本文選題：白藜蘆醇合酶(RS)基因 + 白藜蘆醇��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：白藜蘆醇合成酶(Resveratrol Synthase,RS)是白藜蘆醇(Resveratrol,Res)生物合成代謝途徑中的限速關(guān)鍵酶。RS基因無(wú)論是在植物,還是在微生物中都實(shí)現(xiàn)了遺傳轉(zhuǎn)化與生產(chǎn)活性產(chǎn)物,并且在兩種表達(dá)體系中獲得的Res在生物合成代謝途徑和表達(dá)調(diào)控方面均具有生物學(xué)活性。隨著Res的需求日益增大,通過(guò)對(duì)有效成分的直接提取,已經(jīng)遠(yuǎn)遠(yuǎn)不能滿(mǎn)足對(duì)Res的需求,通過(guò)現(xiàn)代生物技術(shù)和基因工程技術(shù)對(duì)植物產(chǎn)生改良或者超量表達(dá)的途徑來(lái)有效的提高生產(chǎn)Res這種活性成分已經(jīng)成為了有效的途徑和可行的策略。因此,利用轉(zhuǎn)基因植物技術(shù)進(jìn)行異源植物生產(chǎn)Res的研究前景廣闊,同時(shí)通過(guò)轉(zhuǎn)化RS基因達(dá)到協(xié)同作用的理念受到了人們廣泛的關(guān)注。本實(shí)驗(yàn)利用基因工程技術(shù)克隆RS基因并進(jìn)行生物信息學(xué)分析,再通過(guò)農(nóng)桿菌介導(dǎo)法將葡萄中克隆的RS基因轉(zhuǎn)入黃芪中,由于黃芪中主要有效成分黃酮和白藜蘆醇的生物合成途徑都從苯丙氨酸合成途徑開(kāi)始,有相同的合成過(guò)程,所以選擇黃芪為受體材料,不僅可以探索RS基因轉(zhuǎn)化異源植物黃芪,達(dá)到異源植物生產(chǎn)白藜蘆醇的可能,并為黃芪轉(zhuǎn)基因育種提供技術(shù)支持;更可以提高黃芪抗性,進(jìn)一步佐證白藜蘆醇合成酶的功能,為中藥現(xiàn)代化提供了有價(jià)值的理論參考。本論文獲得了以下研究結(jié)果:1.RS基因編碼一個(gè)含310個(gè)氨基酸殘基的蛋白質(zhì),經(jīng)Blast2go軟件分析該基因編碼白藜蘆醇合成酶。2.成功構(gòu)建了RS基因的表達(dá)載體:p BR121-RS并利用農(nóng)桿菌遺傳轉(zhuǎn)化法對(duì)黃芪外植體進(jìn)行感染,共獲得抗性芽204棵,經(jīng)過(guò)生根培養(yǎng)、煉苗、移栽后,共獲得11株轉(zhuǎn)基因陽(yáng)性黃芪植株。3.經(jīng)DNA、Southern blot雜交以及RT-PCR檢測(cè),獲得3株轉(zhuǎn)基因黃芪陽(yáng)性植株,被證明RS基因完全整合到黃芪基因組中并表達(dá)產(chǎn)生白藜蘆醇。4.經(jīng)過(guò)比較轉(zhuǎn)基因陽(yáng)性植株和非轉(zhuǎn)基因植株中的白藜蘆醇含量,發(fā)現(xiàn)3株具有Southern blot雜交信號(hào)的陽(yáng)性植株比非轉(zhuǎn)基因植株的白藜蘆醇含量高,其中黃芪非轉(zhuǎn)基因植株的白藜蘆醇含量為2029.46μg/g,而三顆黃芪轉(zhuǎn)基因植株的白藜蘆醇含量分別為2998.32μg/g、3854.66μg/g、3989.77μg/g,分別提高了48.23%、91.27%和96.59%。本研究建立了黃芪再生及轉(zhuǎn)化體系,并將來(lái)源于巨峰葡萄的RS基因成功的轉(zhuǎn)入黃芪中進(jìn)行表達(dá),獲得黃芪轉(zhuǎn)基因陽(yáng)性植株,這不僅可以探索RS基因轉(zhuǎn)化異源植物黃芪,達(dá)到異源植物生產(chǎn)白藜蘆醇的可能,并為黃芪轉(zhuǎn)基因育種提供技術(shù)支持;更可以提高黃芪抗性,進(jìn)一步佐證白藜蘆醇合成酶的功能,為中藥現(xiàn)代化提供了有價(jià)值的理論參考。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2

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