利用RNAi抑制cre1基因转录提高里氏木霉表达纤维素酶
Improving cellulase expression of Trichoderma reesei by RNAi- mediated repression of cre1 transcription
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摘要: 本研究采用RNA干扰技术（RNAi）对里氏木霉主要的转录抑制因子cre1基因的表达进行抑制,以期提高里氏木霉生产纤维素酶的能力。通过PCR,从里氏木霉的基因组中扩增得到cbh1启动子、反向的cre1基因片段（568963 bp）、正向的cre1基因片段（655961 bp）和cbh2终止子,利用DNA assembler方法将这些基因连接到pRS424质粒上,构建成p Cre1-i质粒。再将RNAi盒分作两个片段扩增,同时转化里氏木霉并通过PCR鉴定阳性转化子。摇瓶发酵显示其中一株转化子在纤维素诱导培养基中培养4.5 d后,其纤维素酶的滤纸酶活、内切葡聚糖酶活和CBHI酶活为0.67、3.70和0.46 U/m L,较出发菌株分别提高了1.3、1.8和5.6倍。通过实时荧光RT-PCR检测,发现该转化子中cre1基因的转录水平比出发菌株降低了43%。Abstract: The RNA interference( RNAi) technique was used to repress the expression of the main transcriptional repressor cre1 in order to improve the cellulase production of Trichoderma reesei. The cbh1 promoter,a reversed cre1 gene fragment( 568 ~ 963 bp),an ordinary cre1 gene fragment( 655 ~ 961 bp),and cbh2 terminator were all amplified from the genomic DNA of T.reesei by polymerase chain reaction( PCR).The DNA assembler method was used to assemble all these gene fragments in pRS424 to obtain the p Cre1-i plasmid.Two fragments encompassing the RNAi cassette were amplified from the plasmid and transformed simultaneously into T.reesei.PCR was used to verify the positive colonies.In the flask batch culture,one of the transformants displayed 0.67、3.70 and 0.46 U/m L for the filter paper cellulase,endoglucanase,and CBHI activity,which were 1.3,1.8,and 5.6 folds of the parent strain.By using RT-q PCR,the transcript level of cre1 in this transformant was determined to be lowered to 43% of that of the parent strain.
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