茯苓转化条件的优化和Smk基因的研究
发布时间：2018-11-01 11:30
【摘要】：茯苓Wolfiporia cocos是我国传统中药材之一,具有极高的药用价值和膳食营养价值。本课题选取茯苓菌种GIM5.219,对其原生质体的制备与再生条件开展了系统的研究;对PEG介导的原生质体茯苓转化体系进一步优化;运用现代分子生物学技术及方法研究参与调控菌核形成的相关基因Smk,以达到在茯苓种植过程中减少对松木资源的依赖,精准调控茯苓菌核发育形成条件并提高产量,促进茯苓种植产业化发展的最终目的。实验结果如下:1、结果表明:GIM5.219菌株在固体和液体培养基上0-2d之间生长缓慢,固体上2-6d内菌丝生长增快,从第7d开始生长趋于稳定;液体中2-7d菌丝球生长较快,8d后菌丝球生长趋于稳定。2、从8个方面对茯苓原生质体制备与再生条件进行筛选研究,得出了最优条件:采取培养6d的茯苓菌丝,在p H为5.6-6.2的3.5%溶壁酶+1%崩溃酶的混合酶液中,34-36℃静置酶解2.5-3h制备原生质体,在马铃薯再生培养基上采用双层涂布法进行再生,原生质体数最高能达到39.20×107/g,再生率最高为32.7%。3、从6个方面对PEG介导的原生质体茯苓转化体系进行筛选实验,得到了最优条件:采用液体培养4d的菌丝球所制备的原生质体,用MTC溶液(0.6mol/L甘露醇,50mmol/L Ca Cl2,10mmol/L Tris-HCl、p H7.5)将其浓度调节为1×108个/ml,向原生质体中加入2ug DNA/(100μl原生质体),用45%PEG4000进行转化,所得的抗性转化子数最多,为27个。将其进行PCR验证,所有的转化子均能扩增出hph特异片段,表明外源DNA已经插入到茯苓基因组中,且能够随着茯苓传代而稳定的遗传。4、构建了Smk基因沉默重组载体p CIT-Smk,经PEG介导转入到茯苓原生质体中,成功验证了进一步优化的PEG介导的茯苓原生质体转化体系的可行性。5、筛选出了继代培养5代后仍能稳定沉默Smk基因的转化菌株,表明转化子具有遗传稳定性。相对荧光定量实验结果表明:未进行转化的茯苓菌株GIM5.219中Smk基因的表达量是转化子的28133.5倍,说明转化子中的Smk基因已被沉默,随着茯苓传代而稳定地遗传。6、沉默Smk基因的转化子菌株在培养过程中,菌丝的生长速度以及菌落的形态特征都发生了明显变化,表明Smk基因可能是调控菌丝生长发育、菌核形成的重要基因。本研究进一步完善了目前茯苓原生质体制备与再生的研究工作,优化了茯苓原生质体转化体系。研究了茯苓菌丝生长发育、菌核形成的相关基因Smk,为茯苓栽培、品种选育工作提供了理论基础,为指导茯苓遗传种植工作具有重要意义。
[Abstract]:Poria cocos Wolfiporia cocos is one of the traditional Chinese medicinal materials, which has high medicinal value and nutritional value. The preparation and regeneration conditions of protoplasts of Poria cocos strain GIM5.219, were studied systematically, and the transformation system of protoplast Poria cocos mediated by PEG was further optimized. Modern molecular biological techniques and methods were used to study the related gene Smk, involved in the regulation of sclerotia formation in order to reduce the dependence on pine resources during the cultivation of Poria cocos, to accurately regulate the conditions for the development and formation of the sclerotia of Poria cocos and to increase the yield. The ultimate aim of promoting the industrialization of Poria cocos planting. The results were as follows: 1. The results showed that the growth of GIM5.219 strain was slow between 0 and 2 days on solid and liquid medium, and the mycelium growth increased within 2 to 6 days on solid, and the growth tended to be stable from the 7th day. After 8 days, the mycelium growth tended to be stable. 2. The preparation and regeneration conditions of Poria cocos protoplast were studied from 8 aspects, and the optimum conditions were obtained as follows: culture for 6 days of Poria cocos mycelia. The protoplasts were prepared in the mixture of 3.5% wall-lysing enzymes and 1% crumbling enzymes (pH = 5.6-6.2). The protoplasts were hydrolyzed at 34-36 鈩，
本文编号：2303789
[Abstract]:Poria cocos Wolfiporia cocos is one of the traditional Chinese medicinal materials, which has high medicinal value and nutritional value. The preparation and regeneration conditions of protoplasts of Poria cocos strain GIM5.219, were studied systematically, and the transformation system of protoplast Poria cocos mediated by PEG was further optimized. Modern molecular biological techniques and methods were used to study the related gene Smk, involved in the regulation of sclerotia formation in order to reduce the dependence on pine resources during the cultivation of Poria cocos, to accurately regulate the conditions for the development and formation of the sclerotia of Poria cocos and to increase the yield. The ultimate aim of promoting the industrialization of Poria cocos planting. The results were as follows: 1. The results showed that the growth of GIM5.219 strain was slow between 0 and 2 days on solid and liquid medium, and the mycelium growth increased within 2 to 6 days on solid, and the growth tended to be stable from the 7th day. After 8 days, the mycelium growth tended to be stable. 2. The preparation and regeneration conditions of Poria cocos protoplast were studied from 8 aspects, and the optimum conditions were obtained as follows: culture for 6 days of Poria cocos mycelia. The protoplasts were prepared in the mixture of 3.5% wall-lysing enzymes and 1% crumbling enzymes (pH = 5.6-6.2). The protoplasts were hydrolyzed at 34-36 鈩，
本文编号：2303789
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