玫瑰花香成分生物合成相關(guān)基因RrNUDX1和RrDXR的功能驗(yàn)證研究
發(fā)布時(shí)間：2019-02-12 06:36
【摘要】：玫瑰(Rosa rugosaThunb.)是世界上重要的天然香料植物,也是我國傳統(tǒng)的藥食同源花卉,單萜類化合物中的香茅醇、香葉醇、橙花醇及其乙酸酯類衍生物是玫瑰鮮花及其精油的主要香氣成分。采用基因工程和代謝工程手段,調(diào)控玫瑰單萜類化合物的生物合成途徑,促進(jìn)下游代謝產(chǎn)物的積累,進(jìn)而創(chuàng)制精油含量提高的玫瑰新種質(zhì),具有重要的理論和實(shí)踐意義。本文在實(shí)驗(yàn)室前期研究的基礎(chǔ)上,進(jìn)一步分離單萜代謝相關(guān)的基因,克隆相關(guān)基因的啟動(dòng)子,開展酵母單雜交篩選;并以矮牽牛為試材,開展玫瑰主要花香成分生物合成相關(guān)基因的功能驗(yàn)證研究,旨在挖掘調(diào)控玫瑰花香代謝的關(guān)鍵基因,為接下來創(chuàng)制高精油含量的玫瑰新種質(zhì)奠定基礎(chǔ)。主要研究結(jié)果如下:1.以玫瑰主栽品種'唐紅'盛開期花瓣為試材,采用RACE法克隆獲得了玫瑰Nudix水解酶基因家族成員RrNUDX1的全長cDNA序列,該cDNA全長777bp,具有完整的開放閱讀框,共編碼151個(gè)氨基酸,基因登陸號為KX096710.1。結(jié)合'唐紅'不同花發(fā)育時(shí)期和花器官不同部位香氣成分的測定,我們發(fā)現(xiàn),RrNUDX1基因表達(dá)水平的變化與玫瑰主要芳香成分含量的變化趨勢一致,均先升高后降低。隨著花朵開放進(jìn)程的推進(jìn),RrNUDX1基因的表達(dá)水平逐步上升,且主要芳香成分的含量也隨之上升,除香茅醇及乙酸香茅酯的含量在半開期達(dá)到最高外,其他4種成分均在盛開期含量達(dá)到最高。從部位上看,玫瑰香氣物質(zhì)主要集中在花瓣、雄蕊、雌蕊中,其他部位含量極低,與RrNUDX1基因在花器官不同部位的表達(dá)規(guī)律一致。這表明RrNUDX1基因與玫瑰主要單萜類芳香成分的生物合成密切相關(guān)。2.以'唐紅'半開至盛開期的花瓣為試材,采用invitrogen系統(tǒng),構(gòu)建了玫瑰三框讀碼酵母單雜交文庫,結(jié)果表明,庫容量大于1X107CFU,質(zhì)�？蛰d率小于5%,平均插入片段長度大于1.2Kbp,符合酵母文庫的篩選要求,為下一步通過建立誘餌載體進(jìn)行單雜交篩選,尋找玫瑰花香代謝相關(guān)轉(zhuǎn)錄因子奠定了基礎(chǔ)。3.采用染色體步移技術(shù),分別克隆獲得了玫瑰RrNUDX1和RrDXR基因的啟動(dòng)子序列,結(jié)果表明,RrNUDX1基因共獲得617bp的啟動(dòng)子序列,預(yù)測發(fā)現(xiàn)其中與花香代謝有關(guān)的元件2個(gè),分別為MYB的核心元件MBS(N1TAACTG)和茉莉酸核心元件(N2 TGACG)。RrDXR基因共獲得2213 bp的啟動(dòng)子序列,預(yù)測發(fā)現(xiàn)其中與花香代謝有關(guān)的元件3個(gè):MYBHv1結(jié)合位點(diǎn)(D1CAACGG)和花特異苯丙烷類花香形成重要順式作用原件(D2GACCAACC,D3AACCTACT)。將預(yù)測的5個(gè)順式作用元件分別構(gòu)建在報(bào)告基因的啟動(dòng)子之前,進(jìn)行酵母單雜交篩選,發(fā)現(xiàn)這些元件與RrNUDX1和RrDXR不存在互作,無法進(jìn)一步篩選出調(diào)控順式作用元件的轉(zhuǎn)錄因子,后續(xù)研究需進(jìn)一步擴(kuò)大預(yù)測片段的篩選量,從而獲得相應(yīng)的轉(zhuǎn)錄因子。4.進(jìn)一步構(gòu)建RrNUDX1基因的過表達(dá)載體,采用農(nóng)桿菌介導(dǎo)法將pCAMBIA1304-RrNUDX1和pCAMBIA1304-RrDXR過表達(dá)載體導(dǎo)入矮牽牛,結(jié)果發(fā)現(xiàn),轉(zhuǎn)RrNUDX1基因的矮牽牛植株葉片變寬,植株長勢旺盛,而轉(zhuǎn)RrDXR基因的矮牽牛植株和野生型相比在表型上無明顯差異;花朵GC-MS檢測表明,與野生型相比,轉(zhuǎn)RrDXR基因的矮牽�；ǘ鋼]發(fā)性成分變化不明顯,而轉(zhuǎn)RrNUDX1基因植株花中苯甲酸甲酯等主要香氣成分的含量明顯提高,且感官上能聞到濃郁的香氣,說明過表達(dá)RrNUDX1基因具有增強(qiáng)矮牽牛花香的功能,這為我們接下來利用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種質(zhì)奠定了突破性基礎(chǔ)。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S685.12
本文編號：2420145
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S685.12
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