嗜盐灰绿曲霉耐盐基因的克隆及抗逆功能验证
发布时间：2018-08-24 11:02
【摘要】：嗜盐微生物在适应不同浓度的盐环境的进化过程中形成其特殊的耐盐机制,对其耐盐基因进行功能研究,对有效开发利用嗜盐微生物的基因资源具有重要意义。26S蛋白酶调节亚基7(S7)在26S蛋白酶体中19S调节亚基复合体的一个亚基,具有保守功能域,参与形成ATPase活性中心,为泛素-26S蛋白酶体代谢途径中底物蛋白的去泛素化、蛋白的转运提供能量。26S蛋白酶体在生物体内参与多种生命活动的过程,如细胞分裂、细胞程序性死亡、DNA损伤的修复等,在细胞中异常蛋白的降解过程发挥重要作用。另外,溶血素蛋白具有良好的理化性质,能够在不同p H范围内保持稳定性,并且在医药方面具有应用价值。但这两个基因在抗逆功能方面尚未有报道。本研究中从一株嗜盐曲霉Aspergillus glaucus CCHA的酵母表达文库中筛选出耐盐性较强的两个基因,通过遗传转化,对其基因功能进行了探究。主要研究结果如下:1.成功克隆出嗜盐灰绿曲霉26S蛋白酶调节亚基7的全长序列,并成功获得酵母转化子p YES2-Ag S7和嗜热菌转化子p GFPGUSPlus-Ag S7,对其进行Na Cl、山梨醇胁迫处理,转化子均未表现出抗逆性,说明Ag S7基因无抗逆性功能。2.分别以曲霉c DNA文库和酵母文库菌株质粒为模板成功扩增出的非全长序列n Ag S7、NAg S7,重新转入酵母获得转化子p YES2-n Ag S7、p YES2-NAg S7,其逆境胁迫验证均未表现为胁迫敏感性,无抗逆性功能,因此推测酵母文库中该基因的酵母菌株发生突变。3.基因Ag Aeg S具有溶血蛋白家族保守功能域,获得的酵母表达菌株p YES2-Ag Aeg S及嗜热菌转化子菌株p GFPGUSPlus-Ag Aeg S在高盐渗透胁迫下表现出抗盐性,初步推断Ag Aeg S基因可能参与嗜盐灰绿曲霉对逆境胁迫的应答。以上结果表明26S蛋白酶调节亚基7基因对逆境胁迫表现敏感,未表现出耐盐特性,可能不参与生物体的抗盐机制;通过酵母表达文库筛选转化子可能存在菌株突变的因素。初步推断溶血素蛋白基因可能参与嗜盐灰绿曲霉的高盐、干旱等高渗透环境胁迫的应答。
[Abstract]:During the evolution of halophilic microorganisms to adapt to different concentrations of salt environment, their special salt tolerance mechanisms were formed, and the function of salt tolerance genes was studied. It is of great significance to effectively exploit the genetic resources of halophilic microbes. A subunit of the 19s regulatory subunit complex of the 26s proteasome, which has a conserved functional domain, is involved in the formation of the active center of ATPase in the 26s proteasome, which is a subunit of the 19s regulatory subunit complex in the 26s proteasome. It provides energy for the desuginization of substrate protein in the metabolic pathway of ubiquitin-26S proteasome and the transport of protein. 26s proteasome participates in many life processes in vivo, such as cell division, repair of DNA damage caused by programmed cell death, and so on. It plays an important role in the degradation of abnormal proteins in cells. In addition, hemolysin protein has good physical and chemical properties, can maintain stability in different pH range, and has application value in medicine. However, these two genes have not been reported in terms of stress resistance. In this study, two genes with strong salt tolerance were screened from a yeast expression library of Aspergillus halophilus Aspergillus glaucus CCHA, and their gene functions were investigated by genetic transformation. The main results are as follows: 1. The full-length sequence of 26S protease regulatory subunit 7 of Aspergillus halophilus was cloned successfully, and the yeast transformant p YES2-Ag S7 and the thermophilic transformant p GFPGUSPlus-Ag S7 were successfully obtained. Under the stress of Na Cl, sorbitol, the transformants showed no stress resistance. The results showed that Ag S7 gene had no resistance to stress. Using the plasmid of Aspergillus c DNA library and yeast library as template, the non-full-length sequence of n Ag S7 nagS7 was successfully amplified and transferred into yeast to obtain the transformant of p YES2-n Ag S7, p YES2-NAg S7. The results of stress test showed that the sequence was not sensitive to stress. No resistance to stress, so we speculated that the yeast strains of the gene were mutated. 3. The gene Ag Aeg S has the conserved functional domain of hemolytic protein family. The yeast expression strain p YES2-Ag Aeg S and the thermogenicity transformant strain p GFPGUSPlus-Ag Aeg S showed salt resistance under high salt osmotic stress. It is inferred that Ag Aeg S gene may be involved in the response of Aspergillus halophilus to stress. These results indicated that 26s protease regulatory subunit 7 gene was sensitive to stress and did not exhibit salt tolerance and might not participate in the salt tolerance mechanism of organism. It was preliminarily inferred that hemolysin protein gene might be involved in the response to high salt and drought stress of Aspergillus halophilus.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q93
本文编号：2200614
[Abstract]:During the evolution of halophilic microorganisms to adapt to different concentrations of salt environment, their special salt tolerance mechanisms were formed, and the function of salt tolerance genes was studied. It is of great significance to effectively exploit the genetic resources of halophilic microbes. A subunit of the 19s regulatory subunit complex of the 26s proteasome, which has a conserved functional domain, is involved in the formation of the active center of ATPase in the 26s proteasome, which is a subunit of the 19s regulatory subunit complex in the 26s proteasome. It provides energy for the desuginization of substrate protein in the metabolic pathway of ubiquitin-26S proteasome and the transport of protein. 26s proteasome participates in many life processes in vivo, such as cell division, repair of DNA damage caused by programmed cell death, and so on. It plays an important role in the degradation of abnormal proteins in cells. In addition, hemolysin protein has good physical and chemical properties, can maintain stability in different pH range, and has application value in medicine. However, these two genes have not been reported in terms of stress resistance. In this study, two genes with strong salt tolerance were screened from a yeast expression library of Aspergillus halophilus Aspergillus glaucus CCHA, and their gene functions were investigated by genetic transformation. The main results are as follows: 1. The full-length sequence of 26S protease regulatory subunit 7 of Aspergillus halophilus was cloned successfully, and the yeast transformant p YES2-Ag S7 and the thermophilic transformant p GFPGUSPlus-Ag S7 were successfully obtained. Under the stress of Na Cl, sorbitol, the transformants showed no stress resistance. The results showed that Ag S7 gene had no resistance to stress. Using the plasmid of Aspergillus c DNA library and yeast library as template, the non-full-length sequence of n Ag S7 nagS7 was successfully amplified and transferred into yeast to obtain the transformant of p YES2-n Ag S7, p YES2-NAg S7. The results of stress test showed that the sequence was not sensitive to stress. No resistance to stress, so we speculated that the yeast strains of the gene were mutated. 3. The gene Ag Aeg S has the conserved functional domain of hemolytic protein family. The yeast expression strain p YES2-Ag Aeg S and the thermogenicity transformant strain p GFPGUSPlus-Ag Aeg S showed salt resistance under high salt osmotic stress. It is inferred that Ag Aeg S gene may be involved in the response of Aspergillus halophilus to stress. These results indicated that 26s protease regulatory subunit 7 gene was sensitive to stress and did not exhibit salt tolerance and might not participate in the salt tolerance mechanism of organism. It was preliminarily inferred that hemolysin protein gene might be involved in the response to high salt and drought stress of Aspergillus halophilus.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q93
【参考文献】
相关期刊论文 前5条
1 王丰;施一公;;26S蛋白酶体的结构生物学研究进展[J];中国科学:生命科学;2014年10期
2 刘晓丹;刘金亮;魏毅;田玉平;范飞飞;潘洪玉;张世宏;;一株极端耐盐曲霉的分离、鉴定及生物学特性分析[J];吉林大学学报(理学版);2011年03期
3 田新朋;张玉琴;唐蜀昆;李文均;徐丽华;姜成林;;云南黑井古盐矿可培养极端嗜盐古菌初步研究[J];微生物学通报;2006年06期
4 米琴,张玉琴,陈义光,鲍敏,陈志;青海盐环境下革兰氏阳性中度嗜盐细菌的分离及其生物学特性[J];陕西师范大学学报(自然科学版);2005年03期
5 刘坚,朱悦,李庆国,丁建东;紫膜脂质体的制备及其质子泵功能的检测[J];复旦学报(自然科学版);1999年05期
相关博士学位论文 前1条
1 曹轶;深海微生物多样性和嗜盐古菌乙醇代谢相关酶基因研究[D];浙江大学;2010年
相关硕士学位论文 前3条
1 谢丽霞;极端嗜盐曲霉核糖体蛋白基因SpRPS3ae和SpRPL44的克隆及其抗逆功能研究[D];吉林大学;2013年
2 黄菁菁;海洋耐（嗜）盐真菌及其次级代谢产物的初步研究[D];厦门大学;2008年
3 沈宇强;嗜盐（耐盐）微生物的分离及其在榨菜废水处理中的应用[D];浙江大学;2008年
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