摘要
以成年健康简州大耳羊为实验动物,利用RT-PCR技术克隆山羊ZNF32基因。利用生物信息学方法对其进行表达特性分析,并利用实时荧光定量PCR(real-time quantitative PCR,qPCR)技术检测ZNF32在山羊各组织中和诱导分化不同阶段的皮下脂肪细胞中的表达水平。同时构建过表达载体并利用qPCR技术检测过表达或干扰ZNF32对增殖抑制相关基因表达的影响,探究其对山羊皮下前体脂肪细胞增殖的作用。结果显示,克隆所得山羊ZNF32核苷酸序列长度为1 049 bp,其中CDS区长为822bp,编码273个氨基酸;ZNF32分子量为30 983.03 Da,理论等电点为9.52,亲水性平均值为-0.813;在氨基酸序列组成中,丝氨酸含量为9.2%,占比最高;蛋白质二级结构预测显示,149个氨基酸形成无规卷曲,含量最高;氨基酸同源性比对结果显示,简州大耳羊与绵羊氨基酸序列相似性最高且在进化上亲缘关系最近;组织表达谱表明,ZNF32基因在山羊各个组织中广泛表达,其中在大肠中表达量最高(P<0.01);时序表达谱表明,ZNF32基因表达水平呈上升趋势,在96 h表达量达到最高,极显著高于分化前(P<0.01);qPCR技术检测结果表明,过表达ZNF32基因抑制增殖抑制相关基因p27和p57的表达,干扰ZNF32基因促进p21、p27、p53及p57的表达。以上结果表明,ZNF32基因可能是山羊皮下脂肪细胞分化和增殖的正调控因子。
Having adult healthy Jianzhou big-ear goat as experimental animals,RT-PCR technology was used to clone the goat ZNF32gene. The bioinformatics analysis was used to analyze the expression characteristics,and real-time quantitative PCR(qPCR)was used to detect the expressions of ZNF32 in goat tissues and inducing differentiation of subcutaneous fat cells at different stages. Meanwhile,the overexpression vector was constructed and qPCR was to detect the effects of overexpression or interference of ZNF32 on the expressions of genes related to proliferation inhibition,and its effect on the proliferation of subcutaneous precursor adipocytes was explored. The results showed that the length of the cloned goats ZNF32 nucleotide sequence was 1 049 bp,of which the CDS area length was 822 bp and 273 amino acids were encoded. ZNF32 molecular weight was 30 983.03 Da,the theoretical isoelectric point was 9.52,and the average hydrophilic value was-0.813. In the composition of amino acid sequences,serine content was 9.2%,accounting for the highest proportion. The secondary structure prediction of proteins demonstrated that 149 amino acids formed irregular curls and the highest content. The results of amino acid homology alignment revealed that the Jianzhou big-ear goat shared the highest similarity with sheep,and they were in the closest genetic relationship.Tissue expression spectrum showed that the ZNF32 gene was widely expressed in various tissues of the goat. Among them,the expression in the large intestine was the highest(P<0.01). The timing expression spectrum showed that the expression level of the ZNF32 gene was on the rise,reached the highest expression at 96 h,which was significantly higher than that before differentiation(P<0.01). The detection results of q PCR technology suggested that the overexpression of the ZNF32 gene inhibited the expression of proliferative inhibition-related genes p27and p57,while the interference of ZNF32 promoted the expression of p21,p27,p53 and p57. The above results show that the ZNF32 gene may be a positive regulatory factor for subcutaneous fat cell differentiation and proliferation.
作者
盛雪晴
赵楠
林亚秋
陈定双
王瑞龙
李傲
王永
李艳艳
SHENG Xue-qing;ZHAO Nan;LIN Ya-qiu;CHEN Ding-shuang;WANG Rui-long;LI Ao;WANG Yong;LI Yan-yan(College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041)
出处
《生物技术通报》
CAS
CSCD
北大核心
2022年第12期300-311,共12页
Biotechnology Bulletin
基金
国家自然科学基金项目(82002817)
西南民族大学大学生创新创业训练计划项目(S202110656085)。
关键词
山羊
ZNF32
基因克隆
表达分析
分化
增殖
goat
ZNF32
gene cloning
expression analysis
differentiation
proliferation