糙皮侧耳脲酶基因的克隆和原核表达分析

尿素是现代农业生产中应用较为广泛的一种氮素化肥,而脲酶（EC 3.5.1.5）是尿素分解利用的关键酶。本文以糙皮侧耳Pleurotus ostreatus栽培菌株New 831为试验材料,探究了糙皮侧耳对尿素的利用情况,结果表明：糙皮侧耳可利用尿素作为唯一氮源,平板培养时尿素最适添加量为20mmol/L;在液体摇瓶培养过程中,培养液中的铵根浓度表现为先急剧升高后缓慢降低。通过对P. ostreatus PC15菌株基因组分析,获得了一个功能注释为脲酶的基因,并克隆获得了其全长基因组DNA（gDNA）和编码区（CDS）片段,命名为Pourease。结果表明：Pourease基因的gDNA和CDS长度分别为3 003bp和2 517bp,由10个外显子和9个内含子组成;Pourease蛋白由838个氨基酸组成,预测分子量为90.03kDa,与SDS-PAGE分析结果相符;Pourease蛋白与细菌、真菌和植物来源的脲酶具有52%-82%的一致性,且含有脲酶保守的镍离子结合位点;与洋刀豆脲酶空间结构类似,Pourease蛋白也以同源三聚体的形式存在。

Cloning and prokaryotic expression analysis of urease gene Pourease from Pleurotus ostreatus

Urea is a widely utilized nitrogen fertilizer in modern agricultural production, and urease (EC 3.5.1.5) plays a key role in the degradation and utilization of urea. Using Pleurotus ostreatus New 831 strain as the experimental material, the urea utilization of P. ostreatus was studied. The results showed that P. ostreatus could use urea as the sole nitrogen source, and the optimal dosage was 20mmol/L. The ammonium concentration in liquid culture dramatically increased first and then slowly declined during the flask-shaking cultivation. A gene, annotated as urease coding gene, was obtained by analyzing the genome of P. ostreatus PC15 strain. Its genomic DNA (gDNA) and coding sequence (CDS) fragments were cloned and designated as Pourease. The gDNA and CDS sequence of Pourease were separately 3 003bp and 2 517bp, containing ten exons and nine introns. Pourease protein consisted of 838 amino acids with a calculated molecular mass of 90.03kDa, corresponding to the result of SDS-PAGE analysis. Pourease protein shared 52%-82% identity with ureases from bacteria, fungi and plants, and contained the conserved nickel ions binding sites of urease. Similar to the spatial structure of Jack bean urease, Pourease protein also existed as a homotrimer.