A full-length cDNA encoding 4-aminobutyrate transaminase (designated as MrGABA-T) which catalyzes the conversion of gamma-aminobutyric acid (GABA) to succinic semialdehyde, was isolated from the GABA-rich Monascus ruber Mr-5. The full-length cDNA of MrGABA-T has a 1563 bp open reading frame (ORF) encoding a protein of 520 amino acid residues. The deduced protein has an isoelectric point (pI) of 9.12 and a calculated molecular weight of 57.5 kDa. Phylogenetic tree analysis reveals that MrGABA-T is more closely related to GABA-Ts from Aspergillus than to those of other fungi. In this study, the function of MrGABA-T, a gene regulating GABA production, was also analyzed by the disruption of MrGABA-T in M. ruber Mr-5. The results revealed that the GABA maximum production of MrGABA-T-deleted strain (Delta MrGABA-T) was about 2.8-fold as that of M. ruber Mr-5 (2.67 g/L). This work will make a contribution to enhance GABA production at the molecular level in M. ruber.

A full-length cDNA encoding glutamate decarboxylase (designated as MrGAD) which catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), was cloned from the GABA-rich Monacus ruber Mr-5 (CGMCC NO. M208043) using homology cloning and RACE. The cDNA of MrGAD has a 1536 bps open reading frame (ORF) encoding a protein of 511 amino acid residues. The deduced protein has an isoelectric point (pI) of 6.05 and a calculated molecular weight of 57.8 kDa. The amino acid sequence of MrGAD has more than 80% sequence identity with the GAD genes of some other fungi species as indicated by multiple alignments. Phylogenetic tree analysis reveals that MrGAD is more closely related to GADs from Aspergillus than to those of other fungi. The cloning and characterization of the MrGAD will enable us to enhance GABA production in Monacus ruber by improving the metabolic processes at the molecular level.

In order to improve the γ-aminobutyric acid(GABA) production of Lactobacillus brevis strain L2 and establish a model refl ecting the nonlinear relationship between the factor and yield, the Plackett-Burman(PB) design and central composite design(CCD) were used to optimize the medium component and culture condition. By analyzing the statistical regression, we found peptone, glucose, MSG and initial pH were the most important factors. On this basis, error back propagation neural network(BPN) and genetic algorithm(GA) were applied to determine the optimum fermentation parameters as peptone 21.185 g/L, glucose 3.857 g/L, MSG 48.948 g/L and initial pH 4.05. Ultimately, the GABA production of strain L2 was up to 27.765 g/L, more than doubling the original yield(13.452 g/L), which indicated that using BPN-GA method to optimized fermentation conditions is an effective way.

从浙江省金华市郊水稻内生菌中筛选到1株拮抗水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)P6生理小种的淡色生赤壳菌Bionectria ochroleuca Bo-1菌株.该菌株的菌体和发酵液对Xoo P6生理小种均表现出较强的拮抗能力.以P6生理小种作为指示菌进行活性跟踪,通过分步萃取、硅胶柱层析、高效液相色谱等,分离纯化获得拮抗活性物质结晶;对所获得的结晶物进行核磁共振氢谱分析和质谱分析,通过解析与比对,该物质分子量为344.0835,分子式为C18H16O7,鉴定为松萝酸(usnic acid).将纯化的松萝酸以20％叶枯唑(bismerthiazol)和3％中生菌素(zhongshengmycin)为对照,采用96孔板法测定对Xoo P6生理小种的抑菌活性,最低抑制浓度为200 μg/mL,表明松萝酸具有较强的抑菌活性,但较中生菌素弱,与20％叶枯唑效果相近.

以淡色生赤壳菌(Bionectria ochroleuca)Bo-1菌株发酵液乙酸乙酯粗提物对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)抑菌活性为检测指标,采用单因素试验优化Bo-1菌株产生抗菌物质培养所需的碳源、氮源、无机盐；通过正交试验优化培养基配方和摇瓶发酵条件.研究结果表明,Bo-1菌株产生抗菌物质适宜的碳源、氮源和无机盐分别为淀粉、蛋白胨、MgSO4·7H2O；优化的培养基配方为:淀粉30 g/L,蛋白胨2g/L,MgSO4·7H2O 0.5 g/L；适宜的发酵条件为:温度30℃,转速150 r/min,装液量80 mL/250 mL,pH 6.5.

利用CODEHOP（Consensus-Degenerate Hybrid Oligonucleotide Primers）软件设计了红色红曲霉丝氨酸羧肽酶基因片段的简并引物,选取1对简并引物进行逆转录-聚合酶链式反应（RT-PCR）,得到348 bp的聚合酶链式反应产物,经pMD18-T载体克隆转化至大肠杆菌DH5α中,测序后进行BLASTX比对,发现此DNA产物与其他丝氨酸羧肽酶基因序列有相似性,推断所克隆的产物即为红色红曲霉的丝氨酸羧肽酶基因片段.

Glutamate decarboxylase( GAD) gene was cloned using cDNA of Lactobacillus brevis strain Lb-2 as a template and then sequenced. The on-line analysis tools and corresponding softwares the were adopted to analyse the components of nucleotide and amino acid sequences the GAD gene,predict the physical and chemical properties,signal peptide as well as advanced structure,and establish a phylogenetic tree. The gene sequence was a complete open reading frame( ORF) of 1 407 bp coding 468 amino acids. The theoretically relative predicted molecular weight and isoelectric of GAD was 53 517. 8 u and 5. 42 respectively,without trans-embrane domain,no subcellular localization sequence,and was hydrophilic protein,having the closest evolutionary relationship of GAD with that of Lactobacillus plantarum and L. delbrueckii.

2 transformants of genetic stability from Monascus purpureus transformants library mediated by Agrobacterium tumefaciens,which were screened with significant changes in producing Monascus pigment were studied.Characters of the transformants,including colonial and microscopical morphology,growth rate,genetic stability,hygromycin resistance,pigments and citrinin production capacity were analyzed.The results indicated that the biological characters of transformants were significantly different from the wild strain S.Under liquid ferment,the color value of transformant S62 was 2.71 U/mL,which was 0.04 fold of the origin,and the transformant S158′s was 17.62 U/mL.

A bacterial strain QT-0304 antagonizing paddy pattern blight ( Rhizoctonia solani) was obtained from paddy field soil in Jinhua, Zhejiang Province, adopting flat plate confrontation method, its maximum antagonistic band width was up to 20 mm. According to its morphological features, physiological and biochemical characteristics, 16S rRNA gene sequence comparison as well as genealogical tree construction analysis, the strain QT-0304 was identified as Lac-tobacillus murinus. The antibacterial spectrum experimental results showed that strain QT-0304 has fairly good inhibi-tion against apple decaying disease (Valsa mali) and poplar ulcer (Botryosphaeria dothidea), and the antagonistic band width could reach up to 34. 5 mm and 23. 0 mm, respectively.

根癌农杆菌介导的真菌转化(ATMT),是分离真菌基因和分析基因功能的重要方法.目前有多种农杆菌菌株可运用于ATMT法,例如LB4404、C58C1、EHA104、AGL-1等.其中AGL-1菌株因其具有Vir毒力高、转化效率高、稳定性好等诸多优点,近年来有关AGL-1菌株高效转化丝状真菌的应用越来越多.就其转化机制、转化过程、转化率影响因素的最新研究进展进行概述.

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5' sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNA(Trp) (HmtRNA(Trp) (UCA)) is 113.6 pmol/microg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.