采用小米粒培养基构建香菇遗传转化体系的研究

发布时间：2018-05-11 00:00

本文选题：单核体菌丝 + 农杆菌　；参考：《上海海洋大学》2017年硕士论文

【摘要】：香菇(Lentinula edodes)是世界上最著名的食用菌之一,是世界第二大食药用真菌,产量仅次于双孢蘑菇(Agaricus bisporus),在中国已有近千年的栽培历史[1]。由于其突出的食药作用和价值,人们对香菇的需求量明显上升,从而促进了香菇产业迅猛发展,自1987年香菇总产量第一次超过日本以来,中国一直保持着世界第一香菇大国的地位[2]。不过与发展迅猛的香菇产业相比,香菇的遗传转化研究较为落后。随着两个香菇全基因组测序的完成和公布[3-4],香菇的遗传及功能基因研究将成为热点。遗传转化技术是运用分子生物学相关方法和基因工程相关方法将本来不属于受体菌株基因组的基因片段插入到受体基因组并整合,再通过复制、转录、翻译等一系列过程,最终可以达到定向的改变受体菌株或者细胞生理生化特性的目的。因此,稳定、高效、便捷的遗传转化体系的构建将具有重要意义和价值。本文将探索以农杆菌为介导的遗传转化体系在香菇原生质体单核体菌株中进行随机插入突变的研究。通过香菇菌丝对潮霉素的抗性试验,确定了农杆菌转化初筛浓度为10mg/L,复筛浓度为12mg/L。通过对比在不同头孢噻肟钠Cef浓度下香菇菌丝的生长情况,确定了Cef的最佳抑制农杆菌的浓度为600mg/L。构建的质粒pYN6982和GPiE以潮霉素抗性基因hyg作为筛选标记基因,以增强型荧光蛋白基因egfp作为报告基因,以农杆菌EHA105和LBA4404为介导,同时转化了3个香菇单核体菌株。香菇受体菌丝创新性地采用小米粒培养基进行培养和转化,采用小米粒培养基培养菌丝不需要用打孔器选取直径相同的菌块,每粒小米粒都是大小均匀的个体,可以直接挑取使用,也无需再接种于铺有无菌玻璃纸的MYG固定培养基中,不易污染杂菌。农杆菌侵染菌丝后,小米粒方便挑取,相较于常用的PDA菌块或菌丝球的方法,这种方法可更快捷得到更多的易于分散的受体材料,并可更加方便、快捷的统计转化效率。受体菌丝经过农杆菌侵染,共培养阶段培养,通过潮霉素抗性筛选,经PCR以及测序验证,转化子中含有潮霉素抗性基因的片段。经过荧光显微观察,经转化的香菇菌丝体内可以观测到绿色荧光,而未经转化的对照菌株则没有荧光信号的产生,表明增强型绿色荧光蛋白在转化香菇菌丝中得到了表达,再经过转化子的有丝分裂稳定性试验得到稳定遗传的转化子。对部分香菇转化子进行了在PDA培养基上菌落形态观察试验,与未转化的对照香菇菌丝相比,发现小部分转化子菌落的形态和生长速度有所差异,可能是T-DNA的插入破坏了转化子基因组中与菌落形态相关的功能基因,从而导致了转化子和对照菌丝的差异。本研究探索出一种采用小米粒培养基培养菌丝并进行转化的新方法,并且从菌丝在小米粒培养基上生长时间、农杆菌类型、质粒上启动子类型、农杆菌侵染过程中超声时间、共培养时间和温度等方面进行探索和优化。同时比较了含有不同启动子的质粒在相同农杆菌中转化效率以及同一质粒在不同农杆菌中的转化效率。结果表明菌丝在小米粒培养基上培养15~20天为最佳的农杆菌侵染转化时间;不同的农杆菌EHA105和LBA4404侵染菌丝后的转化效率没有太大的差异;含有双孢蘑菇Agpd的转化效率要高于含有灵芝Gl-gpd启动子的转化效率;农杆菌侵染过程中超声震荡2分钟为最佳时间;共培养阶段加入乙酰丁香酮能够提高转化效率;共培养时间5天为最佳培养时间,共培养最佳温度为25℃。建立了较稳定的农杆菌介导的香菇遗传转化的体系,并获得可稳定遗传和表达的香菇转化子菌株。
[Abstract]:Letinous edodes (Lentinula edodes) is one of the most famous edible fungi in the world. It is the world's second largest edible medicinal fungus. The yield is second only to Agaricus bisporus (Agaricus bisporus). In China, there has been nearly a thousand years of cultivation history in China. Because of its prominent food and drug effect and value, the demand for letinous edodes has risen obviously, thus promoting the rapid development of letinous edodes industry. Rapid development, since the first letinous edodes output in 1987 has exceeded Japan for the first time, China has kept the position of the world's largest letinous edodes country [2]., but compared with the rapid development of letinous edodes industry, the genetic transformation of letinous edodes is relatively backward. With the completion of the complete genome sequencing of two letinous edodes and the publication of [3-4], the genetic and functional gene research of letinous edodes. It will be a hot spot. Genetic transformation technology is the use of molecular biology related methods and genetic engineering related methods to insert the gene fragments originally not belong to the receptor strains into the receptor genome and integrate, and then through a series of processes such as replicating, transcription, translation and so on, in order to achieve directional change of the receptor strain or cell physiology. Therefore, the construction of a stable, efficient and convenient genetic transformation system will be of great significance and value. This paper will explore the random insertion mutation of a genetic transformation system based on Agrobacterium tumefaciens in the single nucleosome of letinous edodes protoplast. The resistance test of hypha to hygromycin by letinous edodes mycelium has determined agriculture. The initial screening concentration of bacilli was 10mg/L, and the concentration of rescreening was 12mg/L. by comparing the growth of letinous edodes mycelium under the concentration of different cefotaxime sodium (Cef) in different cefotaxime sodium. The best inhibition of the concentration of Agrobacterium tumefaciens was determined by 600mg/L. constructed plasmid pYN6982 and GPiE with the hygromycin resistance gene hyg as a screening marker gene, and the enhanced fluorescent protein gene was enhanced. As a reporter gene, EGFP is mediated by Agrobacterium tumefaciens EHA105 and LBA4404, and 3 single nucleosome strains of letinous edodes are transformed. The mycelium of letinous edodes receptor is innovatively used for culture and transformation of small rice medium. The individual, which can be used directly, and does not need to be inoculated in the MYG fixed medium with aseptic glass paper, is not easy to pollute the heterozygous bacteria. After Agrobacterium tumefaciens infect the mycelium, the small rice grains are convenient to pick up. Compared to the commonly used PDA bacteria block or the method of bacterial silk ball, this method can get more easily dispersed receptor materials and can be more convenient. The receptor mycelium was infected by Agrobacterium tumefaciens, co culture stage and screened by hygromycin resistance. The hygromycin resistance gene fragment contained in the transformant was verified by PCR and sequencing. After fluorescence microscopy, the transformed letinous edodes mycelium could observe green fluorescence and unconverted control bacteria. No fluorescence signal was produced, indicating that the enhanced green fluorescent protein was expressed in the transformed letinous edodes mycelium, and then a stable hereditary transformant was obtained through the mitotic stability test of the transformant. The colony morphology of some letinous edodes transformants was observed on the PDA medium and the unconverted control of the control of the mycelium of letinous edodes. It was found that the morphology and growth speed of the small portion of the colonies were different. It was possible that the insertion of T-DNA destroyed the functional genes related to the colony morphology in the transformant genome, which led to the difference between the transformants and the control mycelium. The growth time of mycelia on the medium of rice grain, Agrobacterium type, promoter type, ultrasonic time, co culture time and temperature during Agrobacterium infection process were explored and optimized. Meanwhile, the conversion efficiency of plasmids containing different promoters in the same Agrobacterium and the same plasmid in different Agrobacterium tumefaciens were compared. The results showed that mycelia was the best Agrobacterium infection transformation time in the culture of 15~20 days on the medium of small rice grain, and the conversion efficiency of Agrobacterium tumefaciens EHA105 and LBA4404 was not very different, and the conversion efficiency of Agpd containing Agaricus bisporus was higher than that of the Gl-gpd promoter with Ganoderma lucidum. In the process of infection, the ultrasonic vibration of 2 minutes is the best time, and the conversion efficiency can be improved by adding acetyl syringone in the co culture stage. The optimum incubation time is 5 days, and the optimum temperature is 25. A stable Agrobacterium mediated transformation system of letinous edodes is established, and the transformation of letinous edodes to stable inheritance and expression is obtained. Substrains.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S646.12

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