黑木耳内切葡聚糖酶基因克隆与原核表达

Cloning and Prokaryotic Expression of Endoglucanase Gene in Auricularia auricular-judae

克隆了黑木耳(Auricularia auricular-judae)内切葡聚糖酶(endoglucanase)基因并进行序列分析,此外,在大肠杆菌BL21(DE3)中成功表达。研究基于前期测得的黑木耳转录组数据,同时结合基因组数据(GenBank登录号为NEKD00000000.1),筛选到黑木耳内切葡聚糖酶基因序列并设计特异引物,以黑木耳菌丝总RNA为模板,采用RT-PCR技术克隆黑木耳内切葡聚糖酶基因cDNA全长,命名为Aa-eg,构建了重组原核表达载体pET32a-Aa-eg并成功在大肠杆菌中表达。序列分析表明,cDNA全长为1242bp,编码413个氨基酸,预测该蛋白分子量为43.59ku,理论等电点为4.42,存在信号肽。由于pET-32a载体包含20.0ku的标签,SDS-PAGE分析在45.0ku和66.2ku之间出现目的蛋白条带,与理论预期值大小一致。通过对黑木耳内切葡聚糖酶基因的克隆及分析,为进一步揭示黑木耳内切葡聚糖酶基因功能奠定基础。

The endoglucanase gene was cloned and analyzed from Auricularia auricular-judae, and the recombinant protein was obtained by prokaryotic expression. Combined with the genomic data of Auricularia auricular-judae in NCBI ( The GenBank login number is NEKDOOOOOOOO. 1 ) and the transcriptome data obtained in the early stage, the sequence of endoglucanase gene was screened out and specific primers were designed. The full length of the cDNA of the endoglucanase gene was cloned by RT-PCR and named as Aa-eg. The recombinant prokaryotic expression vector pET32a- Aa-eg was constructed and successfully expressed in E. coli BL21. Sequence analysis showed that the total length of cDNA was 1 242 bp, encoding 413 amino acid polypeptides. The molecular weight of the protein was predicted to be 43. 59 ku, the theoretical isoelectric point was 4. 42, and there was signal peptide in the endoglucanase. Due to the fact that a tag protein with the molecular weight of 20 ku was involved in the vector, the SDS-PAGE analysis showed that the target protein band was located between 45. 0 ku and 66. 2 ku, which was consistent with the theoretical expectation. The cloning and analysis of the endoglucanase gene laid a foundation for further revealing the function of the endoglucanase gene in A. auricular-judae.