A novel combined immobilization technology was introduced in this work to improve the efficiency and stability of a high-solids biocatalysis system. First, a nano biocatalyst based on commercial cellulase immobilized onto the Fe3O4@SiO2-APTES nanoparticles was synthesized utilizing glutaraldehyde as a chemical crosslinking agent with a final immobilization efficiency of 93.0 %. A decrease in the V max and Km values indicates that the nanoparticleimmobilized cellulase enzyme has an increased binding affinity for the cellulose substrate. Next, a recombinant yeast strain was constructed via yeast cell-surface immobilization with laccase and versatile peroxidase for synergistic lignin-degradation. Functional accessibility was detected by using flow cytometry and immunofluorescence microscopy. Finally, the cooperative biocatalysis of nanoparticle-immobilized cellulase and cell- surface immobilized S. cerevisiae strain for SSF were investigated. Fed-batch operation of the solids at a 30.0 % (DW, w/w) final substrate loading was implemented to manage the instantaneous concentration level of inhibitors and enhance the saccharification of undetoxified biomass. The combined immobilization produced a maximum ethanol titer of 79.5 +/- 4.3 g/L and a theoretical ethanol yield of 88.2 % with a low cellulase loading of 10 FPU/g cellulose. This work presented a promising green pathway for achieving efficient cellulosic ethanol production, highlighting potential industrial applications and contributing to manufacturing optimization.

The DNA damage response (DDR) network comprises a range of protein factors and post-translational modifications (PTMs) that cooperate to maintain genomic stability following DNA damage. Lysine crotonylation (Kcr) is an emerging PTM, though its role in the DDR has not been thoroughly explored. We used quantitative proteomics to identify global Kcr substrates and assess their changes in response to DNA damage. Our results revealed 593 Kcr sites on 360 proteins that increased by more than 1.5-fold, while 331 Kcr sites on 233 proteins decreased by more than 0.67-fold following etoposide-induced DNA damage. Alterations in Kcr levels particularly in RNA splicing factors were most markedly pronounced before and after DNA damage. This study presents the first Kcr proteome regulated during the DDR and highlights the potential critical role of RNA-related factors in this process.

Inoculating lignocellulose-degrading microorganisms can accelerate straw decomposition in paddy field; however, the relationship between indigenous and inoculated microorganisms remains unclear. This study explored the effects of microbial inoculation on straw decomposition, microbial community, lignocellulose-degrading consortia, and associated functional genes. After inoculation, straw degradation rate increased by up to 4.9 %, and the rice yield increased by 790 kg/ha. Microbial inoculation restructured soil microbial community, influencing key taxa and interactions within the microbial network. A lignocellulose-degrading consortia consisting 37 genera was established, with a notable increase in the relative abundance of lignocellulose-degrading bacteria following inoculation. Among them, Pseudarthrobacter, with high lignin-degrading enzyme activity, emerged as a key genus after inoculation. Additionally, the abundance of lignin-degrading enzyme genes also increased significantly after inoculation. These findings offer new insights into how microbial inoculation accelerates the in situ decomposition of rice straw by reshaping the structure and function of lignocellulose-degrading consortia within the soil ecosystem.

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-β-1,4-xylanase with the optimal reaction pH of 3.0-4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.

[目的]为进一步了解前纤维蛋白(profilin,PFN)在丝状真菌中的功能,本文以粗糙脉孢菌(Neurospora crassa)为研究对象,进行了前纤维蛋白对其菌落生长和肌动蛋白(actin)聚合特性影响的探究.[方法]通过采用定点突变、同源重组、分生孢子过膜和PCR等技术,获得粗糙脉孢菌前纤维蛋白F78(F78A和F78D)和V113(V113E、V113R和V113W)的点突变体.利用平板生长法、竞争性生长管和显微镜观察检测表型变化,并结合多聚脯氨酸亲和层析纯化、荧光分光光度技术和高速共沉淀等技术分析点突变的前纤维蛋白对肌动蛋白聚合特性的影响.[结果]获得的粗糙脉孢菌前纤维蛋白点突变株F78A、F78D、V113E、V113R和V113W,与对照菌株ku70RIP相比,突变株生长均明显减慢(P＜0.05),其中PFN(F78D)和PFN(V113W)的突变体在生长的12-48 h,菌落直径分别仅为对照的20.0％-75.7％和12.7％-39.2％.竞争性生长管分析表明,PFN(F78D)和PFN(V113W)突变株菌丝生长速度受到显著抑制,分生孢子形成的节律并未发生明显改变.将纯化获得的前纤维蛋白野生型及点突变株F78D和V113W蛋白进行肌动蛋白的成核研究发现,前纤维蛋白能抑制肌动蛋白自发的成核过程,并表现为浓度依赖效应;而点突变蛋白F78D和V113W抑制肌动蛋白自发成核的作用减弱;进一步对肌动蛋白聚合特性的研究发现:野生型的前纤维蛋白在0-5μmol/L范围内可抑制肌动蛋白的聚合,且随浓度上升抑制作用进一步增强,致使上清中单体肌动蛋白含量最高可上升至82％左右;而5 μmol/L点突变蛋白F78D和V113W抑制肌动蛋白聚合的作用均明显减弱,分别使单体肌动蛋白的含量比对照下降12.0％(P＜0.01)和30.7％(P＜0.01).[结论]本研究证实前纤维蛋白在粗糙脉孢菌中起着重要的作用,其F78和V113是重要的活性位点,对于调节肌动蛋白的聚合解聚过程和粗糙脉孢菌的生长发育具有重要作用.

为探讨粗糙脉孢菌丝切蛋白(NcCof)的生化功能,对其进行纯化和微丝解聚活性分析.序列比对结果表明,NcCof N端第4位丝氨酸(S4)高度保守,推测其是NcCof发挥活性的重要位点,三级结构预测结果表明,它含有5个α-螺旋和6个β-折叠,中间的4个β 折叠片处于反平行状态,5个α-螺旋围绕在β 折叠片周围,具有典型的丝切蛋白/肌动蛋白解聚因子家族结构特征.利用点突变、亲和层析纯化得到蛋白NcCof、NcCof(S4A)和NcCof(S4D),进一步通过高速共沉淀等技术对其微丝解聚活性进行研究,结果表明,NcCof具有经典的微丝解聚活性,并具有剂量效应,突变蛋白NcCof(S4A)和NcCof(S4D)也具有解聚微丝的能力,但是NcCof(S4D)的解聚活性明显减弱.结果为进一步探讨NcCof调控肌动蛋白动态特性的分子机制奠定基础.

为了研究驱动蛋白-1的基因(Kin-1)在菌丝极性生长中的功能,本文以粗糙脉孢菌(Neurospora crassa)菌株为材料,利用同源重组技术尝试获得其敲除突变菌株Kin-1KO,并分析菌株87-3、Ku70RIP和Kin-1KO的菌丝生长情况.结果表明,与菌株87-3和Ku70RIP相比,菌株Kin-1KO的菌落生长缓慢、质地致密,菌丝生长速率下降,菌丝直径变粗、分枝增多.本研究成果获得了粗糙脉孢菌Kin-1敲除突变株Kin-1KO,证实Kin-1对于粗糙脉孢菌菌丝的生长至关重要.

Mitogen-activated protein (MAP) kinase pathways function as signaling hubs that are integral for many essential cellular processes, including sexual development. The molecular mechanisms and cross-talk between PR and CWI MAP kinase pathways have been extensively studied during asexual development. However, if these can be extended to sexual development remains elusive. By analyzing genome-wide transcriptional responses to deletion of each of two MAP kinase coding genes mak-2 (PR-MAP kinase pathway) and mak-1 (CWI-MAP kinase pathway) in Neurospora crassa during protoperithecium formation, 430 genes co-regulated by the MAK-1 and MAK-2 proteins were found, functionally enriched at integral components of membrane and oxidoreductase. These genes include 13 functionally known genes participating in sexual development (app, poi-2, stk-17, fsd-1, vsd-8, and NCU03863) and melanin synthesis (per-1, pkh-1, pkh-2, mld-1, scy-1, trn-2, and trn-1), as well as a set of functionally unknown genes. Phenotypic analysis of deletion mutants for the functionally unknown genes revealed that 12 genes were essential for female fertility. Among them, single-gene deletion mutants for NCU07743 (named as pfd-1), NCU02250 (oli), and NCU05948 (named as pfd-2) displayed similar protoperithecium development defects as the Δmak-1 and Δmak-2 mutants, failing to form protoperithecium. Western blotting analysis showed that both phosphorylated and total MAK-1 proteins were virtually abolished in the Δnrc-1, Δmek-2, and Δmak-2 mutants, suggesting that the posttranscriptional regulation of MAK-1 is dependent on the PR-MAP kinase pathway during the protoperithecium development. Taken together, this study revealed the regulatory roles and cross-talk between PR and CWI-MAP kinase pathways during protoperithecium development.

蛋白质分离纯化技术是生化及分子生物学实验技术的重要组成部分,在本科实验教学中占有重要地位.为了探索合适的实验方法和实验条件,优化本科实验教学中的溶菌酶分离纯化实验,以获得更好的纯化结果和教学效果,该文使用离子交换层析、硫铵沉淀、分子筛层析等方法成功地从蛋清中纯化出高纯度的溶菌酶,用SDS-聚丙烯酰胺凝胶电泳方法鉴定了样品纯度,并用溶壁微球菌做为底物测定了纯化酶的活力及比活力.结果 显示,使用改进后的方法能够得到较高纯度和比活力的溶菌酶,整个实验重复性好、易于操作,适合作为本科教学实验项目.

In order to better understand the factors that influence bacterial diversity and community composition in moss-associated bacteria, a study of bacterial communities in four moss species collected in three seasons was carried out via high-throughput sequencing of 16S rDNA and 16S rRNA. Moss species included Cratoneuron filicinum, Pylaisiella polyantha, Campyliadelphus polygamum, and Grimmia pilifera, with samples collected in May, July, and October 2015 from rocks at Beijing Songshan National Nature Reserve. In total, the bacterial richness and diversity were high regardless of moss species, sampling season, or data source (DNA vs. RNA). Bacterial sequences were assigned to a total of 558 OTUs and 279 genera in 16 phyla. Proteobacteria and Actinobacteria were the two most abundant phyla, and Cellvibrio, Lapillicoccus, Jatrophihabitans, Friedmanniella, Oligoflexus, and Bosea the most common genera in the samples. A clustering algorithm and principal coordinate analysis revealed that C. filicinum and C. polygamum had similar bacterial communities, as did P. polyantha and G. pilifera. Metabolically active bacteria showed the same pattern in addition to seasonal variation: bacterial communities were most similar in summer and autumn, looking at each moss species separately. In contrast, DNA profiles lacked obvious seasonal dynamics. A partial least squares discriminant analysis identified three groups of samples that correlated with differences in moss species resources. Although bacterial community composition did vary with the sampling season and data source, these were not the most important factors influencing bacterial communities. Previous reports exhibited that mosses have been widely used in biomonitoring of air pollution by enriching some substances or elements in the moss-tag technique and the abundant moss associated bacteria might also be important components involved in the related biological processes. Thus, this survey not only enhanced our understanding of the factors which influence microbial communities in mosses but also would be helpful for better use and development of the moss-tag technique in the environmental biomonitoring.

微丝骨架在真菌的生长发育过程中发挥着重要的作用,而动力学特性是其实现功能的关键.前纤维蛋白(profilin)是肌动蛋白动态组装的主要调控因子,对其功能研究有助于阐明微丝骨架在真菌生长发育中的机制.本文以丝状真菌模式生物粗糙脉孢菌(Neurospora crassa)为材料,利用定点突变技术和同源重组技术,分别将前纤维蛋白上肌动蛋白结合位点86位酪氨酸(Y86)和88位精氨酸(R88)进行了单突变和双突变,获得了Y86R、R88E和Y86RR86E前纤维蛋白点突变株.进一步利用平板培养和竞争性生长管培养对点突变株的表型进行分析后发现,与野生型相比,3个前纤维蛋白点突变株的菌丝生长均明显减慢.这些结果表明,前纤维蛋白与肌动蛋白的相互作用对于N.crassa的生长和发育至关重要.

微丝骨架在细胞的生命活动中具有重要的功能,而其动态的解聚聚合特性是其实现功能的前提.丝束蛋白(fimbrin/plastin)做为微丝结合蛋白质,是微丝骨架的重要调控因子之一,含有2个肌动蛋白结合结构域,目前对其结合微丝的机制并不清楚.本文以烟草丝束蛋白的肌动蛋白结合结构域2(NtFAbd2)为研究对象,通过原核细胞表达纯化NtFAbd2,利用体外沉淀法分析发现,NtFAbd2能够与微丝结合;利用激光共聚焦扫描显微镜分析发现,在烟草BY-2悬浮细胞内,NtAbd2-GFP与微丝共分布,这些结果为深入分析植物丝束蛋白的作用机制提供了新的数据.

在丝状真菌中,几丁质是真菌细胞壁的主要成分之一,对维持细胞形态和结构起到了重要的作用.几丁质是由几丁质合酶(chitin synthase,CHS)催化合成的,几丁质合酶参与生物钟调节的生理活动.本文以丝状真菌粗糙脉孢菌几丁质合酶1(CHS-1)为研究对象,通过同源重组基因敲除技术、电转化、分生孢子过膜以及PCR鉴定的方法,获得了chs-1缺失突变菌株CHS-1 KO.通过竞争性生长管(racetube)培养分析发现:对照Ku70Rm和突变菌株CHS-1KO(Ku70Rm背景)均具有明显的分生孢子带,但分生孢子带昼夜节律周期缩短,直线生长速率显著减慢,ku70Rw菌丝生长长度是3.4±0.31 cm/24 h,而突变菌株CHS-1KV(Ku70RIP背景)菌丝生长长度是2.07±0.19 cm/24 h.并进一步结合细胞壁染色对细胞形态分析发现:变菌株CHS-1ko菌丝沿生长方向膨胀、分支变短.这些结果表明:几丁质合酶1在粗糙脉孢菌分生孢子带昼夜节律形成和菌丝生长中发挥重要的作用.

微丝骨架是细胞骨架的重要成员,在细胞的多项生理活动中发挥着重要作用.本论文利用荧光标记鬼笔环肽技术和GFP融合蛋白技术,对活体烟草BY-2悬浮细胞中微丝骨架的标记方法进行了探索,结果表明,10nmol/L的Alexa488-Phalloidin为细胞微丝骨架标记的最佳浓度,利用PCR等技术构建pPZP-NtFABD2-GFP植物表达载体后,转化烟草BY-2悬浮细胞,激光共聚焦扫描显微镜观察发现,NtFABD2-GFP融合蛋白能够清晰地显示活体烟草悬浮细胞内的微丝骨架.这些结果为进一步深入研究微丝骨架在活体植物细胞中的功能奠定了基础.

The actin cytoskeleton plays an important role in hyphal polarity growth of filamentous fungi and its dynamic property is critical for actin function.Formin is an actin-binding protein,it has been shown to be involved in the regulation of actin dynamics.Formin-binding protein(FBP)is essential for formin to function.However,the information about FBP function in hyphal polarity is rather limited.In this study,the FBP knockout(FBPKO) mutant was generated via homologous recombination in Neurospora crassa and homokaryotic FBPKO strains were obtained.FBPKO mutants exhibited slower growth rate and abnormal hyphal morphologies within 24 hours of inoculation versus the latter.These observations suggest that FBP plays a role in regulating the early stages of hyphal polarity development of N.crassa.

麦角甾醇是真菌细胞膜的主要固醇类物质,其生物合成是一个复杂的酶促反应过程,其中C24( 28) -甾醇还原酶是麦角甾醇合成途径中的关键酶,对C24( 28) -甾醇还原酶功能的研究有助于阐明麦角甾醇对真菌极性生长的影响.本文对粗糙脉胞菌C24 (28)-甾醇还原酶蛋白(Erg-2基因编码)序列的同源性分析表明,在子囊菌门的3个物种中,C24( 28) -甾醇还原酶具有很高的保守性.根据同源重组基因敲除原理,通过电转化、分生孢子过膜以及PCR鉴定的方法获得了Erg-2基因缺失突变株(Erg-2KO),进一步利用斜面生长法并结合细胞壁染色进行突变株表型分析发现,与野生型相比,Erg-2KO(Ku70RIP背景)在生长初期菌丝生长缓慢,而后期与野生型无显著差异.这些结果表明,C24( 28) -甾醇还原酶对N. crassa早期的生长和发育至关重要.

The actin cytoskeleton is involved in numerous cellular processes including cell division,cell shape maintenance.The organization of the actin network is modulated by actin-binding proteins.Among the actin-binding proteins,fimbrin is an important regulator in actin polymerization and depolymerization.In this study,Ntfimbrin-ABD1 cDNA was successfully cloned through polymerase chain reaction.In order to investigate the interaction between Ntfimbrin-ABD1 domain and actin,pET28a-Fimbrin-ABD1 recombinant expression vector was constructed.Subsequently,the fusion proteins were expressed as inclusion bodies in Escherichia coli.The inclusion bodies were denatured and renatured,then the fusion proteins were purified by Ni-NTA argarose affinity column.The recombinant NtFimbrin-ABD1 proteins were confirmed to have the ability to interact with actin.All these results provide the basis for further study on Ntfimbrin-ABD1 function.

Establishment and maintenance of cell polarity are critical events for differentiation,proliferation and morphogenesis in eukaryote.Ploar growth of filamentous fungi serves as a typical model for polar growth.Furthermore,it has been widely accepted that microtubules play key roles in cell polarity.The present paper reviews the role of microtubules in polar growth of filamentous fungi.

The tobacco suspension cells were treated with different concentrations of DMSO,then were labeled with fluorescent probe and analyzed with agarose electrophoresis.The results showed that 2% DMSO caused chromatin condensation,cytoplasm shrinkage and cell nucleus disintegration;the genomic DNA became fragment at some degree in apoptosis cells detected from agarose electrophoresis,which show the typical characteristic features of the apoptosis cells.In addition,microfilaments cleavaged into short fragments in cells after 2% DMSO treatment.These results could provide references for further study the role of microfilaments in plant cells apoptosis.

文章介绍植物细胞内几类肌动蛋白结合蛋白(如:前纤维蛋白、形成素、肌动蛋白相关蛋白2/3复合体、肌动蛋白解聚因子和成束蛋白)的结构、性质和功能的研究进展。