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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。
Objective To clone genomic DNA of Rhodiola crenulata uridine diphosphate glucosyltransferase from Rhodiola sachalinensis and analyze its coding region and promoter using bioinformatics. Methods The genomic DNA of Rhodiola sachalinensis was extracted by optimized CTAB method and the DNA sequence of Rhodiola rosea UDPGT (RcUDPGT) gene was obtained by using three steps of hiTAIR-PCR. The sequence of RcUDPGT gene was amplified by bioinformatics Learn to analyze it. Results The 2 977 bp DNA sequence of RcUDPGT gene was cloned. It contained 1 499 bp coding region (including 82 bp intron sequence) and 1 500 bp coding region 5 ’upstream flanking region sequence (including promoter region). Bioinformatic analysis proved that the sequence was UDPGT gene, homologous to other plant UDPGT, hydrophilic and localized in the cytoplasm. The tertiary structure prediction indicated that the protein encoded by this gene had a UDPGT functional binding site. Promoter analysis showed that there were photoreactions, anaerobic responses, thermal responses, hypothermia responses, pressure and defense responses, and anthocyanin synthesis response elements. Conclusion The cloned RcUDPGT gene is structurally complete and has typical functional binding sites, providing a reference for molecular biology and metabolic engineering of Rhodiola.