玫瑰花香成分生物合成相关基因RrNUDX1和RrDXR的功能验证研究

发布时间：2019-02-12 06:36

【摘要】：玫瑰(Rosa rugosaThunb.)是世界上重要的天然香料植物,也是我国传统的药食同源花卉,单萜类化合物中的香茅醇、香叶醇、橙花醇及其乙酸酯类衍生物是玫瑰鲜花及其精油的主要香气成分。采用基因工程和代谢工程手段,调控玫瑰单萜类化合物的生物合成途径,促进下游代谢产物的积累,进而创制精油含量提高的玫瑰新种质,具有重要的理论和实践意义。本文在实验室前期研究的基础上,进一步分离单萜代谢相关的基因,克隆相关基因的启动子,开展酵母单杂交筛选;并以矮牵牛为试材,开展玫瑰主要花香成分生物合成相关基因的功能验证研究,旨在挖掘调控玫瑰花香代谢的关键基因,为接下来创制高精油含量的玫瑰新种质奠定基础。主要研究结果如下:1.以玫瑰主栽品种'唐红'盛开期花瓣为试材,采用RACE法克隆获得了玫瑰Nudix水解酶基因家族成员RrNUDX1的全长cDNA序列,该cDNA全长777bp,具有完整的开放阅读框,共编码151个氨基酸,基因登陆号为KX096710.1。结合'唐红'不同花发育时期和花器官不同部位香气成分的测定,我们发现,RrNUDX1基因表达水平的变化与玫瑰主要芳香成分含量的变化趋势一致,均先升高后降低。随着花朵开放进程的推进,RrNUDX1基因的表达水平逐步上升,且主要芳香成分的含量也随之上升,除香茅醇及乙酸香茅酯的含量在半开期达到最高外,其他4种成分均在盛开期含量达到最高。从部位上看,玫瑰香气物质主要集中在花瓣、雄蕊、雌蕊中,其他部位含量极低,与RrNUDX1基因在花器官不同部位的表达规律一致。这表明RrNUDX1基因与玫瑰主要单萜类芳香成分的生物合成密切相关。2.以'唐红'半开至盛开期的花瓣为试材,采用invitrogen系统,构建了玫瑰三框读码酵母单杂交文库,结果表明,库容量大于1X107CFU,质粒空载率小于5%,平均插入片段长度大于1.2Kbp,符合酵母文库的筛选要求,为下一步通过建立诱饵载体进行单杂交筛选,寻找玫瑰花香代谢相关转录因子奠定了基础。3.采用染色体步移技术,分别克隆获得了玫瑰RrNUDX1和RrDXR基因的启动子序列,结果表明,RrNUDX1基因共获得617bp的启动子序列,预测发现其中与花香代谢有关的元件2个,分别为MYB的核心元件MBS(N1TAACTG)和茉莉酸核心元件(N2 TGACG)。RrDXR基因共获得2213 bp的启动子序列,预测发现其中与花香代谢有关的元件3个:MYBHv1结合位点(D1CAACGG)和花特异苯丙烷类花香形成重要顺式作用原件(D2GACCAACC,D3AACCTACT)。将预测的5个顺式作用元件分别构建在报告基因的启动子之前,进行酵母单杂交筛选,发现这些元件与RrNUDX1和RrDXR不存在互作,无法进一步筛选出调控顺式作用元件的转录因子,后续研究需进一步扩大预测片段的筛选量,从而获得相应的转录因子。4.进一步构建RrNUDX1基因的过表达载体,采用农杆菌介导法将pCAMBIA1304-RrNUDX1和pCAMBIA1304-RrDXR过表达载体导入矮牵牛,结果发现,转RrNUDX1基因的矮牵牛植株叶片变宽,植株长势旺盛,而转RrDXR基因的矮牵牛植株和野生型相比在表型上无明显差异;花朵GC-MS检测表明,与野生型相比,转RrDXR基因的矮牵牛花朵挥发性成分变化不明显,而转RrNUDX1基因植株花中苯甲酸甲酯等主要香气成分的含量明显提高,且感官上能闻到浓郁的香气,说明过表达RrNUDX1基因具有增强矮牵牛花香的功能,这为我们接下来利用RrNUDX1基因提高玫瑰主要花香成分,培育高精油含量提高的玫瑰新种质奠定了突破性基础。
[Abstract]:Roses (Rosa rugosa Thunb.) It is an important natural perfume plant in the world, and is also a traditional medicine and food homologous flower in China, and the citronellol, the geraniol, the orange flower alcohol and the acetate derivative thereof are the main aroma components of the rose flower and the essential oil of the rose flower and the essential oil. By means of gene engineering and metabolic engineering, the biological synthesis way of the rose single-chain compound is regulated, the accumulation of the downstream metabolites is promoted, and the rose new germplasm with the improved essential oil content is further developed, and has important theoretical and practical significance. On the basis of the early research of the laboratory, this paper further separates the genes related to the monostatic metabolism, the promoter of the clone-related gene, and the yeast single-hybridization screening; and takes the dwarf cow as the test material, and carries out the function verification and research of the biosynthetic related genes of the main flower components of the rose, The invention aims at digging a key gene for regulating and controlling the metabolism of the rose fragrance, and laying a foundation for the next generation of the rose new germplasm with high essential oil content. The main results are as follows: 1. in that form of a rose 'Tang Hong' The full-length cDNA sequence of RrNUDX1, a family member of the Rose Ndix hydrolase gene family, was cloned by the RACE method. The full length of the cDNA was 777bp. The whole length of the cDNA was 777bp. The whole length of the cDNA was 777bp, with a total of 151 amino acids. The gene log-in number was KX096710. combination 'Tang Hong' The results showed that the change of the expression level of RrNUDX1 gene and the content of the main aroma components of the rose was consistent with the change of the content of the main aroma components of the rose. With the development of the open process of the flowers, the expression level of the RrNUDX1 gene gradually increased, and the content of the main aromatic components increased, and the content of the citronellol and the citronellyl acetate reached the highest in the half-open period, and the content of the other four components reached the highest in the blooming period. The aroma of the rose was mainly concentrated in the petal, the stamen and the pistil, and the content of other parts was very low, which was consistent with the expression of the RrNUDX1 gene in different parts of the flower organ. This indicates that the RrNUDX1 gene is closely related to the biosynthesis of the main monogamous aromatic components of the rose. to 'Tang Hong' The petal of the semi-open to the blooming period is a test material, and an investment system is adopted to construct a three-frame read-code yeast single-hybrid library of the rose. The results show that the library capacity is more than 1X107CFU, the no-load rate of the plasmid is less than 5%, the length of the average insertion fragment is more than 1. 2Kbp, and the screening requirement of the yeast library is met. in that next step, the bait vector is establish to carry out single-hybridization screening, and a foundation is laid for finding the related transcription factors of the rose fragrance metabolism. The promoter sequence of the RrNUDX1 and RrDXR genes was obtained by using the chromosome-shift technique. The results showed that the RrNUDX1 gene obtained the promoter sequence of 617bp, and it was predicted that the two elements related to the floral metabolism were found. The core element MBS (N1TAACTG) and the jasmonic acid core element (N2 TGACG), respectively, are MYB. The RrDXR gene obtained a 2213 bp promoter sequence, and it was predicted that three elements related to the floral metabolism were found: the MYBHv1 binding site (D1CAACGG) and the flower-specific phenyl-propane flower form an important cis-acting original (D2GACCAACC, D3AACCTACT). the predicted five cis-acting elements are respectively constructed on the promoter of the reporter gene, and the yeast single-hybridization screening is carried out, and the interaction of the elements with the RrNUDX1 and the RrDXR is found, and the transcription factor of the cis-acting element can not be further screened, The subsequent studies need to further increase the screening amount of the predicted fragments, thus obtaining the corresponding transcription factor. The overexpressing vector of the RrNUDX1 gene is further constructed, and the pCAMBIA1304-RrNUDX1 and pCAMBIA1304-RrDXR overexpressing vectors are introduced into the petunia by the agrobacterium-mediated method, compared with the wild type, the variation of the volatile components of the petunia flower of the RrDXR gene is not obvious compared with the wild type, while the content of the main aroma components such as methyl benzoate and the like in the RrNUDX1 gene plant flower is obviously improved, and a breakthrough foundation is laid for cultivating rose new germplasm with high essential oil content.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S685.12

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