关键词: 塔里木马鹿; KCNQ1基因; 非同义突变; 生物信息学; 过表达载体

Abstract: 【Objective】 This study aimed to investigate the biological role of the potassium voltage-gated channel subfamily KQT member 1 (KCNQ1) gene and the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on the renal water reabsorption function of Tarim Red deer.【Method】 Based on the previous sequencing data,genotype frequency alignment was performed to screen and obtain the KCNQ1 gene sequence of Tarim Red deer.Similarity alignment and phylogenetic tree construction were performed,and bioinformatics analysis was performed on the KCNQ1 gene sequence using online software.Whole blood DNA from Northeastern Red deer and Tarim Red deer were utilised as the experimental material to clone and construct the overexpression vectors of the wild-type and mutant of KCNQ1 gene.The vectors were then infected with lentiviruses,and the expression of KCNQ1 protein was detected by Western blotting.【Result】 There was a non-synonymous mutation site c.A835G (p.I279V) associated with drought tolerance in KCNQ1 gene CDS of Tarim Red deer.The results of similarity alignment showed that the similarity was the highest (99.41%) between Tarim Red deer and Cervus canadensis Canadensis,and the similarity with Odocoileus virginianus and Dama dama exceeded 97%.The phylogenetic tree showed that Tarim Red deer had the closest genetic relationship with Cervus canadensis Canadensis,but had a distant genetic relationship with Bos taurus and Capra hircus.KCNQ1 protein of Tarim Red deer was consisted of 340 amino acids,the molecular formula was C₁₆₂₄H₂₆₀₇N₅₃₅O₄₅₆S₆,the protein molecular mass was 37.15 ku,an instability coefficient of 52.12,a theoretical isoelectric point of 11.5,and an average hydrophilicity of ―0.708.KCNQ1 protein was predominantly located in the basolateral membrane,with O-glycosylation and phosphorylation sites,but it lacked N-glycosylation sites,signal peptides,and transmembrane regions.PolyPhen-2 software predicted that the mutation of KCNQ1 protein could lead to a benign change,and this mutant site was on the transcription factor HNF-1β,which promoted the transcriptional efficiency of KCNQ1 gene.The secondary and tertiary structures of KCNQ1 protein were primarily composed of alpha helix and random coil.However,a shift from alpha helix to random coil was observed in the mutant site of Tarim Red deer.Western blotting results revealed that the expression of both wild-type and mutant of KCNQ1 protein in Tarim Red deer were extremely significantly higher than that in control group (P＜0.01).Furthermore,the expression of KCNQ1 protein in mutant was extremely significantly higher than that in wild-type (P＜0.01).【Conclusion】 There was a non-synonymous mutation site c.A835G (p.I279V) in KCNQ1 gene CDS of Tarim Red deer.KCNQ1 protein of Tarim Red deer encoded 340 amino acids and had O-glycosylation and phosphorylation sites.Its mutation site had undergone benign changes and was located on the transcription factor HNF-1β.The experiment successfully cloned and constructed the wild-type and mutant lentivirus overexpression vectors of KCNQ1 gene,and the expression of mutant protein was significantly higher than that of wild-type.The results provided a theoretical basis for the mechanism of renal water reabsorption in Tarim Red deer.

Key words: Tarim Red deer; KCNQ1 gene; non-synonymous mutation; bioinformatics; overexpression vector