RT-qPCR检测双峰驼体内CYP2J基因的分布与表达及其体外表达载体的构建
发布时间：2019-01-23 07:10
【摘要】：为探明CYP2J基因在双峰驼各组织中的表达分布情况以及进一步为揭示CYP2J基因与双峰驼抗盐敏感性高血压生物学特性的相关性提供线索,本研究采用实时荧光定量PCR法从mRNA层面检测CYP2J基因在双峰驼各组织中的表达量,并构建双峰驼CYP2J基因的体外表达载体,为后续的蛋白层面的研究奠定基础。根据GenBank中登录的双峰驼CYP2J预测序列(XM_006187584)设计引物,选用ACTB作为内参基因,利用SYBR Green I实时荧光定量PCR进行扩增,采用2-ΔΔCT相对定量法处理数据,从基因层面上对双峰驼肝脏、心脏、脾脏、肺脏、肾脏、小肠、血管、胰腺等8种组织的CYP2J基因及羊肝脏CYP2J基因的表达量进行检测。结果表明CYP2J基因在双峰驼体内以心脏表达量最高,其表达量显著高于羊肝脏(P0.01)；其次是肾脏、肝脏、小肠、胰腺,而在脾脏、肺脏及血管的表达量极低。本研究应用RT-PCR方法扩增出双峰驼CYP2J mRNA的全长1509bp的完整CDs区,胶回收纯化后,进行TA克隆,使目的基因先连于pMD-18T中并转化于大肠杆菌DH5α的感受态细胞中。经HindⅢ和Kpnl双酶切和质粒PCR鉴定后,扩大培养,并用上述内切酶双酶切重组质粒pMD18-T-CYP2J后,连接于经同样的双酶切的原核表达载体pET-32a,同样转化于大肠杆菌DH5α细胞中鉴定阳性克隆,并经HindⅢ和KpnⅠ双酶切、质粒PCR鉴定及测序鉴定重组质粒是否构建成功。结果表明,在重组质粒pMD18-T-CYP2J和pET-32a-CYP2J双酶切电泳图中均能显示约1500bp的特异性目的条带以及分别于2700bp和5900bp处显示2种质粒的特异条带；以2种重组质粒为模板进行PCR扩增后电泳图均在约1500bp处显示CYP2J特异性条带；并且将测序结果与Genbank中公布的序列进行比对,其相似度为99%。以上结果证明双峰驼CYP2J原核表达载体pET-32a-CYP2J构建成功。因此,通过本实验从RNA层面初步揭示了CYP2J基因在双峰驼主要8种组织内的表达与分布,并成功构建了该基因的原核表达载体,为后续的蛋白层面研究奠定了基础。
[Abstract]:In order to investigate the expression and distribution of CYP2J gene in various tissues of Bactrian Camel and to further reveal the correlation between CYP2J gene and salt-sensitive hypertension biological characteristics of Bactrian camel. In this study, real-time fluorescence quantitative PCR was used to detect the expression of CYP2J gene in the tissues of Bactrian Camel from mRNA level, and the expression vector of CYP2J gene in vitro was constructed, which laid a foundation for the further study on protein level. According to the CYP2J prediction sequence (XM_006187584) of Bactrian camel registered in GenBank, primers were designed, ACTB was selected as internal reference gene, SYBR Green I real-time fluorescence quantitative PCR was used to amplify, and 2- 螖 CT relative quantitative method was used to process the data. The expression of CYP2J gene and CYP2J gene in liver, heart, spleen, lung, kidney, small intestine, blood vessel and pancreas of Bactrian camel were detected from gene level. The results showed that the expression of CYP2J gene in the heart was the highest in Bactrian camel, which was significantly higher than that in sheep liver (P0.01), followed by kidney, liver, small intestine and pancreas, but very low in spleen, lung and blood vessels. In this study, the complete CDs region of the full-length 1509bp of CYP2J mRNA of Bactrian Camel was amplified by RT-PCR method. After the gel was recovered and purified, TA was cloned, and the target gene was first linked to pMD-18T and transformed into the competent cells of Escherichia coli DH5 伪. After being digested by Hind 鈪，
本文编号：2413585
[Abstract]:In order to investigate the expression and distribution of CYP2J gene in various tissues of Bactrian Camel and to further reveal the correlation between CYP2J gene and salt-sensitive hypertension biological characteristics of Bactrian camel. In this study, real-time fluorescence quantitative PCR was used to detect the expression of CYP2J gene in the tissues of Bactrian Camel from mRNA level, and the expression vector of CYP2J gene in vitro was constructed, which laid a foundation for the further study on protein level. According to the CYP2J prediction sequence (XM_006187584) of Bactrian camel registered in GenBank, primers were designed, ACTB was selected as internal reference gene, SYBR Green I real-time fluorescence quantitative PCR was used to amplify, and 2- 螖 CT relative quantitative method was used to process the data. The expression of CYP2J gene and CYP2J gene in liver, heart, spleen, lung, kidney, small intestine, blood vessel and pancreas of Bactrian camel were detected from gene level. The results showed that the expression of CYP2J gene in the heart was the highest in Bactrian camel, which was significantly higher than that in sheep liver (P0.01), followed by kidney, liver, small intestine and pancreas, but very low in spleen, lung and blood vessels. In this study, the complete CDs region of the full-length 1509bp of CYP2J mRNA of Bactrian Camel was amplified by RT-PCR method. After the gel was recovered and purified, TA was cloned, and the target gene was first linked to pMD-18T and transformed into the competent cells of Escherichia coli DH5 伪. After being digested by Hind 鈪，
本文编号：2413585
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