酸性蛋白酶基因Asp在红曲霉中的同源表达及序列分析
Homologous expression and sequence analysis of Monascus Asp gene
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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。Abstract: 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.