The Self-activated Experimental of T_(1083) Substitution Mutation Vector pGBKT7-TS in Yeast

[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.

Detection for Transcriptional Activity of Alternaria Tenuissim Protein Elicitor in Yeast Two-hybrid System

The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.

Screening of Extracellular Binding Proteins of Rice Receptor-like Kinase CR4 by the Yeast Two-hybrid

[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling which was cultivated 14 d.[Result] A lot of proteins which included a peroxide B（D26484）,a methionine thioredoxin reductase（ABF96078）and an unknown function protein were gained.[Conclusion] It provided the theory basis for studying the signal transduction mechanism of CR4.

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

AIM： To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS： We constructed p7 protein bait plasmid by cloning the gene of p7 protein into pGBKTT, then transformed it into yeast AH109 （a type）. The transformed yeast was mated with yeast Y187 （α type） containing liver cDNA library plasmid, pACT2 in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/- Trp-Leu-His-Ade） containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics. RESULTS： Fifty colonies were selected and sequenced. Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nudeoporin 214 ku and two colonies were CLL-associated antigens. CONCLUSION： The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its assodated protein.

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.

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 FOXP3-interacted proteins by yeast two-hybrid technique

Objective： To screen the proteins interacting with the Treg specification factor forkhead box protein P3 （FOXP3） by yeast two-hybrid system, Methods： Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells （PBMC） and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results： The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Conclusion： Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.

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.

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.

酵母菌混合发酵乳清液及其乙醇发酵阶段工艺优化

选择马克斯克鲁维酵母CGMCC13907(以下简称马氏酵母CGMCC13907)和酿酒酵母AS2.119为混合发酵剂发酵乳清液制备乳清醋,并采用Plackett-Burman Design(PBD)和Box-Behnken Design(BBD)试验优化乙醇发酵阶段的工艺。结果表明:乙醇发酵阶段的最优工艺为马氏酵母CGMCC13907接种量4.0×10^(6) CFU/mL,酿酒酵母AS2.119接种量4.5×10^(6) CFU/mL,发酵时间151 h,发酵温度40.0℃,发酵pH值5.6,姜粉用量0.09 g/100 mL,在此条件下,发酵液中乙醇浓度可达8.70%vol,比优化前增加了56.2%。因此,选用马氏酵母CGMCC13907结合酿酒酵母AS2.119混合发酵乳清液,可有效解决乳清醋制作过程中的乳糖发酵难题,并获得较理想的乙醇浓度。

Screening and Identifying of Interaction Protein AtL5 in Arabidopsis thaliana

Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.

Interaction between MAPKs and MKPs in hexaploid chrysanthemum illuminates functional paralogue diversification in polyploids

Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.

甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1)cDNA的克隆和功能鉴定

磷脂二酰甘油酰基转移酶（phospholipids：diacylglycerol acyltransferase,PDAT1）是植物三酰甘油（triacylglycerol,TAG）合成的关键酶。本文在甘蓝型油菜湘油15号c DNA中克隆到3个PDAT1全长编码序列（coding sequence,CDS）,经比对分别定位于A02、A10、C09染色体,分别命名为Bn PDAT1-A02、Bn PDAT1-A10和Bn PDAT1-C09,其序列长分别为1998、2002和2005 bp,各自编码665、666、667个氨基酸。预测Bn PDAT1基因编码蛋白定位于细胞质膜,具有典型的PDAT1保守结构域。多序列比对和进化分析表明,Bn PDAT1基因编码蛋白与甘蓝、拟南芥、亚麻芥PDAT1蛋白具有较高的同源性。酵母互补实验证实,该基因编码蛋白具有PDAT1酶活性。Bn PDAT1基因在湘油15号中的表达现先上升后降低趋势,在开花后25~30 d达最大值,但3个拷贝的表达变化规律存在差异。

酒曲中产酯酵母的筛选及培养条件的研究

采用稀释涂布法从酿酒用曲中筛选到16株产酯能力强、形态各异的酵母菌株,从中优选产酯量最高的1株酵母菌为研究对象,进行产酯条件的研究。通过正交试验,确定较优菌株生长的最适培养条件为温度26℃、pH值为7、培养时间18h、葡萄糖含量为4%(w/v)。对产酯酵母进行耐酒精试验结果表明,最高耐受酒精度为6%vol,有望将其应用到果酒生产中提高果酒品质。

酵母菌Y17吸附Cu^(2+)的影响因素及吸附机理研究

以高效吸附Cu2+的酵母菌Y17为材料,对其吸附Cu2+过程中的主要影响因素,包括溶液pH、Cu2+初始浓度、菌体添加量、吸附时间和温度以及吸附机理进行了探讨。结果表明,对吸附过程影响较大的因素依次为吸附液pH值、Cu2+初始浓度、菌体添加量和吸附时间。正交试验得到最佳吸附条件为溶液pH5.0,吸附时间40min,加菌量5.0g湿菌/L时,对初始浓度为8mmol/L的Cu2+达到最佳吸附率为82.7%。通过对Y17菌体不同处理及解吸实验,初步确定Y17吸附Cu2+的位点在细胞壁,细胞壁表面的-NH2,-COOH基团在其吸附过程中起着重要作用。

酵母胞外槐糖脂产生条件优化及其抑菌作用

通过单因子试验和正交试验对筛选得到的1株未见报道的产胞外槐糖脂的酵母菌Y2A菌株的培养基组成及培养条件进行了优化.结果表明,优化的最适产槐糖脂培养基配方为:质量分数6.0%葡萄糖、体积分数8.0%菜子油、质量分数0.2%酵母粉;优化后发酵条件为:初始pH值6.0、接种量体积分数2.5%、发酵时间120h.在此条件下,槐糖脂产量达到39.4gL.然后发酵液经分离纯化并制备,得到纯品槐糖脂.将槐糖脂进行抑菌试验的研究结果表明,它对某些试验菌株的生长有抑制作用.

选育耐高温酒精酵母的研究

通过采用耐热、耐乙醇等处理方法对酒精酵母KJ进行驯化、筛选,分离得到一株于38 ～40℃具有良好发酵特性的耐高温酒精酵母菌KJ-1,并通过正交试验获得了优化的发酵条件。

复合载体固定化酵母生物合成胞二磷酸胆碱

研究了复合载体固定酵母的条件 ,通过正交实验确定了K 卡拉胶 ,魔芋多糖和去离子水用量 .用扫描电子显微镜观察了固定化酵母冷冻前后的形态变化 ,固定化酵母多次使用后 ,其生物合成胞二磷酸胆碱 (CDPC)