β-1，3-葡聚糖酶基因克隆、表达及抗菌活性的研究

Molecular Cloning, Prokaryotic Expression and Antibacterial Activity of β-1,3-Glucanase

通过RT-PCR的方法分别从小麦和水稻的cDNA中克隆获得β-1,3-葡聚糖酶基因(Glu基因),分别命名为TaGlu9507和OsGlu30。它们的序列分析表明这两个克隆均含一个1002bp的开放阅读框(ORF),编码334个氨基酸,各自的N-端含有一个长20和29个氨基酸残基的信号肽序列。将不含信号肽序列的TaGlu9507和OsGlu30编码区DNA片段分别克隆进pET28-a(+)表达载体,并转入大肠杆菌BL21(DE3),经0.5mmol/L IPTG诱导3h后获得了高量表达,表达量分别占大肠杆菌可溶性蛋白的49.7%和26.7%,表达产物对黑曲霉、酵母等真菌生长均有较为明显的抑制作用。本结果更进一步表明β-1,3-葡聚糖酶基因是植物真菌病防治的潜在目的基因群之一。

In this study, two β-1,3-glucanase （Glu） genes were amplified from the cDNAs of Triticum aestivum and Oryza sativa by RT-PCR and designated as TaGlu9507 and OsGlu30, respectively. Sequence analysis showed that both TaGlu9507 and OsGlu30 contained a 1002 bp-length ORF encoding two putative peptides of 334 amino acids with 20 and 29 residues signal peptides in length, respectively. DNA fragments from the coding sequences （CDS） of TaGlu9507 and OsGlu30 genes without their signal peptide sequences were cloned into the E. coli expression vector pET-28a（＋）. The recombinant vectors were transformed into E. coli BL21 （DE3） strain, respectively. The obtained transformants were able to express β-1,3-glucanase in a large scale when they were induced with IPTG at a final concentration of 0.5 mmol/L for 3 hours. SDS-PAGE analysis showed that the contents of the expressed β- 1,3-glucanase in total soluble protein of E. coli were 49.7% and 26.7%, respectively. The expressed β-1,3-glucanase products had an obvious inhibitory effect on fungi such as Aspergillus niger and Pichiapastoris. These results further revealed that β-1,3-glucanase gene was a potential target gene for the prevention and cure of fungal diseases of plants.