响应内生菌侵染的两个地黄茉莉酸合成关键基因的克隆与表达分析

Cloning and Expression Analysis of Two Key Genes of Jasmonic Acid Synthesis in Response to Endophytic Infection from Rehmannia glutinosa

丙二烯氧化物合酶(allene oxide synthase,AOS)、12-氧代植二烯酸还原酶(12-oxophytodienoate reduc⁃tase,OPR)基因是植物中茉莉酸合成关键基因。从地黄与内生真菌GG22互作转录组中筛选响应内生菌侵染的AOS和OPR基因序列,设计特异引物,克隆RgAOS和RgOPR基因的完整开放阅读框,并对该序列及其编码产物进行生物信息学分析,采用实时荧光定量PCR技术分析RgAOS和RgOPR基因在不同组织及内生真菌GG22侵染前后的表达模式。RgAOS基因开放阅读框为1626 bp,编码541个氨基酸,分子量为60.20 kD。RgOPR基因的开放阅读框为1197 bp,编码398个氨基酸,分子量为44.07 kD。QPCR分析发现,RgAOS在根中表达量最高,花中最少,RgOPR在花和叶中的表达量较高。内生真菌GG22能诱导地黄中RgAOS和RgOPR基因的表达。该研究成功从地黄中克隆了RgAOS和RgOPR基因,为进一步研究地黄中茉莉酸类物质的生物活性及探索内生真菌对地黄次生代谢产物调节的分子机制奠定了基础。

Allene oxide synthase(AOS)and 12-oxophytodienoate reductase(OPR)were the key genes of jasmonic acid biosynthesis in the plants.The genes of AOS and OPR responding to endophytic fungus infection were screened from the interaction transcriptome of Rehmannia glutinosa with endophytic fungus GG22.Specific primers were designed to the open reading frame of RgAOS and RgOPR.Bioinformatics analysis of the sequences and theirs encoded product were performed,and real-time fluorescent quantitative PCR was used to analyze the expression patterns of RgAOS and RgOPR in different tissues infection by endophytic fungus GG22.The open reading frame of RgAOS was 1626 bp,encoding 541 amino acids with a molecular weight of 60.2 kD.The open reading frame of RgOPR was 1197 bp,encoding 398 amino acids with a molecular weight of 44.07 kD.QPCR analysis showed that the expression of RgAOS was the highest in roots,the lowest in flowers,and the expression of RgOPR was higher in flowers and leaves,respectively.Endophytic fungus GG22 induced the expression of RgAOS and RgOPR in R.glutinosa.The RgAOS and RgOPR were successfully cloned from R.glutinosa,laying the foundation for further study on the biological activity of Jasmonic acid substances in R.glutinosa and claiming the molecular mechanism of endophytic fungi in the regulation of secondary metabolites of R.glutinosa.