@ 国家自然科学基金

 

2015年度获资助项目:


杨亲正  国家自然科学基金面上项目《多肽修饰碳纳管作为导电通道的高效生物电化学系统构建与电子传递机理》批准号:31570118

 摘要:提高微生物燃料电池外膜蛋白与阳极界面间电子传递速率是提高产电能力的核心问题。目前存在一个难题:即阳极碳纳米管界面与膜蛋白活性中心接触时如何避免蛋白构象失活。本项目参照膜细胞色素C铁卟啉活性,来设计合成活性多肽修饰的碳纳米管复合结构来解决纳米管的破坏性刺入。研究不同活性多肽修饰的碳纳米管分别与正常菌、OmcA缺失菌、以及纯体外OmcA蛋白结合后体系导电能力的差异,构建以活性多肽修饰碳纳米管作为一维导电通道的高效生物电化学系统,并阐明其电子传递机理。


韩 宁  国家自然基金青年基金《葡萄新型N末端截短质膜H+-ATPase活性调控及其在抗碱性盐胁迫中的作用》批准号:31501738

 摘要:葡萄种植受到土壤盐碱化束缚,因此提高葡萄的抗盐碱性对产业发展非常重要。植物抗盐碱胁迫的重要部位为根,其中根质膜H+-ATPase(质子泵)的活性对植物维持盐碱环境中pH稳恒态至关重要。前期研究中,申请人从盐碱抗性较强的克瑞森无核葡萄根cDNA中克隆了质膜质子泵基因 VvPMA1,酵母功能互补已证实了其强功能,随后序列分析和试验发现克隆的VvPMA1可能是尚未报道的剪接变体,将产生具有较高活性的N末端截短的新型质膜质子泵(VvPMA1β),利于葡萄盐碱抗性。为了验证推测,本项目将对VvPMA1β表达、活性及调控进行进一步分析明确碱性盐胁迫下葡萄是否通过选择剪接产生活性和数量不同的VvPMA1剪接变体影响根质膜质子泵总活性;VvPMA1β是否具有组织、抗性品种特异性;VvPMA1β活性如何调节。希望从基因及蛋白水平了解葡萄根质膜质子泵的表达调控方式,提高盐碱胁迫下葡萄根质膜质子泵活性。

 

  王腾飞  国家自然基金青年基金项目《基于分子水平调控枯草芽孢杆菌芽孢表面高效稳定展示海藻糖合酶体系研究》批准号31501413

  摘要:海藻糖是一种稳定的、安全的生物制品保护剂,应用前景广泛。利用海藻糖合酶一步转化麦芽糖生产海藻糖的方法具有工艺简单,符合工业化生产的现实意义。本项目拟建立一个枯草芽孢杆菌芽孢表面高效稳定展示海藻糖合酶体系,克服海藻糖合酶胞外分泌困难且稳定性差的弊端,降低因胞内表达而徒增的细胞破碎和分离成本,并减少了热源物质的残留。研究内容包括,通过分析麦芽糖制备海藻糖转化体系中诱使芽孢萌发因素,结合基因敲除技术敲除与主要诱导因素相对应的关键受体蛋白基因,实现表面展示海藻糖合酶的芽孢特征的稳定,并探究作用机理;利用分子生物学技术克隆芽孢衣壳蛋白基因,单一或交叉融合绿色荧光蛋白展示于芽孢表面,以最大荧光强度展示组合高效展示海藻糖合酶,分析酶学特性,并利用半理性设计策略改造海藻糖合酶氨基酸组成,加强转化体系中芽孢表面展示海藻糖合酶的耐酸性。

 

  张 松   国家自然基金青年基金《食品用SiO2TiO2ZnO纳米材料对肠道益生菌及其菌群的影响》批准号:31501396

  摘要:纳米材料应用于食品生产、加工和包装过程中,逐渐显现出相当的优势,如提高食品的色泽、风味、营养利用率,并且具有抗菌或者指示食物变质的功能。但是研究表明食品用纳米材料会对细胞或组织产生损伤,因此本项目研究了常用的食品用纳米材料SiO2Ti O2ZnO对肠道益生菌及肠道菌群的生物学效应。首先从食品中分离出SiO2TiO2ZnO纳米材料并进行表征,然后从菌体形貌观察、体内外细胞毒性、体内外基因毒性和菌群结构变化等方面探索食品用纳米材料SiO2TiO2ZnO对肠道益生菌及菌群的影响及其规律,在此基础上改变纳米材料SiO2TiO2ZnO的表面性能改善纳米材料的安全性。因此本项目对于纳米材料在食品领域中的应用及食品安全性具有及其重要的意义。

 

 

2014年度获资助项目:

 

张静  国家自然基金青年基金《我国海带栽培品种(系)线粒体基因组遗传分化研究》批准号:31402300

 摘要:海带属藻类是褐藻门关键的进化种和国际重要的经济种类,本项目针对当前海带遗传育种研究中存在的亲本鉴定缺乏有力依据及种群遗传分化机制不明的问题,以真海带(Saccharina japonica)、长叶海带(Saccharina longissima)、糖海带(Saccharina latissima)及我国海带主要栽培品种(系)为研究对象,在线粒体基因组水平上采取比较基因组学的策略,广泛筛选并发掘可有效用于遗传差异研究及亲本鉴定的线粒体DNA序列,并揭示我国海带种群在长期驯化(选育)下的遗传分化特征与机制。

 



 

高水平研究论文


Biotechnol Biofuels(SCI, 5 years IF= 7.368)

 Production of a high-efficiency cellulase complex via beta-glucosidase engineering in Penicillium oxalicum

 


Background

Trichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei.

Results

In our study, we present a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enhanced both β-glucosidase activity and hydrolysis efficiency of the enzyme system on filter paper. We utilised two strategies to over-express β-glucosidase in the strain RE-10 that—although over-producing cellulase, does so at the cost of the cellulase mixture deficiency. The constitutive promoter of gene pde_02864 encoding 40S ribosomal protein S8 was used to over-express three β-glucosidases: BGL1, BGL4, and BGL5. We found that all mutants show significantly enhanced levels of β-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from bgl2 was used to conditionally over-express the β-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enables significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the β-glucosidase activity at a factor of 65-folds, resulting in levels of up to 150 U/ml. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue (DCCR). The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR at only 25 % enzyme load compared to the parental strain RE-10.

Conclusions

In summary, genetically engineering P. oxalicum to significantly improve β-glucosidase activity is a potent strategy to substantially boost the hydrolytic efficiency of the cellulase cocktail, which will ultimately lead to a considerable reduction of cost for biomass-based biofuel.

 草酸青霉(Penicillium oxalicum)是具有纤维素酶工业生产应用价值的一种丝状真菌,其主要特点之一是能高效表达木质纤维素酶,将难以降解的生物质水解为可发酵的寡糖和单糖。草酸青霉表达的纤维素酶系主要包括外切纤维素酶、内切纤维素酶和β-葡萄糖苷酶。这些纤维素酶组分之间合理的表达比例对于提高生物质的降解效率至关重要。在齐鲁工业大学特聘教授曲音波课题组,基于草酸青霉基因组序列,预测出11个β-葡萄糖苷酶编码基因,并构建了这些基因的突变株,筛选出β-葡萄糖苷酶比活力最高的三个β-葡萄糖苷酶编码基因(BGL1,BGL4和BGL5)。利用筛选出的草酸青霉40S核糖体S8蛋白编码基因PDE_02864启动子,分别在前期构建的高产纤维素酶基因工程菌RE-10进行过表达,构建出了纤维素酶活高表达的菌株。在摇瓶发酵条件下,β-葡萄糖苷酶的酶活相比出发菌株提高65倍,达到150 U/ml。本研究论文的研究结果使草酸青霉的纤维素酶表达体系更为合理,这对进一步推广草酸青霉在生物精炼领域的应用具有重要意义。

本论文的相关实验研究主要在山东大学完成。山东大学曲音波教授和齐鲁工业大学副教授李忠海为共同通讯作者。齐鲁工业大学为第二作者单位。

  论文链接:http://link.springer.com/article/10.1186/s13068-016-0491-4/fulltext.html


Water Research SCI IF=5.528

 Preparation and antibacterial activity of lysozyme and layered double hydroxide nanocomposites

It is necessary to develop “green” disinfection technology which does not produce disin-fection by-products. Lysozyme-layered double hydroxide nanocomposites (LYZeLDHs) were prepared by intercalating LYZ in LDH for the first time. Their antibacterial activity was evaluated using staphylococcus aureus as a target. The bacteria removal mechanism was also studied. Characterization of LYZeLDHs by X-ray diffraction and Fourier transform infrared spectroscopy indicated that LYZ was successfully intercalated in LDH, compressed and deformed without secondary structural change. LYZeLDHs showed excellent bacte-ricidal effectiveness against staphylococcus aureus. The antibacterial performance of LYZ eLDHs was found to be affected by the LYZ/LDH ratio and the pH of the bacteria-containing water. The bacteria removal efficiency of LYZeLDHs with LYZ/LDH mass ratio of 0.8 was consistently above 94% over the pH range of 3e9. LYZeLDHs adsorbed bacteria to their surface by LDH and then killed them by the immobilized LYZ. This new material integrated the bactericidal ability of LYZ and adsorption ability of LDH. Moreover, the antibacterial ability of LYZeLDHs was persistent and not limited by the adsorption capacity.

摘要:首次通过溶菌酶插层到LDH制备出溶菌酶层状双氢氧化物纳米复合材料(LYZ-LDHs)。选取金黄色葡萄球菌来评价其抗菌活性。LYZ-LDHs对金黄色葡萄球菌具有优良的杀菌效果。研究发现LYZ-LDHs的抗菌性能受LYZ/LDH的比值及含菌水的pH值的影响。在pH3-9的范围内,质量比为0.8LYZ-LDHs的除菌率始终高于94%。细菌被LYZ-LDHsLDH吸附到表面,被固定化的LYZ杀死。这种新材料整合了LYZ的杀菌能力和LDH的吸附能力。

本论文的相关实验研究主要在北京大学完成,齐鲁工业大学副教授杨亲正为第一作者,北京大学副教授赵华章为通讯作者。

论文链接http://www.sciencedirect.com/science/article/pii/S0043135413006799


Carbon SCI IF=6.196


Direct electron transfer and conformational change of glucoseoxidase on carbon nanotube-based electrodes

To gain insights into the direct electron transfer (DET) mechanism of multi-walled carbon nanotubes (MWCNTs), we investigated the conformational changes that occur in proteins when they interact with MWCNTs. We used glucose oxidase (GOD) as an example. Using cyclic voltammetry measurements, the GOD that was immobilized on the MWCNT-modified carbon paper electrode exhibited apparent direct electrochemistry compared to that on the bare electrode without MWCNTs. The structural transformation of GOD upon adsorption on the MWCNTs was characterized spectrally. GOD was not denatured, and only small shifts of the wavenumber of the β-sheet structure were observed. There was a consistent tendency for the amount of α-helix to decrease and the β-sheet to increase. The -helix content dropped from 21.2% to 19.6% as measured using Fourier transform infrared spectroscopy and from 27.1% to 25.9% as measured using circular dichroism. The reduction in the amount of α-helix led to a less shielded GOD active site and weakened the resistance of the electron transfer. These MWCNT-induced conformational changes could account for the DET between GOD and the MWCNT-modified electrode surface.

摘要:本文以葡萄糖氧化酶(GOD),采用循环伏安法论证碳纸电极经MWCNT修饰后GOD之间明显的直接电子转移现象。结果显示出MWCNTs 吸附的GODβ-折叠的红外光谱波数发生了轻微的移位,而α-螺旋有所减少,Far-UV CD测试也得到了类似的结果,α-螺旋的含量从27.1%下降到了25.9%。密实的α-螺旋结构的减少就在一定程度上导致较为松散的外层结构,及活性中心的暴露,因而降低电子传递阻力,进而促进了电极界面电子转移。

本论文的相关实验研究主要在北京大学完成。北京大学赵华章教授和齐鲁工业大学副教授杨亲正为共同通讯作者。齐鲁工业大学为第二作者单位。

论文链接http://www.sciencedirect.com/science/article/pii/S0008622309008586

Envrionmental microbiology   (SCI IF=6.78)

    Weel1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle-required cyclin-dependent kinase 1 activity

Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here we show that Wee1 and Cdc25 can balance cell cycle-required cyclin-dependent kinase 1 (Cdk1) activity in Beauveria bassiana. The Cdk1 phosporylation signal was strong in cdc25 but very weak in wee1 and absent in wee1 cdc25. Con-sequently, cell cycles, septation patterns and many septation-dependent gene transcripts of these mutants were reversely changed. Hyphal cells were short in wee1, slender in cdc25 and short and swollen in wee1 cdc25. Conidiation was most defective in wee1, followed by cdc25. Their conidia and yeast-like blastospores also altered antagonisti-cally in both size and complexity, accompanied with abnormally branched germlings in wee1 and wee1 cdc25. Conidial thermotolerance and UV-B resistance decreased much more in wee1 cdc25 than in wee1 but significantly increased in cdc25. The double deletion and the point mutation Cdk1T14A/P15F for inhibitory phosphorylation caused most defective virulence, followed by wee1 deletion. All the changes were restored by ectopic gene com-plementation. Virulence changes in all the mutants and control strains were highly correlated to those in blastospore size or complexity. Taken together, Wee1 and Cdc25 control cell cycle, morphogenesis, asexual development, stress tolerance and virulence of B. bassiana by balancing the Cdk1 activity.

摘要:病原真菌细胞周期调控是其侵染宿主和适应环境的关键。在球孢白僵菌中,调控细胞周期必须的Cdk1激酶的活性被证明是受核激酶Wee1和磷酸化酶Cdc25调控的,因为Cdk1的磷酸化信号在Dwee1减弱,在Dcdc25增强,而在双敲除菌株Dwee1Dcdc25中基本检测不到。因此,这些单基因和双基因的敲除株在细胞周期、菌丝分隔方式及形态以及影响隔膜形成的若干效应基因的转录水平方面都发生了不同方向的改变。Dwee1菌丝细胞变短,Dcdc25的菌丝细胞细长,而Dwee1Dcdc25的菌丝细胞既粗短又肿大。产孢缺陷以Dwee1最严重,其次为Dcdc25。此外,突变菌株的分生孢子和芽生孢子在细胞大小和复杂度上也发生了不同的变化,其中Dwee1Dwee1Dcdc25的分生孢子萌发的芽管异常分支。分生孢子耐高温和UV-B辐射的能力在Dwee1Dcdc25下降最多,在Dwee1中次之,而在Dcdc25中却显著增强。双基因敲除和磷酸位点突变Cdk1T14A/P15F的菌株在毒力上受损最严重,其次为Dwee1。所有单基因敲除引起的表型变化都在回补菌株中得到很好的恢复。此外,所有供试菌株的毒力与其芽生孢子的大小和复杂度之间存在显著的线性相关。因此,球孢白僵菌Wee1Cdc25通过平衡Cdk1的活性即其磷酸化和非磷酸化水平而调节细胞周期循环以及同生防潜能相关的多种表型。

本论文的相关实验研究主要在浙江大学完成,齐鲁工业大学邱磊为第一作者,浙江大学教授冯明光为通讯作者。

论文链接http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12530/full

 

 

 

 






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