嗜鹽灰綠曲霉耐鹽基因的克隆及抗逆功能驗證
發(fā)布時間：2018-08-24 11:02
【摘要】：嗜鹽微生物在適應不同濃度的鹽環(huán)境的進化過程中形成其特殊的耐鹽機制,對其耐鹽基因進行功能研究,對有效開發(fā)利用嗜鹽微生物的基因資源具有重要意義。26S蛋白酶調(diào)節(jié)亞基7(S7)在26S蛋白酶體中19S調(diào)節(jié)亞基復合體的一個亞基,具有保守功能域,參與形成ATPase活性中心,為泛素-26S蛋白酶體代謝途徑中底物蛋白的去泛素化、蛋白的轉運提供能量。26S蛋白酶體在生物體內(nèi)參與多種生命活動的過程,如細胞分裂、細胞程序性死亡、DNA損傷的修復等,在細胞中異常蛋白的降解過程發(fā)揮重要作用。另外,溶血素蛋白具有良好的理化性質(zhì),能夠在不同p H范圍內(nèi)保持穩(wěn)定性,并且在醫(yī)藥方面具有應用價值。但這兩個基因在抗逆功能方面尚未有報道。本研究中從一株嗜鹽曲霉Aspergillus glaucus CCHA的酵母表達文庫中篩選出耐鹽性較強的兩個基因,通過遺傳轉化,對其基因功能進行了探究。主要研究結果如下:1.成功克隆出嗜鹽灰綠曲霉26S蛋白酶調(diào)節(jié)亞基7的全長序列,并成功獲得酵母轉化子p YES2-Ag S7和嗜熱菌轉化子p GFPGUSPlus-Ag S7,對其進行Na Cl、山梨醇脅迫處理,轉化子均未表現(xiàn)出抗逆性,說明Ag S7基因無抗逆性功能。2.分別以曲霉c DNA文庫和酵母文庫菌株質(zhì)粒為模板成功擴增出的非全長序列n Ag S7、NAg S7,重新轉入酵母獲得轉化子p YES2-n Ag S7、p YES2-NAg S7,其逆境脅迫驗證均未表現(xiàn)為脅迫敏感性,無抗逆性功能,因此推測酵母文庫中該基因的酵母菌株發(fā)生突變。3.基因Ag Aeg S具有溶血蛋白家族保守功能域,獲得的酵母表達菌株p YES2-Ag Aeg S及嗜熱菌轉化子菌株p GFPGUSPlus-Ag Aeg S在高鹽滲透脅迫下表現(xiàn)出抗鹽性,初步推斷Ag Aeg S基因可能參與嗜鹽灰綠曲霉對逆境脅迫的應答。以上結果表明26S蛋白酶調(diào)節(jié)亞基7基因對逆境脅迫表現(xiàn)敏感,未表現(xiàn)出耐鹽特性,可能不參與生物體的抗鹽機制;通過酵母表達文庫篩選轉化子可能存在菌株突變的因素。初步推斷溶血素蛋白基因可能參與嗜鹽灰綠曲霉的高鹽、干旱等高滲透環(huán)境脅迫的應答。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;Q93
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;Q93
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