摘要
旨在构建敖汉细毛羊Hoxa5、BMPR1B基因的质粒及共转染成纤维细胞后,通过基因表达量变化研究两基因的互作。以敖汉细毛羊为研究对象,采集40日龄的胎羊。首先,通过RNA的提取反转录成cDNA,参照GenBank中Hoxa5、BMPR1B基因序列信息分别设计1对引物,通过PCR反应扩增获得Hoxa5、BMPR1B基因片段,将得到的Hoxa5、BMPR1B基因分别连接到pEASYTM-T1载体,构建pEASYTM-T1-Hoxa5、pEASYTM-T1-BMPR1B重组质粒并转化大肠杆菌(E.coli)DH5α感受态细胞,提取质粒进行酶切鉴定。鉴定正确后构建pcDNA3.1-Hoxa5、pcDNA3.1-BMPR1B重组质粒,转化大肠杆菌(E.coli)DH5α感受态细胞。其次,对敖汉细毛羊的成纤维细胞进行分离培养,并将构建的质粒pcDNA3.1-Hoxa5、pcDNA3.1-BMPR1B共转染成纤维细胞,利用荧光定量PCR技术和Western Blot技术检测Hoxa5、BMPR1B基因单转染和共转染在成纤维细胞中表达量的变化。结果显示,经酶切、测序鉴定质粒pcDNA3.1-Hoxa5、pcDNA3.1-BMPR1B构建成功,并且共转染成纤维细胞BMPR1B基因的表达量明显下降,Hoxa5基因的表达量明显升高且共转染组的表达量极显著地高于单转染组(P<0.01)。成功构建了敖汉细毛羊Hoxa5、BMPR1B基因的质粒,并且成功共转染成纤维细胞,Hoxa5基因的表达量明显升高,BMPR1B基因的表达量明显下降,因而Hoxa5基因抑制了BMPR1B基因的表达,BMPR1B基因促进了Hoxa5基因的表达,结果可为进一步研究其功能奠定基础。
The study aimed to identify the expression of Hoxa5 and BMPR1B gene plasmid and the interaction between the two genes was studied by means of expression changes.Firstly,a pair of primers were desigened by RNA extraction and referring to Hoxa5 and BMPR1B gene sequence information of Aohan fine wool sheep in GenBank,and the Hoxa5 and BMPR1B gene fragment were amplified by PCR method.The obtained Hoxa5 and BMPR1B gene were ligated into pEASYTM-T1 vector to construct pEASYTM-T1- Hoxa5 and pEASYTM-T1- BMPR1B recombinant plasmid and transformed into E.coli DH5α competent cell.The plasind was identified by restriction enzyme digedtion.The recombinant plasimd pcDNA3.1- Hoxa5 and pcDNA3.1- BMPR1B was constructed and transformed into E.coli DH5α competent cell.Then pcDNA3.1- Hoxa5 and pcDNA3.1- BMPR1B plasmid were cotransfection into fibroblasts,and qRT-PCR was used to detect the expression level of its gene.As the results showed that,pcDNA3.1- Hoxa5 and pcDNA3.1-BMPR1B plasmid cotransfection successfully and identified by enzyme and sequencing.The expression level of BMPR1B gene in fibroblasts was lower than in the control group and expression level of Hoxa5 gene in fibroblasts was higher than in the control group.The plasmid was constructed and cotransfection into fibroblasts successfully.The expression level of Hoxa5 gene increased significantly,and the expression of BMPR1B gene decreased.Therefore,Hoxa5 gene inhibited the expression of BMPR1B gene,and BMPR1B gene promoted the expression of Hoxa5 gene.These results would lay a foundation for the further research.
作者
张梦瑶
杨峰
刘积凤
刘开东
贺建宁
柳楠
ZHANG Mengyao;YANG Feng;LIU Jifeng;LIU Kaidong;HE Jianning;LIU Nan(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Qingdao Institute of Animal Husbandry and Veterinary,Qingdao 266121,China;Inner Mongolia Agricultural University,Hohhot 010018,China)
出处
《华北农学报》
CSCD
北大核心
2019年第2期229-238,共10页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金项目(31402047)
国家绒毛用羊产业技术体系专项资金(CARS-39-05)
青岛农业大学高层次人才科研基金(631410)
