橙色红曲菌（AS3.4384）SOD基因的克隆与功能分析
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摘要
红曲菌是我国重要的微生物资源，能够产生多种具有生物活性的代谢产物，具有很高的商业价值。但是由于红曲菌产桔霉素问题的存在，对红曲的安全性提出了怀疑。实验室前期研究克隆得到了八条可能与桔霉素产毒相关的EST序列。经生物信息学分析，发现其中一个基因P5编码的蛋白与Mn-SOD高度同源。
超氧化物歧化酶(superoxide dismutase，SOD)是一类广泛存在于生物体内的金属酶，能够催化超氧阴离子发生歧化反应，具有抗衰老、抗辐射、抗炎等多种生物活性。目前已广泛的应用于医药、食品、化妆品等领域。本研究利用分子生物学技术和方法，初步研究了该基因的功能，主要研究内容如下：
1．通过PCR技术从橙色红曲菌AS3.4384的Fosmid基因组文库中筛选到含有SOD基因的克隆子Q_5E_4，利用限制性内切酶Sal I将其完全酶切后亚克隆至pUC18质粒Sal I位点，筛选到含有SOD基因及侧翼序列的克隆子pUC18-Q5E4，测序后确定该克隆子为反向插入，含有SOD基因上游907bp及下游356bp序列。
2．成功构建了插入型SOD基因打靶载体pUC18-Q5E4-hph，该载体同源序列长度约为1.8kb。
3．将该基因编码框序列插入毕赤酵母分泌表达载体pPIC9K，构建重组表达质粒pPIC9K-SOD，经Sal I酶切线性化后，转化至毕赤酵母GS115(His~-Mut~+)。将经过PCR鉴定整合目的基因且表型为His~+Mut~+的重组菌株进行摇瓶诱导表达。以1％甲醇诱导48h后，重组酵母发酵液上清具有SOD酶活性，初步证实该基因编码SOD。
Monascus, one of the important microorganism resources of China, produces anarray of diverse bioreactive secondary metabolites during the process offermentation. However, it was also noticed that most Monascus strains producecitrinin, a harmful mycotoxin, in the same process. In order to construct Monascusstrain without excreting citrinin by genetic engineering, we have cloned eightputative genes (ESTs) which are gene fragments possibly related to the citrininbiosynthetic pathway in Monascus. Bioinformatics analysis shows that one EST,named P5, is highly homologous to Mn-SOD.
Superoxide dismutase (SOD), a widely presente metal-containing antioxydativeenzyme, is an important free radical scavenger in vivo and exhibits multiplebiological effects such as antiageing, antiradiation and etc. It is widely used inmedical, food and cosmetic products. In this paper, the function of P5 (SOD) hasbeen analyzed. The main contents are illustrated as following:
1. A clone named Q_5E_4 which contains SOD gene was isolated from the forsmidgenomic library of M. aurantiacus AS3.4384 by PCR. After completely digested bySal I, the restriction digestion fragments of Q_5E_4 was subcloned into pUC18. ThenpUC18-Q5E4 which contains SOD gene and its flanking region was constructed. Itwas later confirmed that pUC18-Q5E4 contains 907 bp 5'-flanking sequence and356 bp 3'-flanking sequence.
2. An insertion type of SOD gene targeting plasmid which is composed of a1.8kb homologous fragment and a hygromycin phosphate transferase expressingcassette has been constructed.
3. According to the SOD gene sequence cloned, a pair of specific primer was designed to clone the open reading frame and then subcloned into the secretionexpression vector pPIC9K. After linearized by Sal I, the expressing vectorpPIC9K-SOD was transformed into P. Pastoris GS115 (His Mut~+). The His~+transformants were verified by PCR with specific primers. The positiverecombinants were cultured in flasks to examine their supematant for SODenzymatic activity after been induced by 1% methanol.
超氧化物歧化酶(superoxide dismutase，SOD)是一类广泛存在于生物体内的金属酶，能够催化超氧阴离子发生歧化反应，具有抗衰老、抗辐射、抗炎等多种生物活性。目前已广泛的应用于医药、食品、化妆品等领域。本研究利用分子生物学技术和方法，初步研究了该基因的功能，主要研究内容如下：
1．通过PCR技术从橙色红曲菌AS3.4384的Fosmid基因组文库中筛选到含有SOD基因的克隆子Q_5E_4，利用限制性内切酶Sal I将其完全酶切后亚克隆至pUC18质粒Sal I位点，筛选到含有SOD基因及侧翼序列的克隆子pUC18-Q5E4，测序后确定该克隆子为反向插入，含有SOD基因上游907bp及下游356bp序列。
2．成功构建了插入型SOD基因打靶载体pUC18-Q5E4-hph，该载体同源序列长度约为1.8kb。
3．将该基因编码框序列插入毕赤酵母分泌表达载体pPIC9K，构建重组表达质粒pPIC9K-SOD，经Sal I酶切线性化后，转化至毕赤酵母GS115(His~-Mut~+)。将经过PCR鉴定整合目的基因且表型为His~+Mut~+的重组菌株进行摇瓶诱导表达。以1％甲醇诱导48h后，重组酵母发酵液上清具有SOD酶活性，初步证实该基因编码SOD。
Monascus, one of the important microorganism resources of China, produces anarray of diverse bioreactive secondary metabolites during the process offermentation. However, it was also noticed that most Monascus strains producecitrinin, a harmful mycotoxin, in the same process. In order to construct Monascusstrain without excreting citrinin by genetic engineering, we have cloned eightputative genes (ESTs) which are gene fragments possibly related to the citrininbiosynthetic pathway in Monascus. Bioinformatics analysis shows that one EST,named P5, is highly homologous to Mn-SOD.
Superoxide dismutase (SOD), a widely presente metal-containing antioxydativeenzyme, is an important free radical scavenger in vivo and exhibits multiplebiological effects such as antiageing, antiradiation and etc. It is widely used inmedical, food and cosmetic products. In this paper, the function of P5 (SOD) hasbeen analyzed. The main contents are illustrated as following:
1. A clone named Q_5E_4 which contains SOD gene was isolated from the forsmidgenomic library of M. aurantiacus AS3.4384 by PCR. After completely digested bySal I, the restriction digestion fragments of Q_5E_4 was subcloned into pUC18. ThenpUC18-Q5E4 which contains SOD gene and its flanking region was constructed. Itwas later confirmed that pUC18-Q5E4 contains 907 bp 5'-flanking sequence and356 bp 3'-flanking sequence.
2. An insertion type of SOD gene targeting plasmid which is composed of a1.8kb homologous fragment and a hygromycin phosphate transferase expressingcassette has been constructed.
3. According to the SOD gene sequence cloned, a pair of specific primer was designed to clone the open reading frame and then subcloned into the secretionexpression vector pPIC9K. After linearized by Sal I, the expressing vectorpPIC9K-SOD was transformed into P. Pastoris GS115 (His Mut~+). The His~+transformants were verified by PCR with specific primers. The positiverecombinants were cultured in flasks to examine their supematant for SODenzymatic activity after been induced by 1% methanol.
引文
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