葡萄白藜芦醇合酶基因的克隆及转化黄芪的研究

发布时间：2018-05-22 15:36

本文选题：白藜芦醇合酶(RS)基因 + 白藜芦醇　；参考：《吉林大学》2017年硕士论文

【摘要】：白藜芦醇合成酶(Resveratrol Synthase,RS)是白藜芦醇(Resveratrol,Res)生物合成代谢途径中的限速关键酶。RS基因无论是在植物,还是在微生物中都实现了遗传转化与生产活性产物,并且在两种表达体系中获得的Res在生物合成代谢途径和表达调控方面均具有生物学活性。随着Res的需求日益增大,通过对有效成分的直接提取,已经远远不能满足对Res的需求,通过现代生物技术和基因工程技术对植物产生改良或者超量表达的途径来有效的提高生产Res这种活性成分已经成为了有效的途径和可行的策略。因此,利用转基因植物技术进行异源植物生产Res的研究前景广阔,同时通过转化RS基因达到协同作用的理念受到了人们广泛的关注。本实验利用基因工程技术克隆RS基因并进行生物信息学分析,再通过农杆菌介导法将葡萄中克隆的RS基因转入黄芪中,由于黄芪中主要有效成分黄酮和白藜芦醇的生物合成途径都从苯丙氨酸合成途径开始,有相同的合成过程,所以选择黄芪为受体材料,不仅可以探索RS基因转化异源植物黄芪,达到异源植物生产白藜芦醇的可能,并为黄芪转基因育种提供技术支持;更可以提高黄芪抗性,进一步佐证白藜芦醇合成酶的功能,为中药现代化提供了有价值的理论参考。本论文获得了以下研究结果:1.RS基因编码一个含310个氨基酸残基的蛋白质,经Blast2go软件分析该基因编码白藜芦醇合成酶。2.成功构建了RS基因的表达载体:p BR121-RS并利用农杆菌遗传转化法对黄芪外植体进行感染,共获得抗性芽204棵,经过生根培养、炼苗、移栽后,共获得11株转基因阳性黄芪植株。3.经DNA、Southern blot杂交以及RT-PCR检测,获得3株转基因黄芪阳性植株,被证明RS基因完全整合到黄芪基因组中并表达产生白藜芦醇。4.经过比较转基因阳性植株和非转基因植株中的白藜芦醇含量,发现3株具有Southern blot杂交信号的阳性植株比非转基因植株的白藜芦醇含量高,其中黄芪非转基因植株的白藜芦醇含量为2029.46μg/g,而三颗黄芪转基因植株的白藜芦醇含量分别为2998.32μg/g、3854.66μg/g、3989.77μg/g,分别提高了48.23%、91.27%和96.59%。本研究建立了黄芪再生及转化体系,并将来源于巨峰葡萄的RS基因成功的转入黄芪中进行表达,获得黄芪转基因阳性植株,这不仅可以探索RS基因转化异源植物黄芪,达到异源植物生产白藜芦醇的可能,并为黄芪转基因育种提供技术支持;更可以提高黄芪抗性,进一步佐证白藜芦醇合成酶的功能,为中药现代化提供了有价值的理论参考。
[Abstract]:Resveratrol Synthase RSs is a rate-limiting key enzyme in the biosynthesis pathway of resveratrol. RS gene realizes genetic transformation and production of active products in plants and microorganisms. Res obtained from the two expression systems has biological activity in biosynthetic metabolic pathway and expression regulation. With the increasing demand of Res, the direct extraction of active components is far from satisfying the demand for Res. It has become an effective way and feasible strategy to improve the production of Res by modern biotechnology and genetic engineering. Therefore, the research prospect of using transgenic plant technology to produce Res from heterologous plants is promising, and the idea of achieving synergistic effect by transforming RS gene has been paid more and more attention. In this study, RS gene was cloned by genetic engineering and bioinformatics analysis was carried out. The RS gene was transferred into Astragalus membranaceus by Agrobacterium tumefaciens. Since the biosynthesis pathway of flavonoids and resveratrol, the main active components of Astragalus membranaceus, starts from the phenylalanine synthesis pathway and has the same synthesis process, the selection of Astragalus membranaceus as receptor material can not only explore the transformation of RS gene into Astragalus membranaceus. It is possible to produce resveratrol from heterologous plants and provide technical support for the transgenic breeding of astragalus, which can enhance the resistance of astragalus, further prove the function of resveratrol synthase, and provide valuable theoretical reference for the modernization of traditional Chinese medicine. In this paper, the following results were obtained: 1. RS gene encodes a protein containing 310 amino acid residues, and the gene encodes resveratrol synthase by Blast2go software. The expression vector of RS gene:% p BR121-RS was successfully constructed and infected with Agrobacterium tumefaciens. A total of 204 resistant buds were obtained. After rooting, seedling cultivation and transplanting, 11 transgenic Astragalus membranaceus plantlets were obtained. Three transgenic Astragalus membranaceus positive plants were obtained by Southern blot hybridization and RT-PCR analysis. It was proved that RS gene was fully integrated into the genome of Astragalus membranaceus and expressed resveratrol. 4. By comparing the resveratrol content in transgenic plants and non-transgenic plants, it was found that the resveratrol content of three positive plants with Southern blot hybridization signal was higher than that of non-transgenic plants. The resveratrol content of Astragalus membranaceus non-transgenic plants was 2029.46 渭 g / g, while the resveratrol content of the three transgenic plants was 2998.32 渭 g / g ~ 3854.66 渭 g / g ~ 3989.77 渭 g / g, which increased 48.23% and 96.59% respectively. In this study, the regeneration and transformation system of Astragalus membranaceus was established, and the RS gene derived from Jufeng grape was successfully transferred into Astragalus membranaceus to obtain the transgenic plants of Astragalus membranaceus, which can not only explore the transformation of RS gene into Astragalus membranaceus. It is possible to produce resveratrol from heterologous plants and provide technical support for the transgenic breeding of astragalus, which can enhance the resistance of astragalus, further prove the function of resveratrol synthase, and provide valuable theoretical reference for the modernization of traditional Chinese medicine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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