【摘要】：根據已知的甜瓜蛋白激酶類基因CmPKC的部分序列,設計帶有酶切位點的引物,采用RT-PCR方法獲得該基因的完整cDNA序列,并對其蛋白進行功能預測和理化性質分析;采用實時熒光定量PCR分析不同白粉病抗性材料、不同處理時間點該基因的表達量變化情況;將該基因完整的ORF連接到原核表達載體p EASY-E1上,轉化大腸桿菌(E.coli)BL21(DE3),通過不同濃度IPTG誘導表達,獲得適宜的誘導表達體系,SDS-PAGE檢測表達產物。結果表明克隆獲得的CmPKC基因長約為2 830 bp,開放閱讀框為2 493 bp,編碼831個氨基酸。生物信息學預測該蛋白的跨膜區(qū)域有4個信號肽;在甜瓜白粉病不同抗性材料上相對表達量變化趨勢不同,在抗性材料上表達豐度出現(xiàn)的時間較感病材料早,且在不同材料上相對表達量變化趨勢與已報道的一個甜瓜感病相關MLO家族基因一致,推測我們所獲得的基因也可能與感病及開花、生長發(fā)育基因相關,且屬于早期應答基因。該蛋白適宜的誘導體系是100 mg/m L IPTG誘導4 h,原核表達產物與預期大小一致,表明該基因能夠成功表達,為深入探索該基因的功能提供了研究依據。
[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.
【作者單位】： 新疆農墾科學院生物技術所;作物種質創(chuàng)新與基因資源利用兵團重點實驗室;新疆兵團農六師農業(yè)科學研究所;
【基金】：973前期研究專項(2014CB160317)資助
【分類號】：S652

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