摘要
研究旨在对鸡胚胎干细胞(Embryonic stem cell,ESC)向鸡精原干细胞(Spermatogonial stem cells,SSC)分化过程中显著差异基因——丝裂原活化蛋白激酶8(Mitogen-activated protein kinase 8,MAPK8)进行生物信息学分析,验证MAPK8慢病毒干扰载体活性及干扰效率,为后期验证MAPK8在ESC向SSC分化过程中的调控方式以及功能研究奠定基础。试验采用RNA-seq和qRT-PCR分析和检测鸡ESC、PGC和SSC的差异表达基因;利用生物信息学在线预测软件对其进行理化性质及结构特征分析;比较鸡MAPK8基因序列与其它物种的同源性并进行组织表达谱检测;验证MAPK8慢病毒干扰载体的活性与干扰效率。结果显示:MAPK8在SSC上特异高表达;生物信息学分析显示鸡MAPK8是一种亲水性的无信号脂溶性蛋白,存在2个N-糖基化位点、13个O-糖基化位点和45个磷酸化位点,与其它物种高度同源(序列一致性达到80%),保守性较高;表达谱分析发现其在性腺中高表达;MAPK8慢病毒干扰载体能够稳定表达EGFP荧光蛋白且shRNA-1干扰效率最佳。该结果为深入探究MAPK8在鸡ESC向SSC分化过程中的功能奠定了基础。
In this study mitogen-activated protein kinase 8(MAPK8) was characterized as a potential mediator during the differentiation of chicken embryonic stem cells(ESCs) into primordial germ cells(PGCs) and then spermatogonial stem cells(SSCs).High-throughput RNA sequencing was performed to identify genes differentially expressed among ESCs,PGCs and SSCs in chicken.RNA-seq data demonstrated the high transcript level of MAPK8 in SSCs specifically,further confirmed by increasing of MAPK8 mRNA level in SSCs compared to ESCs by qPCR.Bioinformatics online prediction software was used to predict the physical and chemical properties,and structural characteristics of MAPK8.Bioinformatics analysis suggested that chicken MAPK8 was a hydrophilic and non-signal fat-soluble protein with 2 N-glycosylation sites,13 O-glycosyls sites and 45 phosphorylation sites.Around 80% identity of MAPK8 gene sequence in chickens and other species with high conservation was determined using Full Alignment.Tissues expression profile detection results revealed the high expression of MAPK8 in gonads.Chicken MAPK8 and non-target control shRNA lentiviral particles were generated and verified in DF-1 cel l,MAPK8 lentiviral interference vector could stably express EGFP fluorescence protein and shRNA-1 had the best interference efficiency.This work provided profound insights into the chicken embryonic cell differentiation research and built a foundation for further investigating the functional role of MAPK8 in the differentiation from chicken ESCs to SSCs.
作者
胡菜
王颖洁
靳锴
吴宇慧
赵宗仪
李熠
张亚妮
HU Cai;WANG Yingjie;JIN Kai;WU Yuhui;ZHAO Zongyi;LI Yi;ZHANG Yani(Key Laboratory of Animal Genetic Breeding and Molecular Design,College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009;The Joint International Research Laboratory of Agriculture&Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009;College of Science and Technology,Wenzhou-Kean University,Wenzhou,Zhejiang 325000)
出处
《中国家禽》
北大核心
2022年第3期17-22,共6页
China Poultry
基金
江苏高校优势学科建设工程资助项目。
