梅花鹿KGF的基因克隆及在不同时期顶端茸皮组织的表达分析

Cloning of KGF gene from sika deer and expression analysis of antler tip skin tissues at different stages

为分析梅花鹿KGF基因的生物学特性及其在鹿茸生长不同阶段顶端茸皮组织的表达差异,以雄性梅花鹿茸皮组织为试验材料克隆梅花鹿KGF基因,并采用荧光定量PCR技术对KGF基因在梅花鹿鹿茸顶端不同生长阶段的茸皮组织中的表达量进行差异分析。结果表明:1)成功克隆获得梅花鹿KGF基因的完整编码区,全长为585bp,共编码194个氨基酸;KGF基因编码的蛋白质是具有跨膜结构的不稳定的亲水蛋白,其相对分子质量为22489.17,理论等电点pI为9.35;二级结构以无规则卷曲为主;2)通过Blastp功能比对显示梅花鹿KGF基因与马鹿、白尾鹿、牛、野牛、山羊的相似性均在99%以上;3)荧光定量PCR结果显示,KGF基因的表达量在梅花鹿鹿茸前期与中期、前期与后期的顶端茸皮组织中差异显著(P<0.05),其在中期的表达量是前期的(2.1585±0.0141)倍,后期的表达量是前期的(1.7285±0.2253)倍;其表达量在中期与后期差异不显著(P>0.05)。综上,本研究发现梅花鹿KGF基因在进化中的保守性较高,对鹿茸的生长发育与组织修复有重要作用,其在不同时期茸皮组织中的相对表达量存在差异。本研究为进一步探索鹿茸生长的分子机理奠定基础,为哺乳动物的组织创伤修复提供参考。

In order to analyze the biological characteristics of KGF gene and its expression differences in the skin tissue at the tip of sika deer in different stages,the KGF gene of sika deer was cloned from the tip skin tissue of male sika deer.Fluorescent quantitative PCR was used to analyze the expression difference of KGF gene in the tip skin tissue of sika deer antler at different growth stage.The results showed that:1)The complete coding region of KGF gene was successfully cloned.The KGF gene was 585bp in length encoding 194amino acids.The KGF protein was an unstable hydrophilic protein with transmembrane structure.Its relative molecular mass was 22489.17and the theoretical isoelectric point pI value was 9.35.The secondary structure was dominated by random coils.2)The Blastp comparison showed that the similarity of sika deer of KGF gene with red deer,white-tailed deer,cattle,bison,and goat was more than 99%.3)The expression of KGF gene in the antler tip skin were significantly different(P<0.05)between the early and mid-term stage,the early and late stage,and the expression level in the mid-term stage was(2.1585±0.0141)times higher than that in the early stage.The expression level in the later stage was(1.7285±0.2253)times higher than that in the early stage;the expression level was not significantly different between the middle and late stages(P>0.05).In conclusion,the KGF gene of sika deer is highly conserved in evolution and plays an important role in the growth and development of antler and tissue repair.The relative expression levels of KGF gene in antler skin tissues are different in different stages.This study lays a foundation for further exploring the molecular mechanism of velvet antler growth and provides reference for tissue injury repair in mammals.