融合疏水蛋白在银耳中的异源表达

为改善疏水蛋白的性质,充分利用疏水蛋白和银耳的可食性优势,将平菇疏水蛋白基因hyd与去除终止密码子的hyd拼接,形成融合疏水蛋白H-H,并构建其表达载体pEGH-H-H。通过电击法将pEGH-H-H导入到根癌农杆菌EHA105中,并转化到银耳芽孢。转化子经潮霉素抗性筛选,PCR检测后,随机挑取10个转化子进行Southern blot,筛选获得高拷贝且能稳定遗传的转化子。实时荧光定量PCR分析显示5号转化子的疏水蛋白相对表达量是野生型的11.4倍。最后提取5号转化子中的融合疏水蛋白,经SDS-PAGE检测,证明融合疏水蛋白在银耳中成功异源表达。起泡性和乳化性实验表明,转化子融合疏水蛋白较平菇疏水蛋白有一定的提升。

Heterologous Expression of Fusion Hydrophobin in Tremella fuciformis

In order to improve the properties of hydrophobin,and to expand the potential application of hydrophobin and the edibility of Tremella fuciformis,the Pleurotus ostreatus hydrophobin gene(hyd)was ligated with hyd without the termination codon,which was the fusion hydrophobin,named H-H,and then the expression vector pEGH-H-H was constructed. The vector was transferred into Agrobacterium tumefaciens EHA105 by electroporation,and then transformed to the yeast-like cells of T. fuciformis.After hygromycin selection and PCR detection,10 transformants were randomly selected and analyzed by Southern blot to screen multi-copy and stable genetic transformants. The quantitative real-time PCR analyzed showed that the relative expression level of transformant No.5 was 11.40-fold higher than that of the wild. Finally,we extracted the fusion hydrophobin from the transformant No.5,which proved the fusion hydrophobin can be expressed in the T. fuciformis successfully. The properties of foamability and emulsibility showed that fusion hydrophobin was better than P. ostreatus hydrophobin.