尾巨桉不同类型愈伤组织代谢组学分析及相关基因表达差异研究

Metabolomics analysis and differential expression of related genes in different callus of Eucalyptus urophylla×E.grandis

桉树是世界著名的三大速生经济林树种之一,具有极高的经济价值。本研究以尾巨桉无菌苗茎段为外植体,诱导得到白色、淡黄色、红色以及绿色4种愈伤组织材料,通过生物信息分析,预测了6个CKX和IPT基因的生物信息学特征及亚细胞定位;基于LC-MS/MS对尾巨桉4种不同形态愈伤组织的生长素和细胞分裂素进行靶向代谢组学检测,分析代谢物差异影响愈伤组织差异分化的作用机制;结合实时荧光定量PCR分析了不同类型愈伤组织中CKXs与IPT基因的相对表达差异。结果表明CKX F具有一个信号肽序列,为分泌蛋白;代谢组数据分析后发现尾巨桉外植体的内源性细胞分裂素浓度相对较高时,更易形成高活性胚性愈伤组织,促进后续培养过程中不定芽的发生。在红色愈伤组织中,CKX F基因上调表达明显。从愈伤组织的形态特征来看,推测IPT及CKX A、CKX B基因和叶绿素的形成与积累密切相关。研究探讨了造成尾巨桉不同愈伤组织差异分化的生理代谢及细胞分裂素合成及其氧化酶相关基因的表达差异,为后续进一步探索提高愈伤组织及不定芽的发生率提供了借鉴与参考。

Eucalyptus is one of the world’s three famous fast-growing economic forest species with high economic value.In this study,the stems of Eucalyptus urophylla×E.grandis seedlings were used as explants to induce white,light yellow,red and green calli.The bioinformatics characteristics and subcellular localization of six CKXs and IPT genes were predicted by bioinformatics analysis.And based on LC-MS/MS,targeted metabolomics detection of auxin and cytokinin in four different forms of calli from E.urophylla×E.grandis was carried out to analyze the mechanism of metabolites difference affecting callus differentiation.The relative expression of CKX and IPT genes in different forms of calli were analyzed by real-time quantitative PCR.The results showed that CKX F had a signal peptide sequence,which was a secreted protein.After the analysis of metabolic group data,it was found that when the endogenous cytokinin concentration of E.urophylla×E.grandis explants was relatively high,it was more likely to form highly active embryogenic calli,and promote the occurrence of adventitious buds in the subsequent culture process.In red callus,CKX F gene was up-regulated significantly.From the morphological characteristics of callus,it was speculated IPT and CKX A,CKX B genes were closely related to the formation and accumulation of chlorophyll.This study explored the differences in physiological metabolism,cytokinin synthesis and expression of oxidase-related genes causing differential differentiation from calli in E.urophylla×E.grandis,which provided some references for further exploration to improve the incidence of adventitious buds in callus.