水稻基因编辑技术体系建立及深海细菌醛脱氢酶的水稻转化研究

发布时间：2018-04-22 00:08

  本文选题：基因编辑 + 水稻　； 参考：《国家海洋局第三海洋研究所》2017年硕士论文

【摘要】：第一部分水稻基因编辑技术体系建立及深海细菌醛脱氢酶的水稻转化研究水稻是一种重要的粮食作物,获得优良的水稻品种对农业发展具有重要意义。新兴的CRISPR/Cas9基因编辑技术在近几年内飞速发展,其构建方法简单、打靶效率高以及基因编辑的靶向性使得它在育种工作中具有极大的应用潜力。在植物育种寻求新突破的探索中,深海微生物中具有特殊功能的极端酶基因可能成为功能基因的巨大宝库,将这些酶基因导入植物内,可能可以对植物的生长以及抗逆性等方面产生积极的影响或可将植物变为生产极端酶的“工厂”。本研究利用水稻建立基因编辑体系,首先使用CRISPR/Cas9技术对控制水稻分蘖性状中的关键调控基因D27进行定点敲除,成功获得了突变体植株,在T0代植株基因的靶位点附近检测出了碱基的插入、替换和缺失,并使得突变体植株叶部D27的表达量较野生型明显下降,同时T1代突变体植株出现了明显的矮化、多分蘖以及叶片变窄变短的表型变化。接着,选取对植物抗逆性紧密相关且具有醛类物质解毒害作用的醛脱氢酶作为突破口,将深海来源的盐单胞菌Halomonas axialensis中的醛脱氢酶基因通过农杆菌介导的转化技术导入水稻基因组,在获得的转基因愈伤组织中检出转入的外源醛脱氢酶基因已成功表达。本实验对深海微生物资源在植物中的开发利用做出了初步的探索,并利用CRISPR/Cas9技术初步建立了后期转基因功能验证的评价体系。第二部分深海海水中间苯二甲酸二甲酯降解菌的多样性分析由于塑料制品的广泛使用和大量生产,邻苯二甲酸酯类(PAEs)化合物的污染已遍布全球。研究表明,PAEs对生物体具有致癌致畸、造成组织损伤等危害,同时也是一类环境激素,对动物的生殖及胎儿发育具有诸多危害。因此PAEs的污染状况亟待治理。目前PAEs类化合物降解方式主要分为非生物降解的水解和光解以及生物降解两种,由于自然条件下的水解和光解方式的降解速率极慢,因此生物降解是PAEs类降解的主要方式。本研究选取PAEs类化合物中的间苯二甲酸二甲酯(DMI)做为降解底物,在我国南海2000 m深海处分别采集原位富集和实验室富集两种样品,通过未培养和实验室培养两种方式对深海海水中DMI降解菌的多样性进行分析。聚类分析结果显示,原物富集与实验室富集样品中的优势菌群有较大不同,推测这种现象是由于两种富集方式中海水含氧量的不同造成的。同时由于培养方式和培养条件的局限性,未培养和实验室培养方式获得的优势菌群也存在较大差异。本实验中通过不同的富集方式和培养方式共分析得到优势菌株Sulfurovum、Corynebacterium、Thalassospira等8个属,其中大部分菌株均有报道称可降解多环芳烃或石油等物质。上述菌株的获得对DMI污染的治理提供了一定的参考价值。
[Abstract]:The first part is the establishment of rice gene editing technique system and the transformation of deep-sea bacterial aldehyde dehydrogenase. Rice is an important food crop. It is of great significance to obtain good rice varieties for agricultural development. With the rapid development of CRISPR/Cas9 gene editing technology in recent years, its simple construction method, high efficiency of targeting and targeting of gene editing make it have great application potential in breeding work. In the search for new breakthroughs in plant breeding, extreme enzyme genes with special functions in deep-sea microbes may become a huge repository of functional genes, which can be introduced into plants. It may have a positive effect on plant growth and resistance, or turn plants into "factories" that produce extreme enzymes. In this study, rice gene editing system was established. Firstly, CRISPR/Cas9 technique was used to knockout the key regulatory gene D27 in rice tillering traits, and the mutant plant was successfully obtained. The insertion, substitution and deletion of the bases were detected near the target site of the gene of generation T0. The expression of D27 in the leaves of the mutant was significantly lower than that in the wild type, and the plant of the T1 generation was obviously dwarfed. Phenotypic changes of multiple tillers and narrow and shorter leaves. Then, aldehydes dehydrogenase, which is closely related to plant stress resistance and has the ability to detoxify aldehydes, is chosen as the breakthrough point. Aldehyde dehydrogenase gene from deep-sea Halomonas axialensis was introduced into rice genome by Agrobacterium tumefaciens mediated transformation, and the transferred aldehydes dehydrogenase gene was successfully expressed in transgenic callus. This experiment made a preliminary exploration on the exploitation and utilization of deep-sea microbial resources in plants, and established a preliminary evaluation system for the validation of transgenic functions in the later stage by using CRISPR/Cas9 technology. The second part the diversity analysis of dimethyl phthalate degradation bacteria in deep-sea seawater. Due to the widespread use and mass production of plastic products the pollution of phthalate ester PAEs-based compounds has spread all over the world. Studies have shown that PAEs have carcinogenic teratogenicity, tissue damage and other hazards, but also a class of environmental hormones, which has a lot of harm to the reproduction and fetal development of animals. Therefore, the pollution of PAEs is in urgent need of treatment. At present, the degradation methods of PAEs compounds are mainly divided into non-biodegradable hydrolysis, photolysis and biodegradation. Because the degradation rate of natural hydrolysis and photolysis is very slow, biodegradation is the main way of PAEs degradation. In this study, dimethyl isophthalate (Dimethyl isophthalate) from PAEs compounds was selected as the degradation substrate, and two samples were collected in situ enrichment and laboratory enrichment at the depth of 2000 m in the South China Sea. The diversity of DMI degrading bacteria in deep sea water was analyzed by uncultured and laboratory culture. The results of cluster analysis show that the enrichment of raw material is different from the dominant flora in the sample enriched in laboratory. It is speculated that this phenomenon is caused by the difference of oxygen content in seawater between the two enrichment modes. At the same time, because of the limitation of culture methods and culture conditions, the dominant flora obtained by uncultured and laboratory culture was also different. In this experiment, 8 genera of dominant strain Sulfurovum Corynebacterium Thalassospira were obtained by different enrichment and culture methods. Most of the strains were reported to be degradable polycyclic aromatic hydrocarbons (PAHs) or petroleum and other substances. The above-mentioned strains provide some reference value for the control of DMI pollution.
【学位授予单位】：国家海洋局第三海洋研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511;Q943.2
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本文编号：1784708