草菇基因枪法遗传转化体系的建立

 草菇菌株V1308对潮霉素、卡那霉素、遗传霉素、膦丝菌素的抗性测验结果表明, 该菌株对潮霉素敏感, 而对其它三者不敏感。进一步测定结果显示, 潮霉素的最低筛选浓度在PDSA固体培养基上为75 μg·mL ^{- 1} , 在PDSB液体培养基中为50μg·mL ^{- 1}。以含有35S启动子、gus报告基因、潮霉素抗性基因的质粒pCAMB IA1301为表达载体, 采用基因枪法对草菇菌丝体进行遗传转化。结果表明, 基因枪的最佳轰击参数为: 氦气压力1 100 Psi, 真空压力87.88 kPa, 靶距离6 cm, 轰击次数1次。Southern杂交结果显示, gus基因已整合进V1308基因组中。GUS组织化学检测结果证明, gus基因在V1308菌丝体中获得了表达。草菇出菇试验结果显示, 转基因草菇和对照都能正常形成子实体, 子实体组织分离长出的菌丝体经GUS组织化学检测, 证明了外源基因gus能够在转基因草菇的菌丝体中稳定表达。     

草菇不同菌株的生物学特性研究

本试验选用草菇的六个不同菌株进行了菌株间的拮抗性测定，同时比较了菌丝生长速度、同工酶谱、CMC酶相对活性和抗霉性。已测结果表明：六个不同菌株中，菌株V35与V112亲缘关系最近，其次是V118，但都是不同的菌株；其次，菌株V35、V112与V118菌丝生长速度和CMC酶相对活性也明显的高于其它菌株，其中V112生长速度最快，长势最好，但V35抗霉性和CMC酶相对活性较其它菌株好。     

稻草床栽草菇培养料优质发酵方法研究

简易发酵、一次发酵和二次发酵稻草培养料床栽草菇的试验结果表明,二次发酵法栽培的草菇生物学效率达16.3%,显著高于一次发酵及简易发酵,且能较好地控制病虫害的发生,提高了草菇的产量和质量。     

草菇冷诱导Cor3基因实时荧光定量PCR标准品质粒和标准曲线的构建

A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.     

草菇噬菌体基因文库的构建

采用CTAB/NaCl法提取草菇基因组DNA,通过限制性内切酶Sau 3AI对纯化的草菇基因组DNA进行部分酶切,获得大小为20kb左右的DNA片段.这些DNA片段再次纯化后,与经Eco R I和Bam H I双酶切消化的Lambda GEM-11噬菌体DNA载体臂相连,以大肠杆菌(E.coli)LE392为寄主细胞,构建了草菇(Volvariella volvacea)基因文库,所构建基因文库效价为1.5×105 pfu/mL.     

草菇染色体核型的鉴定

利用等高锁状同源电场凝胶电泳（CHEF）,获得草菇的电泳核型.综合两组脉冲电泳的电泳条件、溴化乙锭染色以及DNA杂交分析结果,草菇染色体DNA至少可分为9个谱带.这一结果和以前用光学显微镜观察草菇的染色体数目的结果相一致.以混浊酿酒酵母和粟酒裂殖酵母等的相对迁移率为基准,草菇染色体长度估计在1.32～5.4Mb之间,染色体组总长约25Mb.     

Heterologous expression of the multi-functional cellulase gene (mfc) from the mollusc Ampullaria crossean,in Volvariella volvacea

Volvariella volvacea, or straw mushroom, is a widely cultivated and important edible fungus. In this paper, we used straw mushroom as a host to express an exogenous mfc gene encoding a multi-functional cellulase (MFC) cloned from the mollusc Ampullaria crossean. The purpose was to increase expression of MFC in the new host, as well as to improve cellulose degradation and increase future yields of V. volvacea. MFC is an enzyme with exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-xylanase activities. The A. crossean mfc gene was expressed in V. volvacea under the control of an endogenous promoter (gpd-Vvs) using the expression vector pgVvs-mfc, constructed by ligating the gpd-Vvs promoter with the mfc gene. The expression plasmid, pgVvs-afp, containing an afp gene encoding an anti-freeze protein (AFP) cloned from spruce budworm as the selective marker gene, was co-transformed into protoplasts of V. volvacea along with the vector pgVvs-mfc using polyethylene glycol (PEG)-mediated transformation. Putative transformants of V. volvacea were obtained by exposing the mycelia to regeneration medium (200 g l^?1 potato extract, 20 g l^?1 dextrose, 20 g l^?1 agar, and 0.8 M D-mannitol) at 0°C. PCR and Southern blotting were used to identify positive transformants. The results indicated that the mfc gene had been integrated into the genome of V. volvacea. The total cellulose activity, based on filter paper degradation, and carboxymethyl cellulase and xylanase activities in the transformants were 3.92, 4.75, and 58.99 Units ml^?1, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed an approx. 46 kDa band, which was equivalent to the expected size of the MFC protein of A. crossean. Thus, the mfc gene appeared to have been expressed heterologously in V. volvacea.     

浅谈草菇栽培

本文详述了草菇的生物学特性;生长发育所需的条件;良种的选择与栽培技术。     

草菇ras启动子区域的克隆及其序列分析

根据Kajiwara S等报道的ras启动子的序列设计并合成一对特异引物，以草菇菌丝的总DNA为模板，通过PCR扩增，获得一条长约0．75kb片段。DNA序列测定结果表明，该片段长度为751bp。采用Blast软件和Promoter predictions软件进行启动子序列结构分析，结果表明，该片段中243-293bp和539-589bp两区域为ras启动子的基础启动子区。在283bp和579bp处有两个转录起始位点，在244～247bp和292～295bp之间有2个TATAbox，在36～39bp、377～380bp、434-437bp和716～719bp之间有4个CAATbox。此外，该片段中还含有9个Gbox、12个Ibox、2个Pbox以及3个重要顺式作用元件：ABRE元件、WUN-motif(基元)和HSE元件。     

不同温度处理对草菇、真姬菇蛋白酶活性变化的影响

以草菇V884及真姬菇的子实体为供试材料,在4、15、30℃下进行保鲜储藏．结果表明：蛋白酶比活峰值出现愈迟,菇类保鲜期愈长;相反,出现愈早,保鲜期愈短．所以草菇在15℃下可保鲜3d;真姬菇在4℃下可保鲜7d,而在其他的温度处理下,保鲜时问很短(1d),说明草菇、真姬菇两种食用菌的腐烂变质和蛋白酶活性密切相关．