[关键词]
[摘要]
目的 克隆大花红景天Rhodiola crenulata尿苷二磷酸葡萄糖基转移酶基因组DNA,并利用生物信息学对其编码区与启动子进行分析。方法 以大花红景天为样品,采用优化的CTAB法提取大花红景天基因组DNA,并利用hiTAIR-PCR技术经过3次步移获得大花红景天UDPGT(RcUDPGT)基因DNA序列,并利用生物信息学对其进行分析。结果 克隆得到RcUDPGT基因的DNA序列2 977 bp,其中包含1 499 bp的编码区域(包括82 bp的内含子序列)和1 500 bp的编码区5’上游侧翼域序列(包括启动子区域)。生物信息学分析证明该序列为UDPGT基因且与其他植物UDPGT同源,具有亲水性且定位于细胞质中,三级结构预测表明该基因编码的蛋白具有UDPGT功能结合位点。启动子分析表明其存在光响应、厌氧响应、热响应、低温响应、压力与防御响应和花色素苷合成响应元件等。结论 克隆的RcUDPGT基因结构完整,具有典型的功能结合位点,为红景天分子生物学与代谢工程研究提供参考。
[Key word]
[Abstract]
Objective To obtain the genomic DNA sequence of uridine diphosphate (UDP)-glucosyltransferase (GT) gene from Rhodiola crenulata and to analyze its DNA sequence at the level of bioinformatics. Methods The optimized CTAB method was used to extract the genomic DNA from R. crenulata, and after three times genomic walking, the genomic DNA sequence of UDPGT in R. crenulata (RcUDPGT) was obtained by hiTAIR-PCR. The DNA sequence was analyzed by bioinformatics method. Results The 2 977 bp DNA sequence of RcUDPGT was obtained, which contained the 1 499 bp gene sequence (including the 82 bp intron sequence) and 1 500 bp 5’ upstream flanking sequence of coding region (including promoter sequence). The bioinformatic analysis showed that the RcUDPGT was hydrophilic, located in the cytoplasm, and had high homology with UDPGTs of other plants. The tertiary structure of RcUDPGT indicated that protein had UDPGT functional sites. Promoter analysis showed that it contained cis-acting elements responding to light, heat, pressure, temperature, anaerobic, anthocyanins biosynthesis sequences, etc. Conclusion The structure of RcUDPGT gene is integral and has functional sites. The research provides the reference for the molecular biology and metabolic engineering of R. crenulata.
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[基金项目]
国家科技支撑计划项目(2011BAI13B06);重庆市科学技术委员会自然科学基金计划资助项目(2010BB5296)