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 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.

MsWRKY33转录调控MsACS2影响紫花苜蓿耐盐性的研究

乙烯作为重要的植物激素之一,在植物逆境胁迫应答中发挥重要作用。ACS基因是乙烯合成过程中的关键限速酶(ACC合成酶)基因,本研究通过探究紫花苜蓿MsWRKY33转录因子对ACS基因的调控关系,为MsWRKY33参与紫花苜蓿耐盐胁迫响应的具体调控机制奠定理论基础。利用同源克隆、载体构建、酵母单杂交、酵母双杂交、qRT-PCR等技术,验证MsWRKY33对AtACS2和MsACS2的转录调控,并对盐胁迫下转基因株系中MsACS2表达模式进行分析。结果显示:MsWRKY33转录因子可以与W-box元件特异性结合,且对AtACS2和MsACS2具有转录调控作用;MsWRKY33转基因株系中MsWRKY33基因的表达量显著高于对照,转基因株系中MsACS2的表达是在MsWRKY33基因大量表达后呈上升趋势,进一步说明紫花苜蓿MsACS2基因的表达可能受MsWRKY33转录因子的正向调控。因此,紫花苜蓿受到盐胁迫时可诱导MsWRKY33的表达,MsWRKY33转录因子通过正向调控MsACS2表达,可能影响乙烯合成,从而影响紫花苜蓿的盐胁迫响应。

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.

芍药种子休眠解除过程的酵母杂交cDNA文库构建

【目的】芍药具有极佳的观赏价值,但其种子的双重休眠难以解除,严重阻碍芍药的育种进程。本研究利用gateway技术构建芍药种子休眠解除关键时期的酵母杂交cDNA文库,可用于筛选调控芍药种子休眠解除相关基因的转录因子及互作蛋白。本文库的构建可为丰富芍药种子休眠调控研究提供科学支撑,能为芍药栽培育种和野生资源保护及利用奠定分子理论基础。【方法】本研究以休眠解除过程中5个关键时期的芍药种子为试验材料,进行总RNA的提取和m RNA的分离,使用gateway方法构建cDNA初级文库,再通过LR重组,将初级文库重组到pGADT7-DEST次级文库载体上,构建出次级文库,最后经过酵母转化试验,将次级文库质粒转化到酵母Y187感受态细胞中,构建出芍药种子休眠解除过程的酵母文库。【结果】经鉴定,cDNA初级文库库容量为1.28×10^(7) CFU,平均插入片段长度在1000 bp以上,重组率为100%;cDNA次级文库库容量为1.12×10^(7) CFU,平均插入片段长度在1000 bp以上,重组率为100%;酵母文库滴度为7.0×10^(7) CFU/mL,平均插入片段长度在1000 bp以上,重组率为96%。23个阳性克隆测序结果经NCBI比对后,按照功能将20个已知蛋白序列划分为8类,其中7个序列已被报道参与种子的休眠与萌发。【结论】芍药种子休眠解除过程的酵母杂交cDNA文库构建的质量较高,具有较完整的基因信息,符合酵母文库筛选试验所需的标准,为构建芍药种子休眠解除过程的分子调控网络提供基础。

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.

分子生物学技术在酵母菌多相分类鉴定中的应用

随着分子生物学的发展和各项新技术的广泛应用,许多新的分子生物学技术被用来进行酵母菌的分类和鉴定。核酸杂交技术、限制性酶切片段长度多态性分析(RFLP)、随机扩增多态性DNA分析(RAPD)、脉冲电泳核型分析(PFGE)、rDNA序列分析、单链构象多态性分析(SSCP)等技术的应用为酵母菌的多相鉴定带来了突破性的进展,结合分子生物学技术的多相鉴定体系将成为酵母菌分类鉴定的最有效手段。

红曲的生产工艺及Monacolin K和桔霉素的检测

综述红曲的生产工艺及MonacolinK和桔霉素的检测方法,指出采用新型生产工艺、优化发酵条件和应用菌种筛选等现代技术是生产既富含活性成分MonacolinK又不含桔霉素的功能性红曲的重要途径,同时预示MonacolinK和桔霉素检测技术的进展将为功能性红曲产品的质量、安全使用及国际市场的开拓提供技术支撑。

仙人掌多糖发酵提取工艺优化及其抗炎功效研究

本文分别采用乳酸菌与酵母菌发酵提取仙人掌多糖,并探究仙人掌多糖的抗炎功效。在三个单因素液料比、时间、pH方面探究仙人掌多糖的提取工艺,得到三因素最适组合,乳酸菌发酵最适提取工艺为发酵时间7 h、液料比25∶1 (mL/g)、pH为5,此工艺下多糖得率为0.26%。黄酒酵母发酵最适提取工艺为发酵时间48 h、液料比25∶1 (mL/g)、pH为3,此工艺下多糖得率为0.53%。应用实时荧光定量PCR法检测仙人掌多糖对IL-6、IL-8炎症细胞因子相关基因表达的影响,得知仙人掌多糖通过抑制IL-8的表达,促进IL-6的表达来抑制炎症的发生和发展。

活性干酵母与酵母培养物对体外瘤胃发酵参数影响的比较

本研究采用体外产气量法,以全日制混合日粮(TMR,精粗比60∶40)为发酵底物,比较了两种典型酵母制剂活性干酵母(active dry yeast,ADY)和酵母培养物(yeast culture,YC)对体外产气量、产气动力学参数、甲烷(CH4)含量及瘤胃发酵参数的影响,同时根据24h产气量预测了酵母制剂对底物代谢能(ME)、有机物消化率(OMD)及微生物蛋白(MP)产量的影响,旨在比较二者在体外瘤胃发酵影响上的差异,为进一步揭示其应用效果的差异机制及其合理应用奠定基础。结果显示,ADY及YC组产气量相近,但YC组12、24、48h的产气量均高于ADY组;ADY组发酵前期的产气速率较对照组降低了15.3%(P>0.05),YC组较对照组升高了19.4%(P>0.05),YC组较ADY组升高了41.0%(P<0.05);与对照组相比,YC显著增加了CH4气体含量(P<0.05),显著降低了H2及CO2的含量(P<0.05)。ADY显著降低了H2含量(P<0.05),但对CH4及CO2气体无显著影响(P>0.05);与对照组相比,ADY和YC对发酵液的pH、总挥发性脂肪酸均无显著影响(P>0.05),但均显著升高了发酵液中氨态氮(NH3-N)的浓度(P<0.05)。ADY对个别挥发性脂肪酸比例无显著影响(P>0.05),YC有升高乙酸比例、降低异丁酸比例的趋势;与对照组相比,ADY及YC对预测的底物OMD、ME及MP产量无显著影响(P>0.05),但YC组各预测指标均显著高于ADY组(P<0.05)。综上所述,YC与ADY在对发酵底物的体外瘤胃产气量、气体成分含量、产气速率及发酵参数的影响上存在差异。

体外法评价活性酵母及其发酵饲料对瘤胃发酵参数的影响

本研究选择7株酿酒酵母菌(Saccharomyces cerevisiae,编号为XR4、YL5、YN7、XL8、BC、SL、AQ)、5株产朊假丝酵母菌(Candida utilis,编号为SC18、SR12、YJ2、YD6、BY)及1株热带假丝酵母(Candida tropicalis,编号为BR),采用体外产气法研究其对体外瘤胃发酵参数的影响。结果显示,与对照组相比,活性酵母菌株对产气量均未产生显著影响(P>0.05);酵母发酵饲料SC18、XR4、BY、AQ和BC组产气量显著升高(P<0.05),且这5组产气量增长率均优于其对应的活性酵母组(P<0.05)。13株酵母菌中的10株活性酵母及其发酵饲料均能使菌体蛋白含量显著增加(P<0.05),且大部分活性酵母菌蛋白增长率均高于其对应的发酵饲料(P<0.05)。活性酵母及其发酵饲料对氨态氮含量均未产生明显影响(P>0.05)。活性酵母YD6、BR、SC18、YL5、XR4、SR12、BY、BC组总挥发性脂肪酸含量显著高于对照组(P<0.05),酵母发酵饲料YD6、BR、YJ2、SC18、SR12、SL、YL5、XR4组总挥发性脂肪酸含量显著高于对照组(P<0.05)。综上所述,活性酵母可显著提高菌体蛋白含量及挥发性脂肪酸含量,其中BY、XR4、BC效果更佳;酵母发酵饲料可显著提高产气量、菌体蛋白含量、挥发性脂肪酸含量,其中SC18、BY、BC效果更佳;酵母发酵饲料在增加产气量、总挥发性脂肪酸含量上效果要优于其活性酵母。

石榴果酒酵母的分离及发酵工艺

为了得到发酵石榴汁的优良菌种,并确定石榴酒发酵的工艺参数,从自然发酵的石榴酒醪中分离、纯化石榴酒酵母菌种;通过单因素和正交试验,确定石榴酒发酵的工艺参数。结果发现,分离纯化的菌种经形态特征和26S rDNA D1/D2区域序列分析鉴定为酿酒酵母。确定的最佳工艺条件为:温度28℃、pH3.8、糖度为17%、二氧化硫的添加量为100mg/L,最终发酵的石榴酒的酒度在10%VoL左右。

结肠癌羟基喜树碱耐药细胞SW1116/HCPT酵母双杂交cDNA文库的构建和鉴定

目的:构建羟基喜树碱耐药结肠癌细胞株SW1116/HCPT细胞的酵母双杂交cDNA文库.方法:用TRIzol从SW1116/HCPT细胞提取总RNA,采用CLONTECH SMART(switching mechanism at 5' end of RNA transcript)技术和同源重组(homologous recombination)的方法构建SW1116/HCPT细胞的cDNA文库.结果:提取的RNA的A260/A280为1.98,甲醛变性琼脂糖凝胶电泳示出28SrRNA、18SrRNA及5SrRNA3条特异性带,28S与18S带浓度及亮度比值约为2.文库dscDNAsmear较长分布在0.1-5kb,成长瀑布条带,而且在接近1kb的区域有密集的带子,为高丰度表达基因.依据生长菌落计数,共获得(1.2-1.9)×106个转化子,重组率达93.7%,文库插入片段大小为0.2-5kb.结论:成功构建了SW1116/HCPT cDNA文库.

控制上面发酵小麦啤酒中高级醇含量的研究进展

高级醇是啤酒中主要的风味物质,适量的高级醇能赋予啤酒丰满的口感,增加酒体的协调性,高级醇过量则会给啤酒带来异杂味并引起人体某些疾病。该文对高级醇的性质,测定方法,在啤酒中的代谢途径,影响其含量的因素以及降低上面发酵小麦啤酒中高级醇含量的研究情况进行了综述。

乳酸菌和酵母菌复合发酵南瓜汁工艺研究

研究乳酸菌和酵母菌复合发酵南瓜汁的工艺,采用正交设计、感官评价等方式进行发酵南瓜汁工艺参数的优选、稳定剂和甜味剂的筛选。结果确定发酵种子培养基配方为:南瓜浆含量40%,葡萄糖2%,豆浆5%和碳酸钙0.4%。南瓜汁最佳发酵工艺为发酵温度36℃,酿酒酵母SY∶乳酸菌群=1∶3(体积比),接种量3%,发酵12 h后南瓜汁中乳酸菌的浓度可达9.20 lg(CFU/m L)。

白癜风患者郎格罕细胞的光、电镜研究

白癜风患者郎格罕细胞的光、电镜研究杜娟①朱铁君①郎格罕细胞（ＬＣ）是抗原呈递细胞，在许多皮肤病的发病中占重要地位，很多文献也报道了ＬＣ在白癜风皮损中的数量变化，但结论很不一致。本研究旨在通过严格的自身平行对照，观察白癜风患者白斑、白斑边缘及远离白斑处...

糯米稠酒发酵工艺的优化研究

通过单因素试验和正交试验对稠酒的生产工艺进行优化,确定了稠酒的最佳工艺条件。结果表明,稠酒生产的最佳液化条件为添加1.0%的液化酶在40℃作用120min;最佳糖化条件为添加0.5%的糖化酶在30℃糖化24h:复合酵母的最佳配比:甜酒曲:黄酒酵母:葡萄酒酵母为0.8%:0.05%:0.1%;最佳的发酵条件为发酵温度30℃,加水量1倍,发酵时间72h,组合酵母按比例添加甜酒曲0.64%、黄酒酵母0.04%、葡萄酒酵母0.08%。在此条件下生产的稠酒风味独特,具有浓郁的醇香滋味。