陆川猪和杜长大猪Nrf2基因克隆及其在背最长肌中的表达分析

Cloning and expression analysis of Nrf2 gene in longissimus dorsi of Luchuan pig and Duroc×Landrace×Yorkshire pig

    摘要
  • 摘要: 【目的】明确核因子NF-E2相关因子(Nrf2)基因生物学特性,并探究其在陆川猪和杜长大猪背最长肌中的表达差异,为深入研究陆川猪优质肌肉抗氧化性能的分子机制打下基础。【方法】以150日龄的陆川猪和杜长大猪背最长肌组织总RNA为模板,分段克隆Nrf2基因编码区(CDS)序列,并通过ExPASy ProtScale、ExPASy ProtParam及SWISS-MODEL等在线软件进行生物信息学分析,同时应用实时荧光定量PCR和Western blotting检测Nrf2基因在150日龄陆川猪和杜长大猪背最长肌中的表达差异。【结果】陆川猪和杜长大猪Nrf2基因CDS序列长1686 bp,与GenBank中已公布的野猪、杜长大猪、牛、绵羊、黑猩猩、猕猴、狼、人类、家鼠、大白鼠、鸵鸟和普通家鸡的Nrf2基因CDS序列的同源相似性分别为99.7%、99.5%、91.2%、90.6%、90.5%、90.1%、89.5%、86.5%、81.9%、81.7%、72.0%和70.6%。陆川猪与杜长大猪的遗传距离最近,与狼的遗传距离最远。陆川猪和杜长大猪的Nrf2蛋白都由561个氨基酸组成,且以丝氨酸含量最高,占比12.5%,色氨酸含量最低,仅占0.4%,均具有较强的亲水性。在Nrf2蛋白二级结构中,杜长大猪Nrf2蛋白的无规则卷曲占57.75%,α-螺旋占34.05%,延伸链占5.35%,β-转角占2.85%;陆川猪Nrf2蛋白的无规则卷曲占57.93%,α-螺旋占33.51%,延伸链占6.06%,β-转角占2.50%。150日龄陆川猪背最长肌中Nrf2基因相对表达量显著高于同日龄杜长大猪背最长肌中Nrf2基因的相对表达量(P<0.05);且陆川猪Nrf2蛋白表达量极显著高于同日龄杜长大猪背最长肌中Nrf2蛋白表达量(P<0.01)。【结论】成功克隆陆川猪和杜长大猪Nrf2基因CDS序列,陆川猪背最长肌中Nrf2基因表达量显著高于同日龄杜长大猪,推测陆川猪肌肉具有比杜长大猪更加优良的抗氧化性能。

     

    Abstract: 【Objective】To clarify biological characteristics of NF-E2-related factor(Nrf2) gene in Luchuan pig and Duroc×Landrace×Yorkshire(DLY) pig,and to explore the expression difference of Nrf2 gene in longissimus dorsi muscle of Luchuan pig and DLY pig,so as to lay a foundation for further study on the molecular mechanism of antioxidant properties of high-quality Luchuan pig muscle.【Method】The coding DNA sequence(CDS),sequence of Nrf2 gene was cloned from the total RNA of longissimus dorsi muscle of 150-day-old Luchuan pigs and the same day old DLY pigs,and the bioinformatics analysis was carried out through online softweres ExPASy ProtScale,ExPASy ProtParam and SWISS-MODEL.The expression difference of Nrf2 gene in the longissimus dorsi muscle of 150-day-old Luchuan pigs and the DLY pigs of the same age was detected by qRT-PCR and Western-blotting.【Result】The length of the CDS of Nrf2 gene of Luchuan pig and DLY pig was 1686 bp,which was compared with the CDS sequences of Sus scrofa,DLY pig,cattle,sheep,Chimpanzee,macaque,wolf,human,domestic rat,Rattus norvegicus,ostrich,ordinary domestic chicken that published in GenBank. The similarity were,99.7%,99.5%,91.2%,90.6%,90.5%,90.1%,89.5%,86.5%,81.9%,81.7%,72.0% and70.6%,respeetively. The genetic distance between Guangxi Luchuan pig and DLY pig was the nearest,and the farthest was Canis lupus. Nrf2 protein of DLY pig and Luchuan pig was composed of 561 amino acids,and serine was the highest in the amino acid content,accounting for 12.5%,and tryptophan was the lowest,accounting for only 0.4%. In the secondary structure of Nrf2 protein,random coil accounted for 57.75%,α-helix accounted for 34.05%,extended strand accounted for5.35%,β-turn accounted for 2.85% in DLY pig Nrf2 protein. The percentage of random coil,α-helix,extended strand andβ-turn were 57.93%,33.51%,6.06% and 2.50% respectively in Luchuan pig Nrf2 protein;The relative expression of Nrf2gene in longissimus dorsi muscle of 150-day-old Luchuan pigs was significantly higher than that of DLY pigs(P<0.05);The expression of Nrf2 protein in the longissimus dorsi muscle of 150-day-old Luchuan pigs was extremely significantly higher than that of DLY pigs(P<0.01).【Conclusion】Nrf2 gene CDS seqnence of Luchuan pig and DLY pig are cloned successfully,and the expression of Nrf2 in Luchuan pig is significantly higher than that in DLY pig at the same age. It is suggested that the muscle of Luchuan pig has better antioxidant activity than that of DLY pig.

     

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