牦牛PRDX4基因的分子特征分析及其在输卵管和卵巢中的表达

Analysis of molecular characteristics of PRDX4 gene in yak and its expression in fallopian tube and ovary

为研究牦牛过氧化物还原酶4(PRDX4)基因在牦牛卵巢和输卵管中的作用,采取繁殖周期不同阶段牦牛卵巢和输卵管样品,采用IHC技术,进行PRDX4蛋白表达的定位检测;进行牦牛PRDX4基因的克隆,并分析其分子特征;采用RT-qPCR技术,检测m RNA在牦牛卵巢和输卵管中的相对表达量;采用Western-blot技术,检测PRDX4蛋白在牦牛卵巢和输卵管中的相对表达量。IHC结果显示,在繁殖周期不同阶段牦牛卵巢中,PRDX4蛋白主要表达于颗粒细胞、卵巢体细胞和粒黄体细胞,且在妊娠期的染色强于黄体期和卵泡期;在繁殖周期不同阶段牦牛输卵管中,PRDX4蛋白主要表达于黏膜上皮细胞,且在卵泡期的染色强于黄体期和妊娠期。成功克隆出牦牛PRDX4基因的CDs区,其保守性较高,与家牛、印度牛、美洲狮及金钱豹等的同源性均在86%以上;分子特征分析结果显示,牦牛PRDX4蛋白的氨基酸数为227,分子式为C_(1146)H_(1854)N_(348)O_(317)S_(15),原子总数为3680,pI为10.58,分子质量为26.06 ku,且其主要分布于粒线体、细胞核、细胞质、细胞骨架,含量分别为69.6%、21.70%、4.30%、4.30%;该蛋白具有Sec/SPI信号肽,其二级结构由3种折叠方式构成,其中含有89个α-螺旋、31个延伸链、107个无规卷曲,占比分别为39.21%、13.66%、47.14%,且具有三级结构;该蛋白有18个丝氨酸磷酸化位点、13个苏氨酸磷酸化位点,且不存在蛋白结合位点、糖基化位点,具有可溶性,且有两个跨膜区。qRT-PCR结果显示,在繁殖周期不同阶段卵巢中,PRDX4基因在卵泡期和妊娠期的相对表达量均显著高于黄体期(P<0.05),且两者无显著差异(P>0.05);在繁殖周期不同阶段输卵管中,PRDX4基因的相对表达量不存在显著差异(P>0.05)。Western-blot结果显示,在繁殖周期不同阶段卵巢中,PRDX4蛋白在卵泡期显著高于黄体期和妊娠期(P<0.05),且在黄体期和妊娠期无显著差异(P>0.05);在繁殖周期不同阶段输卵管中PRDX4蛋白的相对表达量不存在显著差异(P>0.05)。上述结果表明,本研究成功克隆出了牦牛PRDX4基因的CDs区,它具有较高的保守性,且该蛋白具有一段特殊的信号肽结构。PRDX4蛋白在牦牛卵巢中主要表达于颗粒细胞,在输卵管主要表达于黏膜上皮细胞,且其在卵泡期牦牛的卵巢中表达量最高,在不用阶段牦牛输卵管中出现无显著性变化的高表达情况。本文揭示,PRDX4可能参与了牦牛卵巢和输卵管的某些生理学过程。

The aim of this study was to clone the peroxiredoxin 4(PRDX4) gene of yak,and explore its molecular characteristics;and study expression differences of PRDX4 gene and protein in ovary and fallopian tube of yak at different stages of the reproductive cycle,in order to provide evidence for studying its role in ovary and fallopian tube of yak.Samples of ovary and fallopian tube in yak at different stages of the reproductive cycle was taken,and the localization of PRDX4 protein was performed by IHC;its molecular characteristics was analyzed after cloning PRDX4 gene of yak;the relative expression of m RNA was detected by RT-q PCR;the relative expression of PRDX4 protein in the yak ovary and fallopian tube was detected by Western blot.The results of IHC showed that PRDX4 protein was mainly expressed in granulosa cells,ovarian somatic cells and granuloluteal cells in the ovary of yak at different stages of the reproduction cycle,and the staining during pregnancy was stronger than that in the luteal and follicular phases;PRDX4 protein is mainly expressed in mucosal epithelial cells in the oviduct of yak at different stages of the reproduction cycle,and the staining in the follicular phase is stronger than that in the luteal phase and pregnancy.The CDs region of PRDX4 gene of yak was successfully cloned,which was highly conserved and had a homology of more than 86% with domestic cattle,Indian cattle,mountain lions and leopards;the analysis of molecular characteristics showed that the number of amino acids of PRDX4 protein in yak is 227,and its molecular formula is C_(1146)H_(1854)N_(348)O_(317)S_(15),total number of atoms is 3680,pI is 10.58,and molecular weight is 26.06 ku,it is mainly distributed in mitochondria,nucleus,cytoplasm,and cytoskeleton,the content is 69.6%,21.70%,4.30%,4.30%;it has Sec/SPI signal peptide,and its secondary structure is composed of 3 folding modes,which contains 89α-helices,31 extended chains,and 107 random curly,accounting for 39.21%,13.66%,47.14% respectively,and it has a tertiary structure;it has 18 serine phosphorylation sites,13 threonine phosphorylation sites,and it does not have binding sites dots of protein and glycosylation sites,which are soluble,and it has two transmembrane regions.The results of q RT-PCR showed that the relative expression of PRDX4 gene in the follicular and gestational phases was significantly higher than that in the luteal phase in the ovaries at different stages of the reproductive cycle(P<0.05),and there was no significant difference between the two(P>0.05);there was no significant difference in the relative expression of PRDX4 gene among the fallopian tube in the different stages of the reproductive cycle(P>0.05).The results of Western-blot showed that PRDX4 protein was significantly higher in the follicular phase than in the luteal phase and pregnancy in the ovaries at different stages of the reproductive cycle(P<0.05),and there was no significant difference between the luteal phase and pregnancy(P>0.05);There was no significant difference in the relative expression of PRDX4 protein in the oviduct at different stages of the reproductive cycle(P>0.05).In this study,the CDs region of the PRDX4 gene of yak was successfully cloned,which has high conservation and the protein has a special signal peptide structure. PRDX4 protein is mainly expressed in granulosa cells in the ovary of yak,and mainly expressed in mucosal epithelial cells in the oviduct,and its expression is highest in the ovary of yak the follicular stage,and there is no significant change in the high expression in the oviduct of the yak at the different stage. It was revealed that PRDX4 may be involved in certain physiological processes of the yak ovary and fallopian tube.