绵羊SMAD4基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2023.12.001

摘要/Abstract

摘要： 【目的】克隆绵羊SMAD同源物4(SMAD4)基因并探究其在绵羊中的生物学功能,为研究SMAD4基因在毛囊生长发育过程中的调控作用提供依据。【方法】以小尾寒羊皮肤组织cDNA为模板,PCR扩增SMAD4基因CDS区并克隆测序,对其进行生物信息学分析;采用实时荧光定量PCR检测SMAD4基因在小尾寒羊和新吉细毛羊心脏、肝脏、脾脏、肾脏、皮肤和肌肉中的表达量。【结果】试验成功克隆小尾寒羊SMAD4基因CDS区,长1 662 bp,编码553个氨基酸。系统进化树分析显示,绵羊SMAD4基因核苷酸序列与山羊和牦牛的亲缘关系最近,与鸡的亲缘关系最远。生物信息学分析发现,SMAD4蛋白为不含信号肽的非跨膜蛋白,存在磷酸化和糖基化位点,属于亲水性蛋白,主要存在于细胞核中;SMAD4蛋白二级结构以无规则卷曲为主,与三级结构预测结果一致;蛋白互作分析显示,SMAD4蛋白与SMAD2、SMAD1、TGFBR1、TGFBR2、SMAD3等蛋白存在相互作用。实时荧光定量PCR结果显示,SMAD4基因在绵羊中广泛表达;与新吉细毛羊相比,小尾寒羊心脏、脾脏和皮肤中SMAD4基因表达量均显著上升(P＜0.05),而肺脏和肌肉中SMAD4基因表达量均显著下降(P＜0.05)。【结论】试验成功克隆绵羊SMAD4基因CDS区,该基因在绵羊各组织中均有表达,且在小尾寒羊和新吉细毛羊心脏、脾脏、皮肤、肺脏和肌肉中存在显著差异。试验结果为进一步探究绵羊SMAD4基因调控毛囊发育提供理论依据。

关键词: 绵羊; SMAD4基因; 克隆; 生物信息学; 表达

Abstract: 【Objective】 This study was aimed to clone SMAD homolog 4 (SMAD4) gene and explore its biological function in sheep,so as to provide a basis for studying the regulatory role of SMAD4 gene in the growth and development of hair follicles.【Method】 Using the cDNA of skin in Small-tailed Han sheep as a template,the CDS region of SMAD4 gene was amplified by PCR and cloned, and was sequenced for bioinformatics analysis.Real-time quantitative PCR was used to detect the expression of SMAD4 gene in heart,liver,spleen,kidney,skin and muscle in Small-tailed Han sheep and Xinji Fine wool sheep.【Result】 The CDS region of SMAD4 gene in Small-tailed Han sheep was successfully cloned,which contained 1 662 bp and encoded 553 amino acids.Phylogenetic tree analysis showed that the nucleotide sequence of SMAD4 gene in sheep was the closest to Capra hircus and Bos mutus,and the most distant with Gallus gallus.Bioinformatics analysis found that SMAD4 protein was a non-transmembrane protein without signal peptides,with phosphorylation and glycosylation sites,and belonged to hydrophilic proteins,mainly in nucleus.The secondary structure of SMAD4 protein was dominated by random coil,which was consistent with the tertiary structure.The results of protein interaction analysis showed that SMAD4 protein interacted with SMAD2,SMAD1,TGFBR1,TGFBR2,SMAD3 and other proteins.Real-time quantitative PCR results showed that SMAD4 gene was widely expressed in sheep.Compared with Xinji Fine wool sheep,the expression of SMAD4 gene in heart,spleen and skin of Small-tailed Han sheep were significantly increased (P＜0.05),but the expression of SMAD4 gene in lung and muscle were significantly decreased (P＜0.05).【Conclusion】 The CDS region of SMAD4 gene in sheep was successfully cloned,which was expressed in various tissues of sheep,and there were significant differences in heart,spleen,skin,lung and muscle between Small-tailed Han sheep and Xinji Fine wool sheep.The results provided a theoretical basis for further exploration of the regulation of hair follicle development by SMAD4 gene in sheep.

Key words: sheep; SMAD4 gene; clone; bioinformatics; expression

中图分类号:

S826
Q78

王丹, 张新玉, 付佳棋, 贾琪, 张双双, 崔成都, 张立春, 孙福亮. 绵羊SMAD4基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(12): 4783-4792.

WANG Dan, ZHANG Xinyu, FU Jiaqi, JIA Qi, ZHANG Shuangshuang, CUI Chengdu, ZHANG Lichun, SUN Fuliang. Cloning,Bioinformatics Analysis and Tissue Expression of SMAD4 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 4783-4792.

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