水牛PTHLH基因克隆分析及其真核表达载体构建

doi:10.16431/j.cnki.1671-7236.2018.11.001

Abstract

Abstract:

In order to reveal the effect of parathyroid hormone-like hormone (PTHLH) gene on the reproductive ability of buffalo,buffalo PTHLH gene was cloned,and its nucleotide and amino acid sequences were analyzed by bioinformatics.Primers were designed by CE Design according to the sequence of cattle PTHLH gene in GenBank (accession No.:NM_001290949).The coding region of buffalo PTHLH gene was cloned by PCR amplification and sequencing technology.The primary structure,secondary structure,tertiary structure and physicochemical properties of PTHLH protein,and homology were analyzed,and phylogenetic tree of PTHLH was constructed by softwares such as DNAMAN,ProtParam,SOPMA and PSORTⅡ Prediction,et al.The results showed that PTHLH gene in buffalo was cloned,including a 534 bp length coding region and 177 amino acids.Compared with cattle,pigs,horses,goats,sheep and camels,the nucleotide sequence of gene coding region homology of PTHLH gene in buffalo were 98.3%,90.4%,90.1%,98.1%,97.5% and 89.2%,respectively.Thus,there was a high homology among different species,and the phyletic evolution was the same as their genetic relationship.The research indicated that PTHLH gene was conservative in the course of evolution.The physicochemical properties analysis result showed that the molecular formula of PTHLH protein was C₈₉₅H₁₄₅₁N₂₇₁O₂₆₆S₂,the molecular weight was 2 885 u,the half-life was 30 h,the theoretical electrical points (pI) was 10.00,the extinction coefficient of water solution at 280 nm was 23 950,the peptide chain N-terminal was Met,the unstable coefficient was 60.04,thus PTHLH protein belong to the basic unstable protein.The fat coefficient of PTHLH protein was 72.15,and the total average hydrophilicity was -0.928,so it belonged to insoluble protein.The protein subpopulations were located in the nucleus,cytoplasm and mitochondria.The structural domain prediction results showed that buffalo PTHLH protein contained a PTH region and a low complexity region.The secondary structure analysis showed that buffalo PTHLH protein contained 83 alpha helix (46.89%),17 extended chain (9.60%),10 beta turn (5.66%) and 67 random coil (37.85%),and consistent with the tertiary structure prediction.In this study,the eukaryotic expression vector was constructed,and the accuracy of the carrier was verified by electrophoresis and sequencing.In summary,the successful cloning of PTHLH gene and the successful construction of its eukaryotic expression vector provide materials for the future study of the function and genetic characteristics of PTHLH gene in buffalo.

Key words: buffalo; PTHLH gene; cloning; bioinformatics; vector construction

CLC Number:

LU Xingrong, LIANG Shasha, DENG Tingxian, DUAN Anqin, MA Xiaoya, PANG Chunying, LIANG Xianwei. Cloning and Construction of Eukaryotic Expression of PTHLH Gene in Buffalo[J]. , 2018, 45(11): 2979-2988.

References

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Cloning and Construction of Eukaryotic Expression of PTHLH Gene in Buffalo

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