双孢蘑菇酪氨酸酶基因在酿酒酵母中的异源表达及其酶学特性

【目的】在酿酒酵母中异源表达双孢蘑菇来源的酪氨酸酶基因PPO2,并研究酪氨酸酶在酿酒酵母胞内及胞外的酶学特性。【方法】提取双孢蘑菇总RNA,通过RT-PCR克隆酪氨酸酶基因PPO2,构建表达载体pSP-G1-PPO2,并转化至酿酒酵母进行表达,采用镍亲和层析纯化蛋白并研究其酶学性质。【结果】在酿酒酵母中正确表达了大小为65 kDa的酪氨酸酶蛋白。重组酶能催化底物酪氨酸产生黑色素。体外活性测定表明,酪氨酸酶催化最适温度为45°C,以酪氨酸和多巴为底物时最适pH分别为7.0和8.0。在酿酒酵母中测得底物酪氨酸浓度低于2.5 mg/mL时,黑色素的产量与底物浓度呈现正相关性。【结论】来源于双孢蘑菇的酪氨酸酶基因PPO2在酿酒酵母中成功表达,重组酶具有良好的酶学特性。利用酪氨酸酶产物黑色素的产量与底物浓度呈现正相关性这一特性,可将其作为细胞酪氨酸产量的传感器,为高通量筛选酪氨酸高产菌株提供了思路。     

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity.Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.     

双孢蘑菇疣孢霉病的发病过程及病原菌的核相研究

【目的】确定有害疣孢霉的传播途径,明确双孢蘑菇受有害疣孢霉侵染后发病症状和微观形态变化,以及有害疣孢霉的核相。【方法】将有害疣孢霉喷施于培养料及覆土材料的不同深度,观察记录双孢蘑菇的发病情况;将有害疣孢霉接种于不同生长阶段的双孢蘑菇子实体,观察记录其发病情况;使用光学显微镜及扫描电镜观察双孢蘑菇子实体受有害疣孢霉侵染前后的形态变化;通过DAPI(4′,6-二脒基-2-苯基吲哚)染色的方法对有害疣孢霉核相进行观察。【结果】将有害疣孢霉接种于培养料及覆土层的不同深度得到双孢蘑菇发病率如下:覆土层表面覆土层中间覆土与培养料交界处培养料中间层;有害疣孢霉可以侵染双孢蘑菇的任意阶段,将其接种于原基直径小于3 mm子实体表面时,得到不能正常分化的"马勃状"组织;对有害疣孢霉的侵染过程进行观察得到:其孢子可粘附于双孢蘑菇表面,并萌发长出芽管,接种处双孢蘑菇表面产生褐色病斑,双孢蘑菇菌丝体发生质壁分离,最后菌丝体膨大,细胞壁变薄甚至溢裂,菌丝体内部中空;有害疣孢霉产生两种类型的分生孢子,Ⅰ类无隔膜含1个细胞核;Ⅱ类具1隔膜含2个细胞核,2个细胞核被隔膜分开;细胞核的第1次有丝分裂发生于分生孢子母细胞中;厚垣孢子由上下2个细胞构成,上胞中含有2个细胞核。下胞含1–2个细胞核。有害疣孢霉的厚垣孢子萌发可产生1–2个芽管,芽管中细胞核的数目不断变化,一般0–2个细胞核。【结论】双孢蘑菇受其侵染后发生显著的细胞学变化;我们对有害疣孢霉做遗传分析时,进行单孢分离需挑取无隔膜的分生孢子为实验材料进行遗传分析。     

Screening of porous and cellular materials for covalent immobilisation of Agaricus bisporus tyrosinase

We conducted a systematic study of covalent immobilisation of Agaricus bisporus tyrosinase onto typical enzyme carriers. Acrylic beads, two commercial silica gels with different pore structures and mesoporous silica foam (MCFs) beads functionalised using different organosilanes showed that only aminated MCFs offer active preparations with immobilisation efficiencies greater than 100% and a similar ratio of diphenolase (L-DOPA) to monophenolase (L-tyrosine) activities as the free enzyme. The native enzyme was entirely inactivated during incubation at 55°C for 30 min, whereas the enzyme immobilised on acrylic carrier or MCF retained 46 and 35%, respectively, of the initial activity after similar treatment. Susceptibility of native and immobilised tyrosinase to suicide inactivation in the presence of L-tyrosine and L-DOPA was tested in repeated batch tests. However, none of the preparations obtained in the L-DOPA solution was operationally stable enough to be used for practical applications.     

Transformation of the cultivated mushroom,Agaricus bisporus, to hygromycin B resistance

Application of biotechnology to the cultivated mushroom,Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain ofA. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into theA. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomyceteAspergillus nidulans and fromA. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from theA. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.