酸性蛋白酶基因Asp在红曲霉中的同源表达及序列分析
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摘要
从红曲霉基因组中扩增出酸性蛋白酶基因Asp的编码区,构建其同源表达载体p BC-Hygro-Asp并导入到农杆菌GV3101备用。利用根癌农杆菌介导法将重组质粒导入红曲霉,并筛选获得Asp基因同源重组转化子,实现了酸性蛋白酶基因在红曲霉中的同源表达。转化子中酸性蛋白酶基因表达量是野生型菌株的3.30倍,能达到高产酸性蛋白酶的目的。对红曲霉酸性蛋白酶基因序列进行分析,结果显示该基因产物有两个天冬氨酸蛋白酶活性位点,为亲水性分泌蛋白,不参与信号转导,且与赤曲霉酸性蛋白酶有相同进化速率。
In this work,the coding region of Asp was amplified by PCR from the genomic DNA of the Monascus and the homologous vector p BC- Hygro- Asp was constructed and then was introduced into the Agrobactium tumefaciens strain GV3101 for further study. The agrobacterial cells harboring the p BC- Hygro- Asp vector was introduced into the genome of Monascus via Agrobacterium tumefaciens- mediated method and the positive recombinant of Monascus was obtained. The expression level of acid protease gene in transformants strain was3.30 times that of the wild Monascus,which indicated that the Monascus recombinant with high yield of acid protease was successfully achieved. The gene sequence of acid protease showed the gene had two aspartic proteases with active sites and the protease was a hydrophilic secretory protein without performance of signal transduction and keet the same evolution rate as that of Aspergillus ruber.
In this work,the coding region of Asp was amplified by PCR from the genomic DNA of the Monascus and the homologous vector p BC- Hygro- Asp was constructed and then was introduced into the Agrobactium tumefaciens strain GV3101 for further study. The agrobacterial cells harboring the p BC- Hygro- Asp vector was introduced into the genome of Monascus via Agrobacterium tumefaciens- mediated method and the positive recombinant of Monascus was obtained. The expression level of acid protease gene in transformants strain was3.30 times that of the wild Monascus,which indicated that the Monascus recombinant with high yield of acid protease was successfully achieved. The gene sequence of acid protease showed the gene had two aspartic proteases with active sites and the protease was a hydrophilic secretory protein without performance of signal transduction and keet the same evolution rate as that of Aspergillus ruber.
引文
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[11]薛庆中.DNA和蛋白质序列数据分析工具[M].第三版.北京:科学出版社,2014,80-83.
[2]Steven C,Van S,Nicholas I,et al.Aspartic Acid protease from Botrytis cinerea removes haze-forming proteins during white winemaking[J].J Agric Food Chem,2013,61:9705-9711.
[3]Yin L J,Hsu T H,Jiang S T.Characterization of acidic protease from Aspergillus niger BCRC 32720.[J]J Agric Food Chem,2013,61:662-666.
[4]Virginia M G,Alejandro T J,Ainhoa A C,et al.Purification and characterization of the extracellular aspartyl protease APSm1from the phytopathogen fungus Stenocarpella maydis[J].Protein Expres Purif,2015,98:1046-1050.
[5]曹治云.黑曲霉产酸性蛋白酶催化机制和稳定剂研究[D].福建:福建师范大学,2005.
[6]Cai Q M,Jiang D H,JI H,et al.Establishment and Optimization of Agrobacterium-mediated Transform-ation Systems for Monascus purpureus[J].Journal of Microbiology,2010,30(5):68-73.
[7]郭继平,马光.响应面优化米曲霉酸性蛋白酶的固态发酵培养基[J].中国酿造,2009,209(8):125-128.
[8]Radha S,Nithya V J,Himakiran Babu R,et al.Production and optimization of acid protease by Aspergillus spp under submerged fermentation[J].Archives of Applied Science Research,2011,3(2):155-163.
[9]Long C X,Zhang B,Ma S,Research Progress of Molecular Clock[J].Agricultural Science&Technology,2012,13(12):2496-2498.
[10]朱雷,常慧萍,唐欣昀,等.几种酶活抑制剂对红曲霉色素合成的影响[J].微生物学通报,2007,47(4):706-709.
[11]薛庆中.DNA和蛋白质序列数据分析工具[M].第三版.北京:科学出版社,2014,80-83.