Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein

Alzheimer＇s disease（AD） is a neurodegenerative disorder in which amyloid b plaques are a pathological characteristic. Little is known about the physiological functions of amyloid b precursor protein（APP）. Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1,members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease（PMD） and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut(Arachis hypogaea L.)is a thermophilic crop,and low temperature leads to a significant reduction in annual yields.Despite a few cold tolerant germplasms or cultivars have been discovered and developed,molecular mechanisms governing peanut cold tolerance is poorly understood.Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism.In this study,we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system.GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response.Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature.qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress.Individual overexpression of two UDP-glycosyltransferases(AhUGT2 and AhUGT268)in transgenic yeast cells could enhance their tolerance to multiple abiotic stress.In conclusion,this study advances our understanding of the mechanisms associated with the cold stress responses in peanut,and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

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.

Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...

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.

A rapid absorbance-based growth assay to screen the toxicity of oligomer Aβ42 and protect against cell death in yeast

Multiple lines of evidence show that soluble oligomer forms of amyloidβprotein(Aβ42)are the most neurotoxic species in the brain and correlates with the degree of neuronal loss and cognitive deficit in Alzheimer’s disease.Although many studies have used mammalian cells to investigate oligomer Aβ42 toxicity,the use of more simple eukaryotic cellular systems offers advantages for large-scale screening studies.We have previously established and validated budding yeast,Saccharomyces cerevisiae to be a simple and a robust model to study the toxicity of Aβ.Using colony counting based methods,oligomeric Aβ42 was shown to induce dose-dependent cell death in yeast.We have adapted this method for high throughput screening by developing an absorbance-based growth assay.We further validated the assay with treatments previously shown to protect oligomer Aβ42 induced cell death in mammalian and yeast cells.This assay offers a platform for studying underlying mechanisms of oligomer Aβ42 induced cell death using gene deletion/overexpression libraries and developing novel agents that alleviate Aβ42 induced cell death.

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

To further research the regulatory network of pyruvate dehydrogenase kinase （designated as TaPDK） in physiological male-sterility （PHYMS） of wheat induced by chemical hybridizing agent （CHA） SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Ade/-His/-Leu/-Trp） （QDO）, and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor （TanLTP）, polyubiquitin （TaPUbi）, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen （TaPCNA）, CBS domain containing protein （TaCBS）, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.

Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay

ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing by increasing exon skipping and enhancing proximal poly (A) site use. ELL2 is needed to produce the secretory-specific Ig heavy chain mRNA while ELL1 mRNA does not change in abundance with B cell stages. To investigate the potential interactions of other proteins with the ELL1 and ELL2 proteins, we preformed yeast two-hybrid studies. HSP40 and Testin were found to bind to ELL2 in its amino-terminal half. PCNA binds to ELL2 in a region encompassing amino acids 186 - 344. The potent transcription factors HIF1 α and ZNF622 interact with both ELL1 and 2 in the central, proline rich region. Meanwhile, BBS2 and ING3 interact with ELL1 but not ELL2 in this central proline-rich region. Many of the ELL-interacting-proteins uncovered in the two-hybrid screen are tumour suppressors that may work through the ELL: pTEFb complex to suppress or activate sets of genes in plasma cells.

Construction of a Three-frame Yeast Two-hybrid cDNA Library of Fusarium oxysporum

A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.

Screening of Rice Genes Interacting with p5b of Rice BlackStreaked Dwarf Virus

Rice black-streaked dwarf virus （RBSDV） is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA （dsRNA） segments （$1-$10）, in which the fifth genome segment （$5） contains two open reading frames （ORFs） with a partially overlapping region. The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function. To reveal the function of p5b, its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay. The bait protein p5b was detected in yeast by western blot, and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast. Then the bait protein p5b was used for screening the cDNA expression libraries of rice. Gene fragments of some pivotal enzymes involved in photosynthesis, respiration and other important metabolic processes, were identified to interact with p5b in yeast, suggesting that these interactions may play roles in symptom development in infected plants.

Cloning of Rice Blast Resistance Gene Pik_2-H4 and Screening of Its Interaction Protein

Abstract The broad-spectrum rice blast resistance gene Pik2-H4 was cloned from the highly resistant line H4. The full-length sequence of Pik2-H4 was 3 066 bp, which was different from the corresponding allele sequence of Nipponbare. Pik2-H4 belonged to NBS-LRR genes and coded a protein containing NB-ARC domain and leueine-rieh repeats. To find the interaction proteins with Pik2-H4 from the rice, the yeast two-hybrid system was used and three important proteins （ LOC- Os08939300, LOCOs03g25960 and LOC-Os09929130）were identified. The results could provide some new information for the mechanism of rice blast resistance mediated by Pik2-H4.

新疆传统发酵乳制品调研及其发酵微生物种质资源分析

为挖掘新疆传统发酵乳制品的微生物菌种资源,对不同地区传统发酵乳的制作方式及其发酵微生物进行了调研。采集了全疆12个地州及乌鲁木齐周边共45个县165个乡村483份家庭作坊生产的传统发酵乳制品,并对其进行了发酵微生物筛选。从采集的不涉及任何商品发酵剂的新疆传统发酵乳中,共筛选出497株乳酸菌和364株酵母菌,并首次发现了假肠膜明串珠菌、汉逊德巴利酵母和巴氏醋杆菌等菌。此研究成果可为开发新疆传统发酵乳制品的优质菌种资源提供基础。

酸面团中酵母菌的筛选及其在全麦馒头制作中的应用

为充分挖掘酸面团中优良酵母菌菌种,缓解酸面团因微生物复杂而导致馒头品质不一的问题。以自然发酵的酸面团为研究对象,对酸面团中的酵母菌进行初步筛选及发酵特性研究,并将获取的优良酵母菌应用于全麦馒头的制作。结果发现酸面团中有5株(S1、S2、S3、S4、S5)酵母菌菌株,选出特征明显的S1、S2、S5株菌株进行酵母菌产酒精、繁殖能力及发酵能力研究,分析发现S2和S5产酒精能力最强、S1和S5繁殖能力最强、S2和S5发酵能力最强。通过对3株酵母菌的形态特征及发酵特性的综合评价,确定S5为酸面团中的优势酵母菌。将筛选的优势菌S5与安琪酵母分别添加到全麦面团中,并对2种馒头的外表性质进行测定与感官评定,确定S5发酵的全麦馒头的口感和风味优于安琪酵母全麦馒头。由此可知,S5优势酵母菌对全麦馒头风味物质的积累有显著作用,以期为全麦馒头的品质改良提供有价值的菌种资源和技术参考。

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。

高产乙酸乙酯酵母的筛选鉴定及发酵条件优化

该研究通过传统培养分离方法及测定乙酸乙酯产量从清香型白酒大曲中分离筛选高产乙酸乙酯的酵母菌株,通过形态观察及分子生物学技术对其进行菌种鉴定,并对其耐受性进行分析。以乙酸乙酯产量为响应值,采用单因素试验及响应面试验对筛选菌株的发酵条件进行优化。结果表明,分离筛选得到1株高产乙酸乙酯的酵母菌株,编号为YM001,经鉴定,其为异常威克汉姆酵母(Wickerhamomyces anomalus)。该菌株具有较强的耐受性,在乙醇体积分数10%,乙酸含量0.08%、温度50℃、pH 2、乙酸乙酯含量30 g/L、NaCl含量150 g/L、葡萄糖含量50 g/L的条件下仍能生长,能够较好地适应白酒酿造环境。异常威克汉姆酵母YM001产乙酸乙酯的最优发酵条件为:接种量4%、乙醇体积分数4%、乙酸添加量0.02%、初始糖度9°Bx、初始pH值6、转速210 r/min、发酵温度22℃、第2阶段发酵时间36 h。在此发酵条件下,异常威克汉姆酵母YM001的乙酸乙酯产量最高,达(17.39±0.362)g/L。

降解氨基甲酸乙酯酿酒酵母的筛选及其应用

以宁夏5个产区的酿酒葡萄为原料筛选可以降解氨基甲酸乙酯(Ethyl carbamate,EC)且具有优良酿酒特性的红葡萄酒和白葡萄酒酿酒酵母。初筛试验获得了10株酵母,复筛试验以EC作为唯一氮源筛选出6株EC降解能力较好的酵母菌株,最后通过产气试验和耐受性试验筛选出了2株发酵性能较好的菌株。经过18S rDNA分子生物学鉴定后确定这2个菌株为酿酒酵母。使用筛选出的2个菌株发酵红葡萄酒和白葡萄酒,并以商业酵母作为对照,测定其理化指标及EC含量;再通过GC-MS分析其香气物质并结合感官评价最终筛选出具有优良酿酒特性和降解EC功效的红葡萄酒酿酒酵母(Saccharomyces cerevisiae)NXSC2和白葡萄酒酿酒酵母(Saccharomyces cerevisiae)NXSC1。

高产乙酸乙酯酵母菌的筛选、鉴定及发酵条件优化

该研究以产酯酵母为研究对象,通过气相色谱-质谱联用(GC-MS)技术测定菌株的乙酸乙酯产量并筛选高产乙酸乙酯的酵母菌株,通过形态学观察和分子生物学技术对其进行菌种鉴定,并以乙酸乙酯产量为响应值,通过单因素及响应面试验对其发酵条件进行优化。结果表明,筛选得到一株高产乙酸乙酯的酵母菌株P-3,乙酸乙酯产量为134.32 mg/L。菌株P-3被鉴定为库德里阿兹威毕赤酵母(Pichia kudriavzevii),其产乙酸乙酯的最佳发酵条件为:发酵温度30℃,乙醇体积分数18%,发酵时间4 d,在此条件下,乙酸乙酯产量达到281.47 mg/L,比优化前提高109.55%。

黄泡野生酵母菌的筛选鉴定及其发酵性能研究

为筛选黄泡果酒酿造专用优良酵母菌,该研究采用传统培养分离方法结合形态学观察从黄泡发酵液中分离酵母菌株,进一步通过产气、产乙醇、产酯、嗜杀能力及筛选优良酵母菌株并通过分子生物学技术进行鉴定,并以商业酿酒酵母ADT为对照,对筛选菌株的耐受性进行分析。结果表明,分离纯化得到18株酵母菌,从中筛选得到2株优良酵母菌,编号分别为WQ-3和WQ-8。经鉴定,菌株WQ-3和WQ-8分别属于库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)和盔形毕赤酵母(Pichia manshurica)。这2株酵母菌均具有良好的耐受性,均能耐受乙醇体积分数12%、pH 2.5、低温4℃、高温40℃、SO_(2)质量浓度400 mg/L、葡萄糖质量浓度400 g/L,该研究结果为黄泡果酒酿造提供酵母资源。

酵母强化大曲接种量优化及其生产应用

为提高泥坑浓香型白酒中高温大曲综合生产性能,该研究从中高温大曲中分离筛选菌株,采用2,3,5-氯化三苯基四氮唑(TCC)初筛,发酵实验复筛,获得发酵性能良好的酵母菌,并以Z综合得分为响应值,通过单因素试验及响应面试验对筛选菌株接种量进行优化,并将其用于强化大曲生产。结果表明,共分离纯化出8株菌株,其中,3株产酒性能良好的菌株Y1、Y2、Y3,其最佳接种量分别为7.0×10^(8)CFU/mL、3.0×10^(8)CFU/mL、1.5×10^(8)CFU/mL,在此优化复配条件下,主成分Z综合得分为513,所生产的强化大曲发酵力、糖化力和液化力分别为3.46 g/(g·72 h)、994 mg/(g·h)、1.12 g/(g·h),比优化前提高了424%、6.7%和5.7%。

低酸泡菜发酵乳酸菌与酵母菌筛选及其共发酵研究

该研究以分离自不同发酵蔬菜的异型发酵乳酸菌(编号为L1~L6)和酵母菌(编号为M1~M6)为研究对象,通过酸、盐耐受性和发酵性能研究筛选优良菌株,并以自然发酵芥菜为对照,通过优良菌株组合发酵芥菜,考察发酵芥菜的理化性质和感官特性,筛选适用于低酸泡菜的复合发酵菌剂。结果表明,肠膜明串珠菌(Leuconostoc mesenteroides)L5在pH 4.5和4%NaCl含量下耐受性较好且24 h产酸量较少;少孢哈萨克斯坦酵母(Kazachstania exigua)M1、库德里阿兹威毕赤酵母(Pichia kudriavzevii)M2和二孢接合酵母(Zygosaccharomyces bisporus)M4在p H 4.0和4%NaCl含量下耐受性较好且不产气、不产璞。将菌株L5分别和菌株M1、M2、M4组合发酵芥菜时,菌株M1在体系中生长较好,发酵10 d时活菌数达(2.41±0.06)lg(CFU/mL),而菌株M2、M4在发酵4 d后未检出;发酵结束时菌株L5和M1共发酵组乳酸菌活菌数[(5.71±0.02)lg(CFU/m L)],显著低于其他组(P<0.05),总酸含量(2.30±0.03)g/kg和亚硝酸盐含量(1.35±0.02)mg/kg也低于其他两个接菌发酵组,感官评分(85.00±2.12)最高,且有独特坛香味;自然发酵组泡菜未成熟,具有明显的生涩味。