基于转录组挖掘‘云香’水仙萜类合成基因及NaIDI的克隆
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引用本文:李 琳,肖瑶宇,李 科,李 欢,陈晓静.基于转录组挖掘‘云香’水仙萜类合成基因及NaIDI的克隆[J].西北植物学报,2019,39(8):1379~1388
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李 琳1,2,肖瑶宇1,2,李 科1,2,李 欢3,陈晓静1,2* (1 福建农林大学 园艺学院福州 3500022 福建农林大学园艺植物遗传育种研究所福州 3500023 厦门大学 环境与生态学院,福建厦门 361102) 
基金项目:福建省种业项目(2014S1477 7)
中文摘要:为深入挖掘中国水仙转录组中萜类合成相关基因,了解中国水仙萜类代谢途径和分子调控机制,该研究以‘云香’水仙为材料,在其盛花期花瓣与副冠转录组测序的基础上,筛选已注释的萜类合成途径基因并利用NCBI blastn进行再注释,通过对部分候选基因的表达量与生物信息分析进一步筛选出代表基因,采用RT PCR技术克隆了水仙异戊烯基焦磷酸异构酶基因NaIDI,并对其蛋白序列与特异性表达进行分析。结果表明:(1)Blast比对筛选得到52 个与萜类合成上游途径相关的Unigenes,二次筛选获得11个显著差异表达的代表基因。(2)NaIDI基因开放阅读框(ORF)长度为858 bp,编码285个氨基酸,其氨基酸序列与‘金盏银台’水仙相似度97.19%;亚细胞定位预测显示该基因定位在叶绿体;系统进化分析表明其与芦笋亲缘关系比较近。(3)实时荧光定量分析结果表明,NaIDI基因在水仙开花的不同时期和不同组织器官中差异表达显著,且在盛花期时副冠中的表达量最高,与中国水仙挥发性萜类化合物在开花不同时期和不同组织器官中的表达规律一致,表明NaIDI在萜类代谢中发挥着一定作用。
中文关键词:‘云香’水仙  转录组  萜类代谢  异戊烯基焦磷酸异构酶
 
Search for Related Genes of Terpenes Synthesis and Cloning of NaIDI Based on the Transcriptome of Narcissus tazetta‘Yunxiang’
Abstract:In order to further explore the genes related to terpenoid synthesis in the transcriptome of Narcissus tazetta var. chinensis, and to understand the metabolic pathways and molecular regulation mechanisms of N. tazetta, the study used the N. tazetta ‘Yunxiang’ as the material, screened the annotated terpenoid synthesis pathway genes and re annotated them with NCBI blastn based on the sequencing of the petal and corolla transcripts at the flowering stage. The representative gene was further screened by the expression of some candidate genes and the biosignal analysis. The isopentenyl pyrophos phate isomerase gene NaIDI was cloned by RT PCR, and analyzed for its protein sequence and specific expression. The results showed: (1) blast alignment screened 52 Unigenes associated with the upstream pathway of terpenoid synthesis, and secondary screening obtained 11 significant differentially expressed genes. (2) The open reading frame (ORF) of NaIDI gene is 858 bp in length and encodes 285 amino acids. The amino acid sequence was 97.19% similar to that of N. tazetta ‘Jinzhanyintai’. The subcellular localization is predicted in chloroplast. The phylogenetic analysis shows that it was closely related to asparagus. (3) The results of real time fluorescence quantitative analysis showed that NaIDI was significantly expressed in different flowering periods and different tissues and organs of N. tazetta, and it had the highest expression in the corolla during the flowering stage, and it was consistent with the regularity of volatile steroids in different periods of flowering and different tissues and organs,indicating that NaIDI plays a certain role in the metabolism of terpenes.
keywords:Narcissus tazetta ‘Yunxiang’  transcriptome  terpenes synthesis  isopentenyl diphosphate isomerase (IDI)
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