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为开发以病原菌激发子为主要成分的新型生物农药,根据核盘菌的全基因组数据信息,采用逆转录聚合酶链式反应(reverse transcription-PCR,RT-PCR)技术从核盘菌菌株NGA4克隆到编码角质酶基因的全长c DNA序列,在不同条件下进行原核表达,通过Ni2+亲和层析重组蛋白HisSs Cut,并进行激发子活性检测。序列分析表明,克隆的基因编码核盘菌角质酶开放阅读框为609 bp,预测编码202个氨基酸,并将基因命名为Ss Cut(Sclerotinia sclerotiorum cutinase)。含Ss Cut/p ET32a的表达菌株BL21经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导,于28、30和37℃均能产生44.3 k D左右的融合蛋白,其中30℃诱导3 h,融合蛋白表达量最高,浸润的烟草出现过敏性坏死反应。经纯化的蛋白His-Ss Cut和细菌His-Harpin分别浸润烟草叶片出现类似的细胞死亡。表明His-Ss Cut具有激发子的活性。
In order to develop a new type of biopesticide based on the elicitor of pathogen, based on genome-wide data of S. sclerotiorum, a reverse transcription-polymerase chain reaction (RT-PCR) The full-length c DNA sequence encoding the cutinase gene was prokaryotic expressed under different conditions, and the recombinant protein HisSs Cut was analyzed by Ni2 + affinity chromatography and the activity of the elicitor was tested. Sequence analysis showed that the cloned gene encoded a Sclerotinia sclerotiorum open reading frame of 609 bp, predicted to encode 202 amino acids, and the gene named Ss Cut (Sclerotinia sclerotiorum cutinase). The strain BL21 containing Ss Cut / p ET32a was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced a fusion protein of about 44.3 kD at 28,30 and 37 ℃, Among them, the highest expression level of fusion protein was induced at 30 ℃ for 3 h, and the hypersensitive necrosis reaction occurred in infiltrated tobacco. Similar cell death occurred in tobacco leaves by the purified protein His-Ss Cut and the bacterial His-Harpin, respectively. His-Ss Cut showed elicitor activity.