牦牛FHL2基因的克隆、组织表达与蛋白定位分析

旨在获得牦牛FHL2基因序列,阐明该基因序列、蛋白质结构与性质、mRNA的组织表达模式、在雌性生殖器官及颗粒细胞的蛋白表达特性。本研究以5头健康空怀成年母牦牛的心、肝、脾、肺、肾、卵巢、子宫、输卵管组织及5头妊娠2个月左右母牦牛的子宫、输卵管和卵巢为研究材料,利用逆转录PCR(RT-PCR)技术克隆FHL2基因,并使用在线分子生物学软件分析其生物信息学特性;利用实时荧光定量PCR(RT-qPCR)技术分析FHL2基因的组织表达特性;应用免疫组化(IHC)和免疫荧光(IF)技术检测FHL2蛋白在雌性生殖器官的分布和颗粒细胞上的亚细胞定位。结果发现,FHL2基因CDS区全长840 bp (ON456866),编码279个氨基酸,FHL2蛋白属于弱碱性亲水不稳定蛋白,没有跨膜结构。不同物种的FHL2氨基酸序列比对和进化树分析表明,FHL2基因编码区在物种进化中比较保守。RT-qPCR结果显示,FHL2基因在检测的组织都有表达,在心的表达量极显著高于其他检测组织(P<0.01);在肺、卵巢、输卵管和子宫中的表达量极显著高于肝、脾和肾(P<0.01)。FHL2基因在妊娠期子宫中的表达...

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

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.