfully cloned. The CDS sequence of the Tibetan sheep HSP27 gene was 618 bp in length and encoded 203 amino acids. The Tibetan sheep HSP27 gene overexpression and silencing vectors were successfully constructed; the constructed vector was transfected into the ovary granular cells, the cell morphology of each transfection group changed with the increase of culture time, the cells of the overexpression vector group gradually changed from a long spindle to an irregular polygon, the nucleus deformed and decomposed, and the pseudopodia of the silent vector group decreased, the cells shrank and had a lot of apoptosis. The cell count results showed that, after 72 h incubation, the number of cells in the silent vector group was significantly lower than that in the other 3 groups (P<0.01), and the number of cells in the overexpression vector group was significantly lower than that in the blank group and the negative control group (P<0.05). RT-PCR results showed that the expression of HSP27 mRNA in the overexpression vector group was significantly higher than that in the blank group (P<0.01), and the expression of HSP27 mRNA in the silent vector group was significantly lower than that in the negative control group (P<0.01), the expression of GDF9, BMPR-1B, Erβ mRNA in the overexpression vector group was significantly higher than that in the blank group (P<0.01), and the expression of Erβ mRNA in the silent vector group was significantly lower than that in the negative control group (P<0.01). It can be preliminarily inferred that when HSP27 gene is overexpressed, the granulosa cell proliferation and differentiation was promoted. Silencing HSP27 gene may trigger granulosa cell apoptosis and affect follicular development. The above research results can lay the experimental foundation for the function research of HSP27 gene in the follicular development process of Tibetan sheep.

Key words: Tibetan sheep, heat shock protein 27, gene cloning, sequence analysis, vector construction, follicular development

CLC Number:

S826.2

ZHANG Chunmei, JIA Jianlei, XIE Wen, REN Hao, ZHANG Huaixia, ZHANG Yingying, CHEN Qian. Construction of Tibetan Sheep HSP27 Gene Overexpression and Silencing Vectors and Preliminary Analysis of Its Function on Follicular Development[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3413-3425.

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Construction of Tibetan Sheep HSP27 Gene Overexpression and Silencing Vectors and Preliminary Analysis of Its Function on Follicular Development

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