猪PFKL基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2020.02.001

摘要/Abstract

摘要： 试验旨在探究猪精液6-磷酸果糖激酶（6-phosphofructokinase，PFKL）基因的理化性质和结构特点，以期阐明糖酵解在精子发生过程中的生理作用及调控机制。通过RT-PCR技术扩增PFKL基因，将获得的基因片段插入到pUC57载体上进行克隆测序，并结合生物信息学方法预测和分析其氨基酸序列、跨膜结构、修饰位点、二级结构、三级结构、抗原位点、功能注释等。结果表明，PFKL基因全长2 349 bp，编码782个氨基酸，蛋白质分子质量为83.819 ku，等电点为6.96，为酸性蛋白质，分子式为C₃₆₇₄H₅₈₉₇N₁₀₅₁O₁₁₀₉S₄₀。猪源PFKL基因与鼠、鸭、羊、牛、猩猩、人的参考序列同源性均高达80.0%以上。该蛋白不存在跨膜结构和信号肽区域，含有2个N-糖基化修饰位点、14个O-糖基化位点、35个磷酸化位点。PFKL蛋白三维建模结构主要由无规则卷曲和α-螺旋构成，与肌肉型磷酸果糖激酶和血小板型磷酸果糖激酶的同源性分别为68.24%和70.84%。PFKL基因含有的27个抗原位点强弱不同，柔韧性分布较均匀，有较高的表面可塑性，且存在PKC、PKA特异性蛋白激酶的结合位点，为具有较多潜在B细胞抗原表位的亲水性蛋白。GO功能注释分析结果表明，PFKL基因具有碳水化合物激酶活性、磷酸果糖激酶活性，能够参与己糖代谢和单糖代谢。本研究结果为改善精子品质和提高精卵结合发生率奠定了分子生物学基础。

关键词: PFKL基因; 糖酵解; 生物信息学分析

Abstract: This study was aimed to explore the physicochemical properties and structural characteristics of porcine semen 6-phosphofructokinase (PFKL) gene,elucidate the physiological role and regulatory mechanism of glycolysis in spermatogenesis.PFKL gene was amplified by RT-PCR,the obtained gene fragment was inserted into pUC57 vector for cloning and sequencing,and its amino acids sequence,transmembrane structure,modification site,secondary structure,tertiary structure,antigen site and functional annotation were predicted and analyzed by bioinformatics methods.The results showed that the total length of PFKL gene was 2 349 bp,encoding 782 amino acids,the molecular weight of protein was 83.819 ku,the isoelectric point was 6.96,and the molecular formula was C₃₆₇₄H₅₈₉₇N₁₀₅₁O₁₁₀₉S₄₀.The homology of porcine PFKL gene was more than 80.0% compared with the reference sequences of Mus musculus,Iumenta anatis,Ovis aries,Bos taurus,Pongo abelii and Homo sapiens.The protein structure had no transmembrane structure and signal peptide region,and contained 2 N-glycosylation modification sites,14 O-glycosylation sites and 35 phosphorylation sites.The structure of PFKL protein was mainly composed of random coil and alpha helix,and its homology with muscle phosphofructokinase and platelet phosphofructokinase was 68.24% and 70.84%,respectively.At the same time,the 27 antigenic sites were different in strength,flexibility,uniform distribution and high surface plasticity,and there were binding sites of PKC and PKA specific protein kinase,which were hydrophilic proteins with more potential B cell epitopes.The results of GO functional annotation analysis showed that PFKL gene had carbohydrate kinase activity and phosphofructokinase activity,and could participate in hexoses metabolism and monosaccharides metabolism.In conclusion,this reaults laid a molecular biological foundation for improving sperm quality and the incidence of sperm-egg binding.

Key words: PFKL gene; glycolysis; bioinformatics analysis

中图分类号:

葛晨玲, 李珣, 王晓晔, 梁莹莹, 陈志英, 杜倩, 唐胤晟, 黄明光, 胡传活. 猪PFKL基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(2): 315-325.

GE Chenling, LI Xun, WANG Xiaoye, LIANG Yingying, CHEN Zhiying, DU Qian, TANG Yinsheng, HUANG Mingguang, HU Chuanhuo. A Cloning and Bioinformatics Analysis of Porcine PFKL Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 315-325.

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