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

摘要/Abstract

摘要： 为了深入探究HSP27基因在藏羊卵泡发育过程中的作用，本研究选择健康的（3~4岁）经产藏母羊6只，屠宰获得卵巢组织进行藏羊HSP27基因克隆，利用生物信息学方法分析HSP27基因CDS区序列，构建HSP27基因过表达及沉默载体，分离培养藏羊卵巢颗粒细胞，将构建的载体按不同分组进行细胞转染试验即空白组、过表达载体组、沉默载体组、阴性对照组，每组设3个复孔。显微镜观察各转染组培养0、24、48、72 h的细胞形态变化及细胞计数，RT-PCR检测各转染组HSP27、GDF9、BMPR-1B、Erβ基因mRNA表达量。结果，试验成功克隆藏羊HSP27基因，其CDS序列长度为618 bp，编码203个氨基酸。试验成功构建藏羊HSP27基因过表达及沉默载体；将构建的载体转染至卵巢颗粒细胞，随着培养时间的递增各转染组间细胞形态发生改变，过表达载体组细胞由长梭状逐渐变为不规则多边形，细胞核变形分解，沉默载体组细胞周边伪足伸出减少，细胞皱缩大量凋亡；细胞计数结果显示，转染后培养72 h时，沉默载体组细胞数量极显著低于其他3组（P<0.01），过表达载体组细胞数量显著低于空白组和阴性对照组（P<0.05）。RT-PCR结果显示，过表达载体组细胞HSP27基因mRNA表达量极显著高于空白组（P<0.01），沉默载体组细胞HSP27基因mRNA表达量极显著低于阴性对照组（P<0.01），过表达载体组细胞的GDF9、BMPR-1B、Erβ基因mRNA表达量均极显著高于空白组（P<0.01），沉默载体组细胞Erβ基因mRNA表达量极显著低于阴性对照组（P<0.01）。由此可初步推测，当HSP27基因过表达时能够促进颗粒细胞增殖分化，当沉默HSP27基因时可能会触发颗粒细胞凋亡进而影响卵泡发育，以上研究成果可为HSP27基因在藏羊卵泡发育过程中的功能研究奠定试验基础。

关键词: 藏羊, 热休克蛋白27, 基因克隆, 序列分析, 载体构建, 卵泡发育

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

中图分类号:

S826.2

张春梅, 贾建磊, 谢雯, 任昊, 张怀霞, 张莹莹, 陈倩. 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[J]. 畜牧兽医学报, 2021, 52(12): 3413-3425.

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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