茉莉花芳樟醇生物合成关键基因的克隆与表达分析
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引用本文:陈 笛,陈雪津,郭永春,王鹏杰,岳 川,陈桂信,叶乃兴.茉莉花芳樟醇生物合成关键基因的克隆与表达分析[J].西北植物学报,2019,39(8):1344~1352
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陈 笛,陈雪津,郭永春,王鹏杰,岳 川,陈桂信,叶乃兴* (福建农林大学 园艺学院茶学福建省高校重点实验室福州 350002) 
基金项目:福州市科技计划项目(2017N0018);
中文摘要:芳樟醇是芳香植物的重要挥发性化合物之一,橙花叔醇/芳樟醇合酶基因(NES/LINS)是芳樟醇化合物生物合成的重要调控基因。该研究以茉莉花花瓣为材料,基于茉莉花花瓣转录组测序结果,分别采用RT PCR和染色体步移技术,克隆获得NES/LINS基因的全长cDNA序列及其启动子序列,并应用SPEC GC MS和qRT PCR技术检测茉莉花开放过程中芳樟醇化合物的相对含量及JsNES/LINS基因的表达水平,并分析JsNES/LINS在植物激素处理后的表达水平,为进一步揭示JsNES/LINS基因在茉莉花芳樟醇的生物合成调控中的作用提供理论依据。结果表明:(1)获得的JsNES/LINS基因(登录号为:MH513857.1)cDNA全长为1 843 bp,开放阅读框(ORF)全长为1 755 bp,编码584个氨基酸,为亲水性稳定蛋白;NCBI BLAST分析显示,JsNES/LINS与油橄榄OeNES、金鱼草AmNES/LINS1和AmNES/LINS 2以及山茶花CsLINS的相似性分别为56%、54%、54%和50%。(2)亚细胞定位结果显示JsNES/LINS定位于细胞质中;获得了JsNES/LINS起始密码子上游1 141 bp启动子序列,PlantCare预测结果显示,该序列包含多种与植物激素和光照等响应相关的顺式作用元件。(3)GC MS分析显示,芳樟醇化合物的相对含量在茉莉花未开放18:00 时最低,半开放22:00时达到最大,随后呈下降趋势。(4)qRT PCR结果显示,JsNES/LINS的相对表达量变化与芳樟醇化合物的相对含量变化趋势一致;经IAA、GA、ABA及MeJA处理后JsNES/LINS的表达量受到不同程度的诱导表达,其中MeJA的诱导作用最为明显,诱导作用由大至小分别为:MeJA>GA>ABA>IAA。研究推测,JsNES/LINS在芳樟醇化合物的生物合成过程中具有重要调节作用,且受植物激素调控表达。
中文关键词:茉莉花  芳樟醇  JsNEL/LINS基因  启动子  表达分析
 
Cloning and Expression Analysis of JsNEL/LINS from Jasminum sambac
Abstract:Linalool is one of the most important volatile compounds in aromatic plants. The nerolidol/linalool synthase gene is an important regulatory gene for linalool biosynthesis. In this study, based on the transcriptome results of jasmine petals, we cloned the full length cDNA and promoter sequence of NEL/LINS using RT PCR and Genome Walking Technique, respectively. The content of linalool was detected by GC MS, and the expression level of JsNEL/LINS during the opening process and after treatments of IAA, GA, ABA and MeJA, which provided a theoretical basis for further study of the role of JsNES/LINS gene in the biosynthesis regulation of jasmine linalool. The results showed that: (1) the full length cDNA of JsNEL/LINS (GenBank accession number:MH513857) was 1 843 bp,contained 1 755 bp open reading frame (ORF), encoding a protein of 584 amino acids, and belonged to hydrophilic and stable protein. The results of NCBI BLAST showed that JsNES/LINS had 56%,54%,54% and 50% similarities with Olea europaea OeNES, Antirrhinum majus AmNES/LINS1,AmNES/LINS 2 and Camellia saluenensis CsLINS, respectively. (2) The result of subcellular localization showed that JsNEL/LINS was located on the cytoplasm. 1 141 bp promoter sequence of the JsNES/LINS was isolated, and the result predicted that it contained several cis acting elements involved in plant hormones and light response,indicating that the expression of JsNEL/LINS was regulated by plant hormones and light. (3) The results of GC MS analysis showed that the relative content of linalool was the lowest when jasmine was not opened at 18:00, and reached the maximum when it was half open at 22:00, and then decreased. (4) The analysis of qRT PCR showed that the relative expression level of JsNES/LINS was consistent with the change of the relative content of linalool compounds during the flowering process. The expression of JsNES/LINS was induced by IAA, GA, ABA and MeJA, and the induction of MeJA was the most obvious, and the induction from high to low were MeJA>GA>ABA>IAA. It is speculated that JsNES/LINS plays an important role in the biosynthesis of linalool compounds and is regulated by plant hormones.
keywords:Jasminum sambac  linalool  JsNEL/LINS  promoter  expression analysis
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