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目的:获得白花丹参参与乙烯和多胺合成的顺式还原酮加双氧酶(acireductone dioxygenase,ARD)基因(命名为SmARD)序列,并进行生物信息学分析和初步的表达特性研究。方法:利用全长cDNA文库技术,从两年生白花丹参根中获得目的基因SmARD序列。利用BLAST进行序列比对,ORF Finder寻找该基因的开放读码框,prosite分析蛋白质的基本结构域。用半定量RT-PCR检测其在白花丹参幼苗根、茎、叶和成花中的表达情况。结果:得到688 bp的SmARD基因全长序列。具有一个591 bp的开放读码框,编码196个氨基酸,预测蛋白质相对分子质量23.27 kDa。使用预测的SmARD蛋白序列在NCBI中进行蛋白保守域分析,发现SmARD与ARD/ARD′家族具有很高的同源性。半定量RT-PCR表明,SmARD在白花丹参幼苗根、茎、叶和成花等组织中均有转录水平的表达,但是在根部表达最强。水分亏缺处理3 d,150 mmol.L-1NaCl处理1 d,4℃低温处理1 d和100μmol.L-1ABA处理1 d均抑制SmARD的表达,100μmol.L-1茉莉酸甲脂(MJ)和10μmol.L-1乙烯利(ETH)处理1 d诱导SmARD的表达。结论:首次得到白花丹参的顺式还原酮加双氧酶(ARD)基因序列,为其参与白花丹参响应逆境和次生代谢产物合成的信号调节功能研究奠定了基础。
OBJECTIVE: To obtain the sequence of the salidroside dioxygenase (ARD) gene (named as SmARD) involved in the synthesis of ethylene and polyamine in Salvia miltiorrhiza Bunge and analyze its bioinformatics and preliminary expression characteristics. Methods: The full-length cDNA library was used to obtain SmARD sequence from the biennial Salvia miltiorrhiza root. Using BLAST sequence alignment, ORF Finder looking for the open reading frame of the gene, prosite analysis of the basic protein domain. Semi-quantitative RT-PCR was used to detect the expression of Danshen in root, stem, leaf and flower of Salvia miltiorrhiza. Results: The 688 bp SmARD gene full-length sequence was obtained. It has a 591 bp open reading frame encoding 196 amino acids and predicted protein relative molecular mass of 23.27 kDa. Using the predicted SmARD protein sequence for protein conserved domain analysis in NCBI, SmARD was found to have high homology to the ARD / ARD ’family. Semi-quantitative RT-PCR showed that SmARD expressed at the transcriptional level in roots, stems, leaves, flowers and other tissues of Salvia miltiorrhiza, but the expression was the strongest at the roots. After 3 days of water deficit treatment, 150 mmol.L-1 NaCl for 1 day, SmARD expression was inhibited 1 day after 4 ℃ low temperature treatment and 100μmol.L-1ABA treatment for 1 day, 100μmol.L-1 jasmonic acid methyl ester (MJ) And 10μmol.L-1 ethephon (ETH) for 1 d to induce SmARD expression. CONCLUSION: The cis-reductone and dioxygenase (ARD) gene sequence was obtained for the first time in Salvia miltiorrhiza, which laid the foundation for its involvement in the response regulation of Salvia miltiorrhiza and the signal regulation of secondary metabolites.