甜瓜蛋白激酶类基因CmPKC的克隆及原核表达载体的构建
发布时间：2018-11-12 21:21
【摘要】：根据已知的甜瓜蛋白激酶类基因CmPKC的部分序列,设计带有酶切位点的引物,采用RT-PCR方法获得该基因的完整cDNA序列,并对其蛋白进行功能预测和理化性质分析;采用实时荧光定量PCR分析不同白粉病抗性材料、不同处理时间点该基因的表达量变化情况;将该基因完整的ORF连接到原核表达载体p EASY-E1上,转化大肠杆菌(E.coli)BL21(DE3),通过不同浓度IPTG诱导表达,获得适宜的诱导表达体系,SDS-PAGE检测表达产物。结果表明克隆获得的CmPKC基因长约为2 830 bp,开放阅读框为2 493 bp,编码831个氨基酸。生物信息学预测该蛋白的跨膜区域有4个信号肽;在甜瓜白粉病不同抗性材料上相对表达量变化趋势不同,在抗性材料上表达丰度出现的时间较感病材料早,且在不同材料上相对表达量变化趋势与已报道的一个甜瓜感病相关MLO家族基因一致,推测我们所获得的基因也可能与感病及开花、生长发育基因相关,且属于早期应答基因。该蛋白适宜的诱导体系是100 mg/m L IPTG诱导4 h,原核表达产物与预期大小一致,表明该基因能够成功表达,为深入探索该基因的功能提供了研究依据。
[Abstract]:According to the known partial sequence of muskmelon protein kinase gene CmPKC, a primer with restriction endonuclease site was designed, the complete cDNA sequence of the gene was obtained by RT-PCR method, and the function and physical and chemical properties of the protein were predicted and analyzed. Real-time fluorescence quantitative PCR was used to analyze the expression changes of the gene in different powdery mildew resistant materials and different treatment time points. The intact ORF was ligated to the prokaryotic expression vector p EASY-E1 and transformed into Escherichia coli (E.coli) BL21 (DE3). A suitable expression system was obtained by different concentrations of IPTG, and the expression product was detected by SDS-PAGE. The results showed that the length of the cloned CmPKC gene was about 2 830 bp, and the open reading frame was 2 493 bp, encoding 831 amino acids. Bioinformatics predicted that there were four signal peptides in the transmembrane region of the protein. The change trend of relative expression amount on different resistant materials of melon powdery mildew was different, and the time of expression abundance on resistant materials was earlier than that on susceptible materials. The variation trend of relative expression in different materials is consistent with that of a reported MLO family of muskmelon susceptible to disease. It is speculated that our obtained genes may also be related to susceptible, flowering, growth and development genes, and belong to early response genes. The suitable induction system was 100 mg/m L IPTG for 4 h, and the prokaryotic expression product was consistent with the expected size, which indicated that the gene could be successfully expressed, which provided a basis for further study on the function of the gene.
【作者单位】： 新疆农垦科学院生物技术所;作物种质创新与基因资源利用兵团重点实验室;新疆兵团农六师农业科学研究所;
【基金】：973前期研究专项(2014CB160317)资助
【分类号】：S652
[Abstract]:According to the known partial sequence of muskmelon protein kinase gene CmPKC, a primer with restriction endonuclease site was designed, the complete cDNA sequence of the gene was obtained by RT-PCR method, and the function and physical and chemical properties of the protein were predicted and analyzed. Real-time fluorescence quantitative PCR was used to analyze the expression changes of the gene in different powdery mildew resistant materials and different treatment time points. The intact ORF was ligated to the prokaryotic expression vector p EASY-E1 and transformed into Escherichia coli (E.coli) BL21 (DE3). A suitable expression system was obtained by different concentrations of IPTG, and the expression product was detected by SDS-PAGE. The results showed that the length of the cloned CmPKC gene was about 2 830 bp, and the open reading frame was 2 493 bp, encoding 831 amino acids. Bioinformatics predicted that there were four signal peptides in the transmembrane region of the protein. The change trend of relative expression amount on different resistant materials of melon powdery mildew was different, and the time of expression abundance on resistant materials was earlier than that on susceptible materials. The variation trend of relative expression in different materials is consistent with that of a reported MLO family of muskmelon susceptible to disease. It is speculated that our obtained genes may also be related to susceptible, flowering, growth and development genes, and belong to early response genes. The suitable induction system was 100 mg/m L IPTG for 4 h, and the prokaryotic expression product was consistent with the expected size, which indicated that the gene could be successfully expressed, which provided a basis for further study on the function of the gene.
【作者单位】： 新疆农垦科学院生物技术所;作物种质创新与基因资源利用兵团重点实验室;新疆兵团农六师农业科学研究所;
【基金】：973前期研究专项(2014CB160317)资助
【分类号】：S652
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