农业生物技术学报2017，Vol.25Issue(10)：1643-1652,10.DOI:10.3969/j.issn.1674-7968.2017.10.010
从江香猪PHKG1和PHKG2基因克隆及其mRNA在组织中表达水平
Cloning of PHKG1 and PHKG2 Genes in Congjiang Pigs (Sus scrofa) and Their mRNA Expression in Tissues
摘要
Abstract
Phosphorylase kinase γ1 (PHKG1) and phosphorylase kinase γ2 (PHKG2) genes are important in the glycogen metabolism pathway,and have the function of decomposing glycogen to provide energy for muscle contraction and maintain blood glucose balance.In order to further explore the research on PHKG1 and PHKG2 genes genetic mechanism,the Guizhou Congjiang pig (Sus scrofa) and Large White pig PHKG1 and PHKG2 genes CDS sequences were cloned by the T-A cloning method.PHKG1 and PHKG2 genes structures,physicochemical properties of protein and homology comparision to other species were described by bioinformatic anyalysis software.Real-time quantitative PCR (qRT-PCR) technology was also be used to comparative analysis of these 2 genes in Congjiang pig and the Large White pigs in different tissues (heart,liver,spleen the lung,kidney,stomach,small intestine,large intestine,longissimus muscle and fat) in differential expression level.The results showed that the CDS of PHKG1 gene was 1 167 bp in length,encoding 388 amino acids,and the full sequence ofPHKG2 gene was 1 221 bp in length,encoding 406 amino acids,both of which constituted a transmembrane with S_TKc domain Hydrophilic and non-secreted proteins.The phylogenetic analysis of PHKG1 and PHKG2 protein and phylogenetic tree analysis showed that the two proteins had some homology.The results showed that there were 4 mutations in PHKG1 gene,which were A371G,A525G,T945C and C1050T respectively,but there was a mutation in T945C compared with Large white pig,which did not cause amino acid change.PHKG2 gene sequence analysis showed that G236A,C43 1T,A726G,A807G,A816G and A867G were Large white pig,Congjiang pig and Bama miniature pig (KJ 186785) common mutations.Congjiang pig had a special A614G mutation,and caused 205th glutamic acid into alanine.Sequence analysis revealed that the amino acid phylogenetic relationship of PHKG1 and PHKG2 of Congjiang pig was close to pig(sus scrofa),cattle(Bos taurus),sheep(Ovis aries),ordinary people(Homo sapiens),Mus musculus,Rattus norvegicus (Rattus norvegicus)6 species,the homology was 92％～100％,and zebrafish (Danio rerio) was distantly related,qRT-PCR results showed that PHKG1 gene was present in longissimus muscle with highest ratio while heart,liver,spleen,kidney,stomach,large intenstine,small intenstine showed less expression in Congjiang pig and Large white pig.And in the lung,the expression was significant different (P＜0.05).Relative expression of PHKG2 gene was high indication in spleen,lung,large intenstine,small intenstine,kidney and with lower ratio in heart and longissimus muscle.This study provides a theoretical basis for the follow-up eukaryotic expression of PHKG1 and PHKG2 genes.关键词
从江香猪/PHKG1/PHKG2/克隆/组织表达Key words
Congjiang pig (Sus scrofa)/PHKG1/PHKG2/Cloning/Tissue expression分类
农业科技引用本文复制引用
王圆圆,许厚强,陈伟,周迪,孙成娟,张青青,赵焕平,张鸣,杨洋..从江香猪PHKG1和PHKG2基因克隆及其mRNA在组织中表达水平[J].农业生物技术学报,2017,25(10):1643-1652,10.基金项目
国家科技支撑计划(No.2015BAD03B02-3)和黔科合重大专项(黔科合NY字[2013]6008号) （No.2015BAD03B02-3）
