绵羊磷脂酶C-γ1基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.06.001

Abstract

Abstract: The purpose of this study was to screen and identify the predicted sequence of phospholipase C-γ1 (PLC-γ1) gene in sheep,obtain the gene sequence and analyze its bioinformatics.Four different transcripts given by GenBank (accession No.:NC_040264.1) and Ensembl (accession No.:ENSOARG00000001700.1) were compared by BLAST.Specific primers were designed in the non-homologous region.The total RNA was extracted from sheep ovary and transcribed into cDNA,to amplify the specific fragment by RT-PCR.After gel recovery,the sample was sequenced,and the gene sequence was screened and consistent with the fragment by BLAST comparison.According to the selected primers,the sheep PLC-γ1 gene was amplified by PCR with LA Taq (GC Buffer) fidelity enzyme.The gel recovery product was ligated to PUC57-T vector and transformed into Escherichia coli DH5α competent cells,and the single clone was sequenced.According to the obtained sequence,the gene and the encoded amino acid sequence were analyzed by bioinformatics technology.The results showed that the non-homologous fragment of 330 bp,was successfully amplified,and the predicted sequence of transcript X3 was consistent with it by BLAST analysis.The PLC-γ1 gene fragment of 3 870 bp in sheep was successfully amplified by sequencing.PLC-γ1 gene in sheep was closely related to Capra hircus,and had a particularly strong evolutionary relationship with Bos taurus, Equus caballus, Sus scrofa and other livestock animals.The protein molecular formula was C₆₅₈₂H₁₀₁₆₀N₁₈₁₂O₁₉₆₂S₅₈,and the molecular weight was 147.9 ku.It was a hydrophilic weak acid unstable protein,which existed in cytoplasm and had no transmembrane and signal peptide.The N- and C-terminal GC content of this gene was relatively high,reaching 80%,70% or more.It had highly conserved domains such as SH2,SH3,and C2.The ratio of α-helix,extended chain,β-turn and random coli in the secondary structure was 36.46%,16.14%,5.04% and 42.36%,respectively.There were 112 phosphorylation sites.Y428,Y509 and S514 were the key phosphorylation sites.It provided a theoretical basis for the study of PLC-γ1 protein expression in sheep,and was of great significance for studying the embryonic development of Xinjiang local breed sheep and improving the fertilization rate.

Key words: Ovis aries; phospholipase C-γ1 (PLC-γ1) gene; bioinformatics analysis

CLC Number:

Q781

YUAN Liming, HU Guangdong, LIU Xinjie, WU Xiaoxue, LIU Suping, CHEN Ning, Saiwujiafu. Cloning and Bioinformatics Analysis of PLC-γ1 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1883-1893.

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Cloning and Bioinformatics Analysis of PLC-γ1 Gene in Sheep

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