【摘要】：為探明CYP2J基因在雙峰駝各組織中的表達(dá)分布情況以及進一步為揭示CYP2J基因與雙峰駝抗鹽敏感性高血壓生物學(xué)特性的相關(guān)性提供線索,本研究采用實時熒光定量PCR法從mRNA層面檢測CYP2J基因在雙峰駝各組織中的表達(dá)量,并構(gòu)建雙峰駝CYP2J基因的體外表達(dá)載體,為后續(xù)的蛋白層面的研究奠定基礎(chǔ)。根據(jù)GenBank中登錄的雙峰駝CYP2J預(yù)測序列(XM_006187584)設(shè)計引物,選用ACTB作為內(nèi)參基因,利用SYBR Green I實時熒光定量PCR進行擴增,采用2-ΔΔCT相對定量法處理數(shù)據(jù),從基因?qū)用嫔蠈﹄p峰駝肝臟、心臟、脾臟、肺臟、腎臟、小腸、血管、胰腺等8種組織的CYP2J基因及羊肝臟CYP2J基因的表達(dá)量進行檢測。結(jié)果表明CYP2J基因在雙峰駝體內(nèi)以心臟表達(dá)量最高,其表達(dá)量顯著高于羊肝臟(P0.01)；其次是腎臟、肝臟、小腸、胰腺,而在脾臟、肺臟及血管的表達(dá)量極低。本研究應(yīng)用RT-PCR方法擴增出雙峰駝CYP2J mRNA的全長1509bp的完整CDs區(qū),膠回收純化后,進行TA克隆,使目的基因先連于pMD-18T中并轉(zhuǎn)化于大腸桿菌DH5α的感受態(tài)細(xì)胞中。經(jīng)HindⅢ和Kpnl雙酶切和質(zhì)粒PCR鑒定后,擴大培養(yǎng),并用上述內(nèi)切酶雙酶切重組質(zhì)粒pMD18-T-CYP2J后,連接于經(jīng)同樣的雙酶切的原核表達(dá)載體pET-32a,同樣轉(zhuǎn)化于大腸桿菌DH5α細(xì)胞中鑒定陽性克隆,并經(jīng)HindⅢ和KpnⅠ雙酶切、質(zhì)粒PCR鑒定及測序鑒定重組質(zhì)粒是否構(gòu)建成功。結(jié)果表明,在重組質(zhì)粒pMD18-T-CYP2J和pET-32a-CYP2J雙酶切電泳圖中均能顯示約1500bp的特異性目的條帶以及分別于2700bp和5900bp處顯示2種質(zhì)粒的特異條帶；以2種重組質(zhì)粒為模板進行PCR擴增后電泳圖均在約1500bp處顯示CYP2J特異性條帶；并且將測序結(jié)果與Genbank中公布的序列進行比對,其相似度為99%。以上結(jié)果證明雙峰駝CYP2J原核表達(dá)載體pET-32a-CYP2J構(gòu)建成功。因此,通過本實驗從RNA層面初步揭示了CYP2J基因在雙峰駝主要8種組織內(nèi)的表達(dá)與分布,并成功構(gòu)建了該基因的原核表達(dá)載體,為后續(xù)的蛋白層面研究奠定了基礎(chǔ)。
[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 偽. After being digested by Hind 鈪，

本文編號：2413585