RNA干扰技术降低黑曲霉细胞工厂的内源蛋白背景

利用RNA干扰技术(RNA interference,RNAi)改造黑曲霉细胞工厂,减少其内源蛋白表达背景。研究RNAi载体中反向互补片段特异性对RNA干扰的影响,同步干扰糖化酶基因glaA、淀粉酶基因amyA及蛋白酶调控因子基因prtT,并利用经RNA干扰改造的黑曲霉宿主SH-2表达异源脂肪酶基因tll。研究结果表明,不同长度的靶基因反向互补片段表现出不同程度的RNA干扰效应,载体pAMDS-RNAi-glaA388对糖化酶的干扰效果(94.40%)比载体pAMDS-RNAi-glaA784(70.60%)好;经三基因RNAi载体pAMDS-multi-RNAi改造的黑曲霉菌株中glaA、amyA、prtT的表达量仅为原始菌株的5.30%、17.10%和34.60%;利用经三基因RNAi载体改造的黑曲霉菌株表达脂肪酶tll,最高酶活达到97.27 U/mL,比利用原始菌株表达tll的最高酶活(73.05 U/mL)提高33.16%。因此,RNAi技术可以有效降低黑曲霉宿主内源蛋白的表达背景,有利于提高外源基因在黑曲霉细胞工厂中的表达水平和稳定性。

Reducing the Endogenous Protein Background of Aspergillus niger Cell Factory by RNA Interference Technology

RNA interference (RNAi) technology was used to modify Aspergillus niger cell factory by reducing the expression level of its endogenous proteins. The effect of the specificity of reverse complementary sequence on RNA interference was investigated. Simultaneous interference of glucoamylase gene glaA, amylase gene amyA and protease regulator gene prtT was conducted in A. niger SH-2, and heterologous lipase tll was expressed in the RNAi-modified A. niger SH-2 host. Results illustrated that the reverse complementary sequence of different lengths exhibited RNA interference effect in different extent. RNAi vector pAMDS-RNAi-glaA388 with short reverse complementary sequence owned better interference effect (94.40%) than RNAi vector pAMDS-RNAi-glaA784 with long reverse complementary sequence (70.60%). Simultaneous interference of the three genes by pAMDS-multi-RNAi resulted in reduced expression of glaA (5.30%), amyA (17.10%) and prtT (34.60%) in A. niger SH-2. The expression of lipase tll in such RNAi-modified A. niger SH-2 reached the highest lipase activity of 97.27 U/ml, which was improved by 33.16% compared to tll expression in wild A. niger SH-2 (73.05 U/mL). Thus, RNAi technology could effectively reduce the expression background of endogenous proteins in A. niger SH-2, and could improve the expression level and stability of heterologous genes in A. niger cell factory.