辣椒疫霉漆酶基因Pclac3克隆表达

辣椒疫霉是一种侵染性植物病原,能引起多种茄科及葫芦科植物的病害。利用同源克隆法从辣椒疫霉基因组内克隆了漆酶基因Pclac3,该基因全长1 881个核苷酸,编码626个氨基酸,与已知的真菌漆酶基因具有较高的同源性,并且具备漆酶基因的保守区域。利用PCR将该基因克隆于pPIC9K载体,并转化于毕赤酵母GS115,经过1%甲醇诱导表达获得其异源表达产物。将表达产物进行SDS-PAGE检测,获得分子量大约为90 kDa的特异蛋白。利用ABTS法对Pclac3的表达产物粗酶液进行酶活性分析发现,其活性在第11天时最大,达到45 U/mL。这为研究辣椒疫霉漆酶基因家族及探索卵菌漆酶生物活性及其潜在的应用提供了理论依据。

Cloning and Expression of Laccase Gene Pclac3 from Phytophthora capsici

Phytophthora capsici is an aggressive plant pathogen that affects solanaceous and cucurbitaceous hosts.From this fungus,the laccase gene Pclac3 was cloned.The 1 881 bp full-length cDNA of Pclac3 encoded a mature laccase protein containing 626 amino acids.The deduced protein sequence showed high similarity with the other known fungal laccases and contained conserved domains typical of laccase protein.Heterologous expression of Pclac3 was achieved using the expression vector pPIC9K with the Pichia pastoris expression system induced under 1％ methanol.The recombinant protein migrated as a single band in SDS-PAGE,with an apparent molecular weight of ca.90 kDa.A high activity of recombinant protein （45 U/mL）,induced with methanol with 2,2＇-azino-di（3-ethylbenzothialozin-6-sulfonic acid） （ABTS） as substrate,was observed at the 1 lth dpi.The reported data adds new knowledge to P.capsici laccase multigene family and shed light on the potential function of individual laccase isoforms of oomycetes for biotechnological and potential industrial applications.