Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

外源毒性物质及其在植物抗虫品种培育中的应用概况

对昆虫具有毒性的外源毒性物质广泛存在于微生物、植物和一些动物中。概述了Bt晶体蛋白、蛋白酶抑制剂、植物凝集素、淀粉酶抑制剂、几丁质酶等几种外源毒性物质的特征、杀虫机理及其基因在植物抗虫品种培育方面的应用概况,并对今后转外源毒性基因抗虫植物的主要研究方向作了探讨。     

PGPR mediated management of stem blight of Phyllanthus amarus (Schum and Thonn) caused by Corynespora cassiicola (Berk and Curt) Wei

Bacillus subtilis (BSCBE4), Pseudomonas chlororaphis (PA23), endophytic P. fluorescens (ENPF1) inhibited the mycelial growth of stem blight pathogen Corynespora casiicola (Berk and Curt)Wei under in vitro. All these bacterial isolates produced both hydroxamate and carboxylate type of siderophores. But the siderophore production was maximum with the isolate ENPF1. Delivering of talc based formulation of BSCBE4 through seedling dip and foliar application effectively reduced stem blight disease incidence and increased the dry matter production under pot culture and field conditions. Application of BSCBE4, PA23 and ENPF1 increased the defense related enzymes such as peroxidase, polyphenol oxidase, chitinase and β-1,3 glucanase in P. amarus up to ten days after challenge inoculation with C. cassicola. Native gel electrophoretic analysis revealed that challenge inoculation of pathogen with BSCBE4 and PA23 induced both peroxidase and polyphnol oxidase isoforms.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

链霉菌A048产几丁质酶最佳发酵工艺研究

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺牛产几丁质酶,几丁质酶活力比一步发酵工艺提高1．1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1．8倍,发酵周期缩短54h;在二步发酵工岂中另添加0．4％纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18．52U／mL。采用几丁质和纤维索双因子诱导二步发酵工艺可能是链霉菌A048生产几丁质酶的最佳工艺。     

微生物几丁质酶研究进展及应用*

几丁质由N-乙酰-D-氨基葡萄糖聚合而成,是自然界中仅次于纤维素的第二大类聚合物。微生物几丁质酶来源丰富,是生物降解或利用几丁质的主要媒介。野生型菌株几丁质酶产量低、活性弱,故近年来有关几丁质酶的研究侧重于对其产量及催化活性的提升等方面。此外,几丁质酶具有水解病原真菌细胞壁、破坏害虫体壁、生产N-乙酰氨基葡萄糖寡聚体或单体的应用价值,在医药、农业、食品加工等领域表现出巨大的市场潜力。综述微生物几丁质酶的来源、分类及工程改造,为后续几丁质酶的研究及开发利用提供参考。     

高产几丁质酶巴伦葛兹类芽孢杆菌的筛选和发酵条件优化

【目的】鉴定一株来源于中国南海海水样能够分泌多种胞外几丁质酶的类芽孢杆菌CAU904,并优化其产几丁质酶的发酵条件。【方法】采用形态学观察、16S r DNA序列比对及生理生化实验鉴定;通过碳源、氮源、温度、初始p H、表面活性剂种类以及发酵时间的单因素优化实验获得最佳发酵条件。【结果】菌株CAU904被鉴定为巴伦葛兹类芽孢杆菌(Paenibacillus barengoltzii),其最优发酵产酶条件为:0.5%胶体几丁质,0.2%酵母浸提物,0.1%吐温-80,培养基初始p H 7.0,45°C培养72 h。在最优发酵条件下,该菌株最大产酶水平达到8.2 U/m L,比优化前提高了5.4倍。几丁质酶的酶谱分析表明该菌株能够产生多达11种具有几丁质水解活性的同工酶,其中主要酶谱带对应分子量分别为54、47和38 k D。【结论】实验结果为巴伦葛兹类芽孢杆菌几丁质酶的分离纯化和酶的应用提供了基础。     

Prediction of protein targets of kinetin using in silico and in vitro methods: a case study on spinach seed germination mechanism

Kinetin, a cytokinin which promotes seed germination by inhibiting the action of abscisic acid, is an important molecule known to trigger various molecular mechanisms by interacting with an array of proteins shown from experimental observations in various model organisms. We report here the prediction of most probable protein targets of kinetin from spinach proteome using in silico approaches. Inverse docking and ligand-based similarity search was performed using kinetin as molecule. The former method prioritized six spinach proteins, whereas the latter method provided a list of protein targets retrieved from several model organisms. The most probable protein targets were selected by comparing the rank list of docking and ligand similarity methods. Both of these methods prioritized chitinase as the most probable protein target (ΔG_pred = 5.064 kcal/mol) supported by the experimental structure of yeast chitinase 1 complex with kinetin (PDB: 2UY5) and Gliocladium roseum chitinase complex with 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (caffeine; 3G6M) which bears a 3D similarity of 0.43 with kinetin. An in vitro study to evaluate the effect of kinetin on spinach seed germination indicated that a very low concentration of kinetin (0.5 mg/l) did not show a significant effect compared to control in inducing seed germination process. Further, higher levels of kinetin (>0.5 mg/l) constituted an antagonist effect on spinach seed germination. It is anticipated that kinetin may have a molecular interaction with prioritized protein targets synthesized during the seed germination process and reduces growth. Thus, it appears that kinetin may not be a suitable hormone for enhancing spinach seed germination in vitro.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-015-0135-3) contains supplementary material, which is available to authorized users.     

转几丁质酶基因黑杨的获得

以G1黑杨幼叶为外植体建立了组织培养高频再生体系 ,对传统的农杆菌介导法进行了改进 ,用来自于菜豆的几丁质酶基因 (CH5B)进行转化 ,经PCR和PCR Southern杂交鉴定 ,证实CH5B基因已整合入G1黑杨核基因组中。     

Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate

Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4 × 10⁹ (without chitin) to 14.4 × 10⁹ SBU/L and from 18.2 × 10⁹ (without chitin) to 25.1 × 10⁹ SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7 × 10⁹ SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5 × 10⁹ SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.