甘薯IbP5CS基因的克

发布时间：2018-01-11 23:19

本文关键词：甘薯IbP5CS基因的克隆、拟南芥转化及功能分析　出处：《四川师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：甘薯是我国最主要的粮食作物之一,其产量大、价格低,是重要的食品及工业原料。由于甘薯多种植于贫瘠土地中,产量容易受到非生物胁迫影响,因此对甘薯相关抗逆基因功能的研究具有重要的意义。Δ′-吡咯琳-5-羧酸合成酶(Δ1-pyrroline-5-carboxylate synthetase,P5CS)是植物进行脯氨酸合成的关键酶,许多研究都表明植物P5CS基因与植物抗非生物胁迫能力有着重要联系。研究发现过表达P5CS基因的植株相比对照其非生物胁迫能力有明显的提升,转基因植株中的脯氨酸含量也明显增加。脯氨酸作为一种兼容性小分子在植物体内具有渗透调节、抗氧化、降低毒性及信号传导等功能,当植物受到干旱、高盐和其它非生物胁迫时脯氨酸能够快速大量积累,增强植物细胞抵御逆境胁迫的能力。本研究利用甘薯转录组和NCBI数据库中相关P5CS基因序列,设计特异引物,成功克隆得到甘薯的Ib P5CS基因,利用相关生物信息学软件对其进行了初步的分析。构建Ib P5CS基因原核(p ET32-P5CS)及真核表达载体(p YES2-P5CS),分别转化大肠杆菌BL21和酿酒酵母,在大肠杆菌BL21中经IPTG诱导表达Ib P5CS蛋白,在非生物胁迫(干旱、高盐和高温等)下验证Ib P5CS基因在大肠杆菌和酿酒酵母中的相关功能。结果表明过表达Ib P5CS基因能够提升大肠杆菌和酿酒酵母的抗盐及抗高温胁迫能力,但是在干旱和碱性胁迫下没有明显的增加。为了研究Ib P5CS基因在植物中的功能,本研究将p CAMBIA1302-P5CS质粒转化农杆菌,利用农杆菌侵染拟南芥,研究Ib P5CS基因过表达对拟南芥生长发育的影响,结果发现转基因植株的光合作用和蒸腾作用相比野生型有一定的提升,未来本研究将继续完成后续实验,为深入研究甘薯Ib P5CS基因的功能提供实验依据,为提升农作物的产量打下坚实的基础。
[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.
【学位授予单位】：四川师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S531;Q943.2

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