鸡HMIT基因的克隆与表达分析

doi:10.11843/j.issn.0366-6964.2020.08.005

Abstract

Abstract: The aim of this study was to clone the transcriptional sequence of chicken H+/myoinositol transporter (HMIT) gene, and to detect its expression in different tissues of chickens, and to explore the effect of exogenous insulin treatment on the relative expression of HMIT gene. In this study, AA broilers were used as experimental animals. The full-length coding region of chicken HMIT gene was cloned by RT-PCR technique, and the biological characteristics of its encoded protein were analyzed by bioinformatics technology. The expressions of HMIT gene in brain, liver, abdominal fat, leg muscle, heart, kidney, jejunum and ileum were detected by fluorescence quantitative PCR. The expressions of HMIT gene in kidney and brain were studied after insulin treatment for 120 and 240 min. The results showed that the chicken HMIT gene (1 694 bp, GenBank accession number: MN708211) was successfully cloned by using the chicken HMIT sequence(XM_001232939.5) predicted by NCBI as a template. The cloned full-length coding region of chicken HMIT was 1 380 bp, which was less than the predicted coding sequence(XM_001232939.5) of 561 bp, which contained a protein of 459 amino acids. Nucleotide and amino acid sequence alignment showed that chicken HMIT was highly homologous among species. The collinearity analysis showed that the region of chromosome 1 where HMIT was located was also highly conserved among species. The protein encoded by HMIT was an unstable hydrophilic protein, its secondary and tertiary structure were mainly composed of random coil and α-helix. Transmembrane structure analysis showed that there were 8 distinct transmembrane domains in chicken HMIT. The results of subcellular localization showed that chicken HMIT protein was distributed in plasma membrane (52.3%), endoplasmic reticulum (21.7%), vacuole (17.4%), mitochondria (4.3%) and Golgi apparatus (4.3%). The results of real-time fluorescence quantitative PCR showed that the expressions of HMIT gene were the highest in chicken brain and kidney, which was significantly higher than those in other tissues(P<0.05). At 240 min after insulin treatment, the expressions of HMIT in kidney and brain were significantly lower than that in PBS control group (P<0.05). In this study, the full-length coding region of chicken HMIT was cloned, the results of bioinformatics and tissue expression characteristics analysis laid a foundation for further exploration of the physiological function and regulatory mechanism of chicken HMIT gene.

Key words: HMIT, gene cloning, bioinformatics analysis, tissue expression, insulin

CLC Number:

S831.2

DU Pengfei, CHEN Bo, GAO Lin'ge, ZHU Yao, ZHANG Xiangli, ZHU Xinghao, CHEN Wen, HUANG Yanqun. Cloning and Expression Analysis of Chicken HMIT Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1811-1822.

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Cloning and Expression Analysis of Chicken HMIT Gene

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