橡胶树炭疽菌(Colletotrichum siamense)疏水蛋白Hydr基因的克隆与原核表达

Cloning and Prokaryotic Expression Analysis of Hydrophobin Gene Hydr from Colletotrichum siamense

为了研究Hydr在橡胶树炭疽菌(Colletotrichum siamense)中的功能,本研究利用同源克隆法克隆获得橡胶树炭疽菌疏水蛋白Hydr基因,并利用原核表达系统对其进行表达。结果显示:Hydr基因DNA为520bp,cDNA为417 bp,编码139个氨基酸,包含2个内含子,在蛋白的N端含有由19个氨基酸组成的一段信号肽;分别构建了含信号肽的Hydr-PET-32a和缺失信号肽的Hydr (-18aa)-PET-32a的融合表达载体。利用SDS-PAGE和Western Blot检测蛋白表达情况,结果表明该蛋白在去除信号肽的情况下,供试条件下(IPTG0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L;温度16℃)均可表达,但保留信号肽时,在菌体上清和包涵体以及细胞培养液中均未获得融合蛋白,表明信号肽的存在会影响疏水蛋白Hydr原核表达。本研究结果为进一步验证疏水蛋白与脂滴包被蛋白的互作,以及疏水蛋白在橡胶树炭疽菌的功能分析提供理论依据。

In order to study the function of Hydr in Colletotrichum siamense, the gene of hydrophobin Hydr was cloned by homologous method, and it was expressed by prokaryotic expression system. The results showed that the Hydr gene DNA sequence was 520 bp, and its c DNA sequence was 417 bp. The gene contained two introns and predicted to encode 139 amino acids polypeptide, which contained a signal peptide consisting of 19 amino acids at the N-terminus of the protein;Then both fusion expression plasmid Hydr-PET-32a with sequence encoding a signal peptide and Hydr(-18 aa)-PET-32a without sequence encoding a signal peptide were constructed, respectively.SDS-PAGE and Western blot were used to detect the expression of the fusion protein. The results showed that the protein without signal peptide could be successfully expressed in Eschrichia coli under the induction of IPTG at 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1.0 mmol/L and temperature of 16 ℃. However, no fusion protein was detected in the supernatant, precipitation and cell culture medium when the signal peptide was retained. It showed that the existence of signal peptide affects the Hydr protein prokaryotic expression. This study will lay a foundation for further verification of the interaction between hydro-phobin and lipid-drop coating proteins, as well as the functional analysis of hydrophobins in Colletotrichum siamense.