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[摘要]
目的 克隆苦荞Fagopyrum tataricum类黄酮3’-羟化酶(flavonoid 3’-hydroxylase,F3’H)基因全长cDNA序列,对该基因进行序列分析和原核表达,以及冷胁迫条件下该基因表达量和花青素定量测定。方法 采用同源克隆和RACE技术从苦荞花蕾中克隆苦荞F3’H(FtF3’H);构建FtF3’H原核表达载体pET-30b (+)-FtF3’H,在大肠杆菌BL21(DE3)中进行诱导表达;采用半定量RT-PCR分析FtF3’H基因在冷胁迫下芽期苦荞不同组织的表达量,并采用分光光度计法测量花青素量。结果 苦荞FtF3’H基因含有一个1 470 bp的ORF,编码489个氨基酸,编码蛋白属于细胞色素P450家族;SDS-PAGE分析表明,目的蛋白相对分子质量54 000;芽期苦荞冷胁迫处理前后子叶和胚轴中FtF3’H表达量以及花青素量的变化分析表明,冷胁迫显著增强了FtF3’H的表达和花青素的积累(P<0.05)。结论 成功克隆FtF3’H基因,并实现了FtF3’H基因的异源表达;芽期苦荞在冷胁迫下,可能通过提高FtF3’H基因表达量进而促进花青素的合成,参与其抗逆生理过程。
[Key word]
[Abstract]
Objective To clone and analyze the full-length of flavonoid 3'-hydroxylase (F3'H) gene from tartary buckwheat (Fagopyrum tataricum) and to express it in Escherichia coli. FtF3'H gene expression and anthocyanins accumulation is also to be analyzed in tartary buckwheat sprout under cold stress. Methods Homology cloning and RACE method were used to obtain FtF3'H gene from flower buds of tartary buckwheat. The recombinant vector pET-30b (+)-FtF3'H was constructed and expressed in E. coli BL21 (DE3). Semi-quantitative RT-PCR was used to analyze FtF3'H gene expression when spectrophotometric method was used to determine anthocyanin content. Results FtF3'H gene contains an open reading frame (1 470 bp) encoding 489 amino acids and belongs to cytochrome P450 family. SDS-PAGE analysis of IPTG induced recombinant E. coli BL21 (DE3) showed that a predicted 54 000 Da fusion protein was expressed in the culture. Cold stress significantly enhanced the expression level of FtF3'H and anthocyanin accumulation (P < 0.05). Conclusion FtF3'H gene could be cloned from F. tataricum and efficiently expressed in E. coli. Under cold stress, FtF3'H gene may enhance its expression level to promote anthocyanin accumulation, by taking part in the process of cold-stress resistance of F. tataricum sprouts.
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[基金项目]
四川农业大学“211工程”双支计划(00770106)