Yeast protein-protein interaction network model based on biological experimental data

Duplication and divergence have been widely recognized as the two domi- nant evolutionary forces in shaping biological networks, e.g., gene regulatory networks and protein-protein interaction （PPI） networks. It has been shown that the network growth models constructed on the principle of duplication and divergence can recapture the topo- logical properties of real PPI networks. However, such network models only consider the evolution processes. How to select the model parameters with the real biological experi- mental data has not been presented. Therefore, based on the real PPI network statistical data, a yeast PPI network model is constructed. The simulation results indicate that the topological characteristics of the constructed network model are well consistent with those of real PPI networks, especially on sparseness, scale-free, small-world, hierarchical modularity, and disassortativity.

Analysis of Protein Interactions:Probing the Function of Proteins with Yeast Two-Hybrid System

The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.

Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

Interaction between Live Yeast and Dietary Rumen Degradable Protein Level: Effects on Diet Utilization in Early-Lactating Dairy Cows

Four early lactating Holstein cows were used to study the effect of live yeast (LY, Actisaf®CNCM I-4407, Lesaffre Feed Additives, Marcq en Baroeul, France) supplementation on diet digestive utilization of dairy cows receiving concentrated corn silage-based diets with two rumen-degradable protein (RDP) levels. For a 33 d period, cows were fed a total mixed ration (TMR) containing an adequate level (AL) of RDP or a low level (LL, 30% below AL) by using soybean meal or tanned soybean meal, respectively: for 21 d with no LY addition followed by 12 d during which LY was added to the diet. The pH and redox potential (Eh) were recorded and ruminal fluid samples were collected over 3 consecutive days. Feces were collected individually over 48 h and individual dry matter intake (DMI) was measured for determining apparent nutrient digestibility. The effective degradability of individual feed ingredients composing both diets was evaluated with nylon bags technique. Structure of the ruminal bacterial community was studied and diversity index was calculated. Digestibility of organic matter (OM) and crude protein (CP) were lower for LL than those for AL. With LY, digestibility of OM and CP was increased: +2.4 and +0.8 points, for AL, and +3.7 and +5.9 points for LL, respectively. Live yeast reduced dietary N ruminal degradation with both AL and LL. Ruminal pH and Eh were lower with AL compared to LL: 5.95 and –167 mV vs. 6.13 and –144 mV. Live yeast increased ruminal total volatile fatty acids (VFA) (+8.6%), C2 (+10%), and C4 (+35%) contents for LL and decreased that of C3 (?9.8%) for AL. Neither the structure of bacterial populations of the rumen nor the diversity index (Shannon) was altered by treatments. Those results suggested a specific interest in using LY in RDP deficient diets for early lactating cows.

Dairy Farming Conditions and Utilization Levels of Liquid Brewers’ Yeast in Kenya

Dairy production plays an integral part in supporting smallholder farmers’ livelihoods. The desire to increase the number of dairy cattle is not feasible due to the reduced output of feed resources occasioned by climate change. Consequently, the need to increase productivity per cow is inevitable. Conventional protein supplements are costly;hence, the need to explore affordable nutrientdense alternative feed resources. Liquid brewers’ yeast (LBY), a by-product of the brewing industry, is a rich protein supplement in dairy production. This study aimed to assess the dairy farming conditions and utilization levels of LBY as a feed supplement in Githunguri Sub-county, Kiambu. Semi-structured questionnaires were administered to 457 dairy farmers in a cross-sectional survey. The findings revealed that most farmers (94.2%) fed their cattle on established forage/fodder and crop residues with supplementation. Even though 53.1% of the respondents were aware of the use of LBY, only 30.6% utilized it to supplement dairy cows, most of whom (96.0%) used it fresh without preservation. Membership in farmers’ organizations increased awareness of LBY (r = 0.732). Principal component analysis indicated that the benefits of using LBY outweigh the challenges involved with a loading matrix of 0.891 - 0.954 and 0.681 - 0.807, respectively. The low adoption and use levels of LBY as a source of protein supplements were due to low awareness. There is a need for concerted efforts by stakeholders in the industry to increase farmers’ knowledge base on the utilization and effectiveness of LBY in dairy production.

Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by YeastTwo-hybrid System

[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus （PEDV） N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage （PAM） by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins （FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598） interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation（CoIP） test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.

利用酵母双杂交筛选菠萝AcSWEET11的互作蛋白

SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。

大豆GmNF-YA13互作蛋白的筛选及鉴定

大豆GmNF-YA13蛋白是一个核转录因子Y(NF-Y),在干旱和高盐响应过程中均发挥重要作用。为研究其抗旱和耐盐的作用机理,寻找GmNF-YA13的互作蛋白,构建pGBKT7-GmNF-YA13诱饵载体,采用酵母双杂交筛选大豆酵母文库,并进行X-α-gal染色验证。结果显示:酵母双杂交获得85个阳性克隆,测序分析后得到36个候选的互作蛋白。功能预测显示互作蛋白主要参与生长发育、胁迫响应、能量代谢、转录调控和信号转导等生物过程。选择GmUVR8、GmCML41、GmFbox13和GmFBA与诱饵pGBKT7-GmNF-YA13进行一对一验证,只有GmFBA能与GmNF-YA13发生相互作用,预示GmNF-YA13功能的发挥需要GmFBA的参与。该结果可为NF-YA抗逆分子网络的研究提供基础。

长足大竹象信息素结合蛋白CbuqPBP2互作蛋白的筛选与验证

【目的】筛选长足大竹象(Cyrtotrachelus buqueti)信息素结合蛋白CbuqPBP2的互作蛋白,为实现利用信息物质对长足大竹象种群持续控制提供参考。【方法】利用GST pull-down联合质谱技术,对长足大竹象信息素结合蛋白CbuqPBP2的互作蛋白进行筛选、鉴定和分析,并采用酵母双杂交试验验证了CbuqPBP2与信息素结合蛋白CbuqPBP1的特异性互作。【结果】GST pull-down共筛选出45个与长足大竹象信息素结合蛋白CbuqPBP2特异性结合的候选互作蛋白,包括CbuqPBP1、ND2、CYTB。这些互作蛋白主要参与细胞过程、定位、代谢过程、应激反应以及生物调控等多个生物学过程。使用酵母双杂交体系,构建了pGADT7-PBP1重组猎物质粒与pGBKT7-PBP2重组诱饵质粒,通过诱饵质粒毒性检测和自激活检测,表明重组诱饵质粒对Y2HGold酵母菌无毒性作用。共转化验证结果显示,诱饵质粒pGBKT7-PBP2共转化酵母菌株能够在TDO培养基上生长。【结论】CbuqPBP2和CbuqPBP1之间有相互作用,不同信息素结合蛋白间的互作对深入理解长足大竹象嗅觉感受机制提供了新的思路。

Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins （protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain）, and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.

运动营养食品的营养素、功能及酵母蛋白在其中的应用前景

全民健康意识的不断增强使运动营养食品的市场容量不断扩大,然而产品同质化严重、缺乏创新等因素限制了行业的发展。酵母蛋白营养价值高,富含必需氨基酸和支链氨基酸,具有慢消化特性,目前主要作为蛋白质补充剂和肌肉恢复补充剂等应用于食品加工。酵母蛋白可增强食品中蛋白质的营养价值、部分替代乳清蛋白,并延长饱腹感,在运动营养食品中具有广阔的应用前景。本文从运动营养食品中常见营养素功能、酵母蛋白营养特性和应用现状入手,对酵母蛋白在运动营养食品中的应用前景进行分析总结,为运动营养食品创新发展提供思路。

陆地棉抗黄萎病基因GhENODL6互作蛋白筛选

研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养基上正常生长,但不能生长于QDO/X/A培养基,表明GhENODL6蛋白对酵母宿主无毒害作用,没有自激活活性。将携带pNC-GBKT7-ENODL6诱饵载体的Y2HGold酵母菌与cDNA文库进行杂交筛选,结果获得一个能够显示蓝色的菌落。通过PCR扩增,在蓝色酵母菌中获得一段526 bp的非载体插入片段,与陆地棉基因组内基因WRKY47序列高度一致。通过同源扩增,从陆地棉中克隆到WRKY47开放读码框,全长1 587 bp,编码528个氨基酸残基。再次利用酵母双杂交技术确认了WRKY47与GhENODL6之间存在互作关系。综上,构建了重组载体pNC-GBKT7-ENODL6,并鉴定到与GhENODL6互作的蛋白WRKY47。

菌酶协同法与酶解法制备酵母源肉味香精

该研究以酵母蛋白为主要原料,对比菌酶协同法和酶解法制备得到的酵母蛋白水解液理化指标和美拉德反应后制备得到的香精风味物质及感官评价。结果表明:菌酶协同法相较于酶解法制备的水解液在水解度、蛋白质回收率、总氮含量、肽基态氮含量、氨基酸态氮含量均有明显提高,分别提高48.0%、48.0%、100.5%、122.5%、82.3%;十二烷基硫酸钠⁃聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS⁃PAGE)结果显示,菌酶协同法得到的水解液蛋白质分子量集中在4.6 kDa以下,低于单独酶解法的分子量;氨基酸分析结果表明,两种制备方法制备的水解液游离氨基酸组成差异较大,尤其是必需氨基酸占比,菌酶协同法较酶解法高45%;固相微萃取⁃气相色谱⁃质谱联用(solid⁃phase microextraction⁃gas chromatography⁃mass spectrometry,SPME⁃GC⁃MS)分析结果显示,两种香精检测出的风味物质具有较大差异,尤其是在酸类物质含量及醇类物质的种类上差异较大;感官分析结果表明,菌酶协同法制备的肉味香精的异味和焦糊味显著减少,水果酯香,肉香风味上显著增强(p<0.05)。

玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

水稻OsCYP22互作蛋白的筛选及验证

为了探究水稻亲环素家族基因OsCYP22的功能,构建OsCYP22酵母双杂交诱饵载体,并利用水稻酵母双杂交cDNA文库筛选与OsCYP22相互作用蛋白。自激活试验结果表明,OsCYP22诱饵载体无自激活活性,也对酵母细胞无毒性。通过对酵母双杂交文库的筛选,共得到38个阳性菌落,结合测序结果和生物信息学的方法筛选到20个可能与OsCYP22相互作用的蛋白。进一步对OsCYP22分别与调控植物根系生长相关基因OsCSN5和OsRUB1的全长互作验证,结果显示OsCSN5与OsCYP22在酵母中相互作用。这为深入研究OsCYP22的生物学功能提供了理论依据。

白菜种子cDNA酵母文库的构建及BrTTG1互作蛋白的筛选及分析

【目的】通过构建白菜种子的cDNA文库,筛选WDR 40蛋白TRANSPARENT TESTA GLABRA 1(TTG1)的互作蛋白,探究TTG1参与MBW三元复合体调控种皮原花青素形成的分子机制。【方法】以棕籽白菜自交系‘B147’的种子为材料,提取总RNA并建立cDNA文库,通过gateway技术构建诱饵载体pGBKT7-TTG1并进行酵母双杂交筛库。【结果】酵母文库库容为1.2×10^(7)CFU,文库滴度是5.0×10^(7)CFU/mL,插入片段平均长度大于1000 bp,诱饵载体在酵母中无自激活活性。通过构建的诱饵载体pGBKT7-TTG1与构建的cDNA文库杂交,共获得了38个阳性互作蛋白,功能预测显示其中一个蛋白注释为MYB转录因子,注释为MYB73,序列分析结果显示该基因含有R2R3-MYB型抑制子保守基序C1和C2,推测该基因为白菜中参与种皮颜色形成的R2R3-MYB型抑制子,暗示着白菜中可能存在不同MYB转录因子参与的调控网络,影响着原花青素的形成。【结论】本研究构建了白菜种子组织的酵母双杂交cDNA文库,获得了38个TTG1阳性互作蛋白,首次挖掘到了可能影响白菜种皮颜色原花青素形成的R2R3-MYB型抑制子MYB73,为后期探究白菜种皮原花青素的调控网络奠定良好的基础。

水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析

多蘖矮秆基因dwarf-3(D3)是水稻独脚金内酯信号转导过程中的重要节点基因,拟南芥中的MAX2基因与D3同源,且MAX2参与拟南芥的抗病防卫反应。本研究以水稻泛素连接酶D3为诱饵进行酵母双杂筛库,发现水稻抗病相关蛋白维管植物单锌指蛋白VOZ2与D3存在潜在的相互作用。通过酵母双杂交试验证实,D3与VOZ2存在互作。通过荧光定量PCR证实,接种水稻白叶枯病菌后,VOZ2基因在转录水平上的表达受到显著诱导。利用水稻原生质体开展的亚细胞共定位实验发现,D3与VOZ2共定位于细胞核。双分子荧光互补实验发现,D3与VOZ2在烟草叶肉细胞的细胞核和细胞质均产生较强的荧光,进一步证实了D3与VOZ2的相互作用。研究结果为进一步探究D3和VOZ2在水稻抗病防卫反应中的功能与分子机理奠定了基础。

山药DoWRKY40基因表达特征分析及互作蛋白筛选

【目的】WRKY作为植物特异型转录因子,参与植物生长发育过程。克隆山药DoWRKY40基因,分析其表达模式,通过酵母双杂交技术筛选与DoWRKY40互作的蛋白,探究WRKY40在山药膨大过程中的调控机制。【方法】以‘大和长芋’山药为材料克隆DoWRKY40基因,并进行生物信息学分析、表达模式分析、亚细胞定位,构建山药块茎不同发育时期酵母文库,筛选与DoWRKY40互作蛋白。【结果】DoWRKY40的开放阅读框长为927 bp。DoWRKY40蛋白属于WRKY家族的第II组,含有一个典型的WRKY转录因子保守结构域,定位于细胞核。与几内亚薯蓣亲缘关系较近。DoWRKY40基因在山药块茎生长发育的120 d表达量最高,且在叶、茎和块茎中均有表达,具有组织特异性。山药cDNA文库的库容量为1.484×10^(8);pGBKT7-WRKY40诱饵载体无自激活活性。酵母双杂交法从山药文库中筛选到4类与DoWRKY40相互作用的蛋白,分别参与植物生长发育、细胞周期调节与细胞扩增、信号转导及环境胁迫应答等。【结论】克隆得到DoWRKY40基因,其响应山药的生长发育过程,筛选到的4类互作蛋白表明其在山药细胞膨大及信号转导中起调控作用。

巴西橡胶树HbPSKR2基因克隆及互作蛋白鉴定

磺肽素是一种高等植物特有的小肽类激素,广泛参与植物的生长发育、分裂分化、生物或非生物胁迫等生物学过程。磺肽素受体蛋白(phytosulfokine receptor,PSKR)是磺肽素的直接受体,对于磺肽素信号传导至关重要。本研究采用RT-PCR(reverse transcription-polymerase chain reaction)技术克隆了橡胶树的HbPSKR2基因,并对其进行生物信息学、基因表达模式、互作蛋白筛选及鉴定分析。结果显示HbPSKR2基因的开放阅读框全长3159 bp,编码1052个氨基酸,理论分子量为114.84 kDa,理论等电点为6.34。结构域分析显示HbPSKR2属于典型的跨膜蛋白,前640个氨基酸是由亮氨酸重复组成的天线结构,第692~714个氨基酸是跨膜结构域,第765~1052个氨基酸是膜内激酶结构域。对HbPSKR2膜内激酶结构域以及拟南芥和水稻的PSKR同源序列进行多重比对,结果显示均存在ATP binding site、CaM binding site、Activation segment、GC Centre等保守性位点。表达模式分析显示,HbPSKR2基因在橡胶树形成层区高丰度表达,其表达量在冠菌素处理前期显著上升。通过酵母双杂交技术筛选到12个与HbPSKR2互作的候选蛋白,并对其中的2个蛋白激酶(HbPBL8和HbPIX13)与HbPSKR2的互作关系进行荧光素酶互补成像验证。结果显示,在烟草中共转化HbPSKR2-nLUC/HbPBL8-cLUC和HbPSKR2-nLUC/HbPIX13-cLUC可以观察到强烈的荧光信号,进一步证明了HbPSKR2在体内可与HbPBL8激酶和HbPIX13激酶互作。橡胶树HbPSKR2基因克隆及互作蛋白鉴定将为深入揭示橡胶树乳管分化分子机制提供新的思路。

基于复杂网络理论的酵母菌PPI网络中关键蛋白质与核心蛋白质组的识别

识别关键蛋白质对疾病治疗、药物设计等领域有重要作用。本文首先采用5种节点重要性排序算法,对4种酵母菌PPI网络进行关键蛋白质识别,并通过分析不同网络之间共有的关键蛋白质,构建了关键蛋白质子网。再通过杰卡德相似度指标,筛选出子网中拓扑特征相似的关键蛋白质对,发现4种网络中存在Gavin-EPG 1、Gavin-EPG 2、Babu-EPG 1、Babu-EPG 2、LCMS-EPG、MALDI-EPG共6个核心蛋白质组。尽管它们大都是核糖体蛋白质,然而不同的酵母菌PPI网络中核心蛋白质组中蛋白质的组成差异很大。本文所发现的关键蛋白质以及核心蛋白质组对进一步研究核糖体上蛋白质如何相互作用影响肽链的合成以及折叠提供了重要的理论参考。