本文關(guān)鍵詞：甘薯IbP5CS基因的克隆、擬南芥轉(zhuǎn)化及功能分析　出處：《四川師范大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 甘薯 P5CS 非生物脅迫 生物信息學 擬南芥

【摘要】：甘薯是我國最主要的糧食作物之一,其產(chǎn)量大、價格低,是重要的食品及工業(yè)原料。由于甘薯多種植于貧瘠土地中,產(chǎn)量容易受到非生物脅迫影響,因此對甘薯相關(guān)抗逆基因功能的研究具有重要的意義。Δ′-吡咯琳-5-羧酸合成酶(Δ1-pyrroline-5-carboxylate synthetase,P5CS)是植物進行脯氨酸合成的關(guān)鍵酶,許多研究都表明植物P5CS基因與植物抗非生物脅迫能力有著重要聯(lián)系。研究發(fā)現(xiàn)過表達P5CS基因的植株相比對照其非生物脅迫能力有明顯的提升,轉(zhuǎn)基因植株中的脯氨酸含量也明顯增加。脯氨酸作為一種兼容性小分子在植物體內(nèi)具有滲透調(diào)節(jié)、抗氧化、降低毒性及信號傳導等功能,當植物受到干旱、高鹽和其它非生物脅迫時脯氨酸能夠快速大量積累,增強植物細胞抵御逆境脅迫的能力。本研究利用甘薯轉(zhuǎn)錄組和NCBI數(shù)據(jù)庫中相關(guān)P5CS基因序列,設(shè)計特異引物,成功克隆得到甘薯的Ib P5CS基因,利用相關(guān)生物信息學軟件對其進行了初步的分析。構(gòu)建Ib P5CS基因原核(p ET32-P5CS)及真核表達載體(p YES2-P5CS),分別轉(zhuǎn)化大腸桿菌BL21和釀酒酵母,在大腸桿菌BL21中經(jīng)IPTG誘導表達Ib P5CS蛋白,在非生物脅迫(干旱、高鹽和高溫等)下驗證Ib P5CS基因在大腸桿菌和釀酒酵母中的相關(guān)功能。結(jié)果表明過表達Ib P5CS基因能夠提升大腸桿菌和釀酒酵母的抗鹽及抗高溫脅迫能力,但是在干旱和堿性脅迫下沒有明顯的增加。為了研究Ib P5CS基因在植物中的功能,本研究將p CAMBIA1302-P5CS質(zhì)粒轉(zhuǎn)化農(nóng)桿菌,利用農(nóng)桿菌侵染擬南芥,研究Ib P5CS基因過表達對擬南芥生長發(fā)育的影響,結(jié)果發(fā)現(xiàn)轉(zhuǎn)基因植株的光合作用和蒸騰作用相比野生型有一定的提升,未來本研究將繼續(xù)完成后續(xù)實驗,為深入研究甘薯Ib P5CS基因的功能提供實驗依據(jù),為提升農(nóng)作物的產(chǎn)量打下堅實的基礎(chǔ)。
[Abstract]:Sweet potato is one of the most important food crops in China, its yield is large, the price is low, it is an important food and industrial raw material. Because sweet potato is planted in barren soil, the yield is easy to be affected by abiotic stress. Therefore, it is of great significance to study the function of related stress resistance genes in sweet potato. 螖 -pyrrolidin-5-carboxylic acid synthase (螖 -pyrroliden-5-carboxylic acid synthase). 螖 1-pyrroline-5-carboxylate synthetase. P5CS is the key enzyme for proline synthesis in plants. Many studies have shown that plant P5CS gene has an important relationship with plant resistance to abiotic stress. It has been found that plants that have expressed P5CS gene have significantly improved their abiotic stress ability compared with the control. Proline content in transgenic plants also increased significantly. Proline as a compatibility of small molecules in the plant has osmotic regulation, antioxidant, reduce toxicity and signal transduction functions, when the plant is subjected to drought. Proline can accumulate rapidly under high salt and other abiotic stress, and enhance plant cells' ability to resist stress. In this study, we used the p5CS gene sequence in sweet potato transcriptome and NCBI database. Ib P5CS gene of sweet potato was cloned successfully by designing specific primers. The prokaryotic expression vector of IbP5CS and eukaryotic expression vector pYES2-P5CSwere constructed by using bioinformatics software. E. coli BL21 and Saccharomyces cerevisiae were transformed to express Ib P5CS protein induced by IPTG in Escherichia coli BL21, and the Ib P5CS protein was expressed under abiotic stress (drought). High salt, high temperature, etc.). The function of Ib P5CS gene in Escherichia coli and Saccharomyces cerevisiae was verified. The results showed that overexpression of Ib P5CS gene could enhance the salt resistance and high temperature stress resistance of Escherichia coli and Saccharomyces cerevisiae. In order to study the function of Ib P5CS gene in plants, the p CAMBIA1302-P5CS plasmid was transformed into Agrobacterium tumefaciens. Effects of IbP5CS overexpression on growth and development of Arabidopsis thaliana were studied by Agrobacterium tumefaciens. The results showed that the photosynthesis and transpiration of transgenic plants were higher than those of wild type. In the future, this study will continue to complete follow-up experiments, which will provide experimental basis for further study on the function of Ib P5CS gene in sweet potato, and lay a solid foundation for increasing crop yield.
【學位授予單位】：四川師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S531;Q943.2

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本文編號：1411682