Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Construction of brewing-wine Aspergillus oryzaepyrG- mutant bypyrG gene deletion and its application in homology transformation

pyrG- host cells are indispensable for pyrG- based transformation system. Isolations ofpyrG- host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG- mutant by sitedirected mutation of pyrG gene deletion which would be used as a host for further transformation, pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to con- struct pyrG- mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating thatpyrG was completely excised. The ApyrG mutants were applied as pyrG- host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production ofA. Oryzae. The xdh disrup- tion mutants were efficiently constructed by transforming a pMD-pyrC-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Axdh mutant was three times as much as that of the ApyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG- host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding ofA. Oryzae.     

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed‐batch synthesis of galacto‐oligosaccharides (GOS) from lactose with β‐galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed‐batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L^?1 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g^?1 in fed‐batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g^?1). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS‐5 and GOS‐6) was observed in the fed‐batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:59–67, 2014     

曲酸菌选育及发酵工艺研究

筛选获得产曲酸菌株米曲霉（Aspergillusoryzae）MSA进行⁶⁰Co诱变,并进行了发酵工艺条件研究,以葡萄糖为碳源、豆饼粉为氮源,在pH3.0、33℃摇瓶培养,可达到5d产酸5％以上水平。发酵液采用直接浓缩结晶工艺,脱色与重结晶后获得无色针状晶体,红外图谱检测确证为曲酸。     

互作对水稻白叶枯病菌JXOⅢ和JXOⅤ超氧阴离子释放的调控

水稻白叶枯病菌能够引起水稻的白叶枯病等一系列水稻病害。水稻白叶枯病菌JXOⅢ和JXOⅤ细胞中超氧阴离子的释放水平存在显著的差异,并进一步显示在两者亚细胞组分的超氧阴离子释放水平上。这种差异暗示致病性不同的病原菌中,O2^-可能具有信号传导及毒性因子的不同作用。亲和性不同的植物一病原菌互作过程对病原菌JXOⅢ和JXOⅤ的超氧阴离子释放具有不同的调控作用。对自身O2^-水平很低的JXOⅢ来说,亲和互作和非亲和互作过程都可诱导JXOⅢ中O2^-的释放;然而在自身听水平较高的JXOⅤ中,则随亲和性不同而表现出诱导或抑制的不同调控作用。无论是在JXOⅢ还是在JXOⅤ中,互作后分离得到的病原菌仍然能够显示出互作对其O2^-释放的显著影响,这种调控作用很可能具有一定的遗传稳定性。虽然机制还不清楚,但是推测这种调控可能和互作的走向相关。     

米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

以米根霉菌基因组DNA为模板,根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物,PCR扩增得到963 bp的DNA片段,经序列分析后将其亚克隆到原核表达载体pET30a上,构建成重组质粒pET30a-ldhL.将pET30a-ldhL转化到BL21感受态细菌中,经IPTG诱导表达后进行SDS-PAGE分析,可见约43 kD的与预期大小一致的目的蛋白条带,结果表明ldhL基因在大肠杆菌中进行了表达,经酶活分析产物的酶活力为98 U/mL,证明了表达产物具有预期的酶活性,这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础.     

Co-production of fumaric acid and chitin from a nitrogen-rich lignocellulosic material - dairy manure - using a pelletized filamentous fungus Rhizopus oryzae ATCC 20344

Fumaric acid is widely used as a food additive for flavor and preservation. Rhizopus oryzae ATCC 20344 is a fungus known for good fumaric acid production. It also has been reported that the fungal biomass has high chitin content. This study investigated the possibility of producing both fumaric acid and chitin via R. oryzae fermentation of dairy manure. Co-production of valuable bio-based chemicals such as fumaric acid and chitin could make the utilization of manure more efficient and more profitable. A three step fermentation process was developed which effectively utilized the nitrogen as well as the carbohydrate sources within the manure. These steps were: the culturing of pellet seed; biomass cultivation on liquid manure to produce both biomass and chitin; and fumaric acid production on the hydrolysate from the manure fiber. Under the identified optimal conditions, the fermentation system had a fumaric acid yield of 31%, and a biomass concentration of 11.5 g/L that contained 0.21 g chitin/g biomass.     

Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae

Abstract Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB ) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.     

产L-乳酸米根霉PW352特性及低能离子注入诱变高产菌株研究

为了获得更适于工业生产的产乳酸菌株,以葡萄糖为碳源对PW352菌株特性进行了研究,并在此基础上采用离子束诱变方法,对米根霉PW352进行改良,获得高产L( )—乳酸菌株RE3303,产酸能力达131～136g／L,最高可达140g／L,糖转化率为86％～90％,产酸比PW352提高75％。