刺芹侧耳生长条件和栽培特性的研究

对刺芹侧耳[Pleurotus eryngii(DC.ex Fr.)Quel]生长条件与栽培特性进行了分析.结果表明:刺芹侧耳菌丝生长的适宜pH值为pH 5～pH 7,最适pH值为pH 6;黑暗环境有利于菌丝生长,光照对菌丝生长具有抑制作用;菌丝生长适宜的碳源为可溶性淀粉、葡萄糖和蔗糖,适宜的氮源为蛋白胨和钼酸铵,葡萄糖和蛋白胨的适宜浓度分别为50和1 g·L-1;在马铃薯黄豆粉培养基、黄豆粉玉米粉培养基和马铃薯蛋白胨培养基中可较好地形成菌丝球.采用液体菌种栽培,菌丝的生长速度比使用玉米固体菌种快,其中用马铃薯黄豆粉液体菌种栽培,菌丝生长最快;使用马铃薯蛋白胨液体菌种在木屑棉籽壳混合培养料中栽培,可获得菌柄长菌盖大的子实体,而且产量高.     

刺芹侧耳菌渣对肺形侧耳（秀珍菇）生长和营养成分的影响

以常规棉籽壳培养料为对照,用刺芹侧耳Pleurotus eryngii工厂化生产的废料（简称菌渣）代替部分棉籽壳进行格氏侧耳Pleurotus geesterani栽培试验,并测定格氏侧耳的营养成分。结果表明,供试培养料中菌丝长势均良好,随着菌渣替代比例的增加,菌丝满袋时间、原基出现时间和转潮期缩短,生长周期变短;菌渣代替比例增加,产量呈降低趋势,其中替代比例33%（1:2）和55%（1.2:1）时培养料的生物学效率与对照无显著差异,替代比例55%和78%（3.5:1）时培养料对第四潮和第五潮菇产量影响明显,显著低于对照;供试菌渣培养料栽培格氏侧耳的粗蛋白和总氨基酸含量略高于或显著高于对照,说明添加适量的菌渣栽培格氏侧耳可提高子实体的营养成分。     

刺芹侧耳产漆酶条件优化及对偶氮染料甲基橙的脱色

漆酶是一种含铜的单电子多酚氧化酶,能够催化氧化各种酚类及多种染料,在处理染料废水方面具有巨大的潜力。刺芹侧耳Pleurotus eryngii具有较强的产漆酶能力,但漆酶产量在较大程度上受环境条件限制。本文研究了氮源含量、pH、温度、金属离子等环境条件对刺芹侧耳产漆酶能力的影响,优化了其产漆酶条件,并用其粗酶液对典型偶氮类染料甲基橙进行脱色,结果表明,在氮源0.5%（W/W）、pH 5.5、温度28℃、添加5.0mmol/L Mg ²⁺的培养条件下,刺芹侧耳产漆酶能力最强,培养6d时,漆酶酶活可达78.0U/L。用优化培养的刺芹侧耳粗酶液对偶氮染料甲基橙进行脱色,28h后脱色率可达90%,脱色反应为准一级动力学反应,甲基橙并未完全矿化,而是生成小分子中间产物。     

刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。     

酸法脱酰胺修饰对刺芹侧耳蛋白质消化性和功能特性的影响

为研究酸法脱酰胺修饰对刺芹侧耳蛋白质的功能特性和消化性的影响,本研究以刺芹侧耳粉为原料,通过碱提酸沉法提取刺芹侧耳蛋白质,并采用0.175mol/L盐酸、57.5℃改性,改性时间为121min 5s,制备出脱酰胺修饰蛋白质,探索脱酰胺改性对蛋白质的消化性及功能特性的影响。结果表明：脱酰胺修饰可提高刺芹侧耳蛋白质的溶解性、持油性、起泡性和乳化性,但对其持水性的改善作用不明显;同时脱酰胺修饰蛋白质表面疏水性和荧光强度增大,蛋白质巯基和二硫键含量变化不明显;体外消化率提高,消化产物的多肽含量降低,游离氨基酸含量增大。酸法脱酰胺修饰可在一定程度上有效改善蛋白质的功能特性和提高蛋白质的体外消化水平。     

刺芹侧耳不同发育时期的转录组差异分析

为了探讨刺芹侧耳子实体生长发育时期的基因表达变化,本文利用高通量测序技术对刺芹侧耳不同发育时期（菌丝期、原基期、子实体时期）进行RNA-Seq分析,在转录水平上解析差异表达基因在刺芹侧耳生长发育过程中的作用和功能。KEGG功能富集显示,菌丝期差异表达基因主要富集在碳代谢和氨基酸代谢中,其中三羧酸循环中编码柠檬酸合酶、乌头酸水合酶、异柠檬酸脱氢酶、琥珀酰辅酶A合成酶、琥珀酸脱氢酶、苹果酸脱氢酶的基因表达量均上调,说明碳代谢和氨基酸代谢是菌丝时期的主要能量来源;原基期上调的差异表达基因主要富集在脂肪酸代谢,其中RT-PCR定量结果显示原基期编码脂肪酸合酶的基因和编码脂酰辅酶A合成酶的基因下调,编码超氧化物酶的基因和编码过氧化氢酶的基因上调,表明脂肪酸代谢和抗氧化酶对刺芹侧耳原基期维持机体的稳定和生物应激方面起着重要作用。子实体时期上调的差异表达基因主要富集在剪接体、类固醇的生物合成以及AMPK信号通路中,说明环境因子对子实体时期有一定的影响。     

刺芹侧耳菌丝体固体发酵多糖在斑马鱼模型上的降血脂活性

高脂血症是诱导脂肪肝、高血压、动脉粥样硬化和心脑血管疾病的一个关键风险因素。本课题组前期研究已经在细胞水平、小鼠和斑马鱼动物模型上证实,刺芹侧耳多糖具有抑制体内脂质积累的生物活性。本文利用农林废弃物玉米芯和麦麸作为主要培养原料,通过固体发酵获得刺芹侧耳菌丝体多糖PESF（polysaccharide from Pleurotus eryngii mycelium solid-state fermentation）;进而,采用斑马鱼幼鱼和成鱼高脂动物模型,研究了刺芹侧耳菌丝体多糖在动物体内的降脂效率并解析了可能的降血脂途径和机理。实验结果证实,剂量400μg/mL的PESF不仅可显著抑制高脂饮食引起的斑马鱼幼鱼体内脂质积累,而且也可以有效抑制高脂饮食下斑马鱼成鱼的肝脏和肠道组织内的脂质积累,证实刺芹侧耳多糖具有显著抑制动物体内脂质积累的活性。这些结果建议,刺芹侧耳多糖降血脂的途径可能是通过降低肠道对脂类物质的吸收,从而减少了脂滴在肝脏中的积累。因此,本研究建议刺芹侧耳多糖具有开发成为降脂食品添加剂或者降脂药物原料的潜力。     

农杆菌介导的刺芹侧耳遗传转化体系的建立

以刺芹侧耳菌丝球为受体,潮霉素（Hyg）为筛选标记,应用农杆菌介导法对刺芹侧耳菌丝进行了遗传转化研究。潮霉素敏感性测试结果表明,刺芹侧耳Hyg耐受浓度为50mg/L。农杆菌介导的刺芹侧耳菌丝最佳遗传转化体系为：菌液浓度OD₆₀₀=0.6-0.7,侵染时间30-35min,共培养时间2d,侵染液和共培养培养基中乙酰丁香酮（AS）浓度为1mg/mL;经潮霉素抗性筛选、PCR鉴定和GUS活性的组织化学分析,表明外源基因GUS已转入到刺芹侧耳菌丝中,并获得表达。本实验成功地建立了稳定的农杆菌介导的刺芹侧耳遗传转化体系。     

刺芹侧耳两种保护酶基因的克隆、分析及温度对其表达量的影响

为了探究温度胁迫下刺芹侧耳中过氧化氢酶和铜锌超氧化物歧化酶基因的作用,本研究通过RT-PCR方法对刺芹侧耳转录组数据库检索得到的过氧化氢酶（CAT）和铜锌超氧化物歧化酶（Cu/Zn-SOD）基因进行克隆,并利用荧光定量PCR分析在高低温胁迫下CAT和Cu/Zn-SOD基因的表达量,以探讨刺芹侧耳中这两种保护酶基因在高、低温胁迫下的响应特征。结果表明,克隆得到的CAT和Cu/Zn-SOD基因开放阅读框长度分别为1 581bp和582bp,分别编码526个和193个氨基酸,分子量分别为59.6kDa、19.62kDa,等电点分别为6.35、6.31。在高温（35℃）和低温（4℃）两组处理中,CAT和Cu/Zn-SOD基因的表达量及酶活均在处理48h时达到最高且与对照组呈现显著差异,说明两种基因在刺芹侧耳抵御高低温胁迫的过程中具有重要作用。本研究结果初步研究了温度胁迫下刺芹侧耳中CAT与Cu/Zn-SOD基因的生物学功能,为进一步解析刺芹侧耳抵抗温度胁迫的机理奠定基础。     

湿热预处理对刺芹侧耳蛋白质理化性质、结构和功能特性的影响

以刺芹侧耳Pleurotus eryngii蛋白为研究对象,分别将其在50℃、60℃和70℃下热处理0至30min,探讨了湿热处理对刺芹侧耳蛋白理化性质、结构和功能特性的影响.结果表明,湿热处理后刺芹侧耳蛋白的疏水性先升高后降低(P＜0.05),总巯基含量先降低后升高(P＜0.05),此外,刺芹侧耳蛋白二级结构受到影响...     

Cloning of the glutamine synthetase gene fromspirulina platensis

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.     

Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli.

The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.     

Functional expression of bovine growth hormone gene in Pleurotus eryngii

The expression of the bovine growth hormone (bGH) gene was examined in Pleurotus eryngii, which belongs to the family of oyster mushrooms. The region encoding mature bGH, which has a variety of regulatory effects on growth and metabolic processes, was amplified using designed primers containing initiation and termination codons and then subcloned into pPEV binary expression vector. The recombinant vector (pPEVbGH) was introduced in P. eryngii via Agrobacterium tumefaciens-mediated transformation. Recombinant bGH was expressed in P. eryngii harboring pPEVbGH vector under control of the cauliflower mosaic virus (CaMV) 35S promoter up to a level of approximately 26% of total cell proteins after 6 days of cultivation, after which the recombinant protein was analyzed by SDS-PAGE and Western blotting. Interestingly, the growth rate of P. eryngii mycelia harboring pPEVbGH vector was approximately three times faster than that of control P. eryngii, suggesting that bGH affected the growth of P. eryngii. Biological activities were examined in Sprague-Dawley rats, which were administered regular feed mixed with mycelial extracts containing bGH (0.1 or 0.2 μg of bGH per g of animal feed). Mycelial extracts containing bGH significantly affected growth rates and lipid profiles; total cholesterol, triglyceride, HDL, and LDL levels were improved in rats fed mycelial extracts compared with those administered regular feed containing nontransgenic P. eryngii. This result indicates that P. eryngii harboring pPEVbGH vector could produce biologically active bGH. Further, levels of all growth-related factors increased, resulting in faster growth rates in bGH-treated groups. Accordingly, these data suggest that P. eryngii can be applied to the production of industrially useful proteins using a plant expression vector as an efficient mushroom host system.     

Cloning of the glutamine synthetase I gene from Rhizobium meliloti.

Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium. Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII. As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14. A gene bank of R. meliloti was constructed by using the bacteriophage P4 cosmid pMK318. Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source. One of the cosmids, pJS36, contains an insert of 11.9 kilobases. ET8051(pJS36) grows slowly on minimal media. When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45). Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable. pJS36 hybridizes with the glnA structural gene from Escherichia coli. Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity. This interrupted version of a glutamine synthetase gene was substituted for the normal R. meliloti sequence by homologous recombination in R. meliloti. Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source. These mutants are unaffected in nodulation and nitrogen fixation.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.