Improved methods for cloning and detection in the yeast two hybrid assay

The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning methods were made in the past two decades, the conventional cloning method of restriction-enzyme (RE) digestion followed by ligation is still widely used. Unfortunately, many researchers, especially new-comers, often encounter difficulties in cloning a gene into a desired vector. Secondly, interaction between two proteins is commonly detected by growth of the diploids in specific media. This step takes about two weeks. Here, we describe improved cloning and detection procedures for the Y2H assay that accelerate the research progress. The changes in procedures involve running an agarose gel after the doubly digested vector and insert are ligated in the cloning step to determine the efficiency of RE digestion and ligation, and performing an additional replica-plating on plates for earlier assessment of interaction in the detection step. We show an example of Y2H interaction between Trs23 and Trs120 (respective subunits of TRAPP I and TRAPP II), as a proof of concept. By following the improved methods described here, the chances of successful cloning increased and the time for the whole Y2H experimental process is significantly shorter.

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.

禾虫酶解液脱腥的工艺优化

通过比较活性炭、绿茶、酵母3种不同脱腥处理对禾虫酶解液的脱腥效果,研究最佳的脱腥工艺。以禾虫酶解液为主要原料,进行活性炭吸附脱腥处理、绿茶掩盖脱腥处理、酵母发酵脱腥处理后,测定禾虫酶解上清液的澄清度、蛋白损失率,进行腥味感官评价,根据感官评价的模糊数学法评定出3种脱腥处理后腥味去除效果的强弱顺序。在此基础上,应用正交试验探究脱腥组合的禾虫酶解液脱腥效果,最终确定禾虫脱腥的最优工艺。结果表明,绿茶的脱腥效果最好,但蛋白质损失率较高;酵母的脱腥效果次之,而蛋白质损失率最低;活性炭的脱腥效果差且蛋白质损失率最高。对比绿茶和酵母的正交试验结果,确定禾虫酶解液最佳的脱腥方法是酵母处理,最优工艺为酵母用量0.6 g,温度35℃,时间30 min,经该工艺处理后,得到的禾虫酶解上清液澄清透亮、腥味较小、蛋白质损失率低。

梯度升温发酵工艺对不同盐分稀态发酵酱油微生物变化和风味形成的影响

本文以12%和18%(w/v)两种盐分浓度的稀态发酵酱醪为研究对象,探究梯度升温(15℃-20℃-25℃-30℃)工艺对发酵酱醪微生物计数和风味形成的影响。结果显示:采用梯度升温工艺的低盐分酱醪样品(TC12)的酵母计数在发酵到期时比恒温(30℃)对照组(C12)要高1.0 log CFU/g左右,这表明酵母在梯度升温工艺中维持生长代谢时间较长。这或与发酵前期较低的温度限制了酵母过快的繁殖有关。其中,TC12的发酵到期原油的氨基酸态氮高达1.14 g/100 g,达到我国规定的特级酿造酱油标准。同时,TC12中的鲜味(19.03 g/L)和甜味氨基酸(19.32 g/L)含量较高,共占氨基酸总量约60%,呈现较好的鲜甜滋味。挥发性物质检测数据显示,TC12中的乙醇、苯乙酸乙酯、棕榈酸乙酯、3-甲硫基丙醛和苯甲醛等含量较高,样品整体呈现“水果甜香和酯香”。另一方面,高盐组梯度升温样品(TC18)则测得更多的愈创木酚和甲基麦芽酚,呈现“烟熏香和酱香”。感官鉴评数据显示,低盐分梯度升温发酵样品TC12相比恒温发酵组C12具有更高的香气和滋味得分,综合口感更优。综上所述,梯度升温工艺有利于限制低盐分(12%)发酵酱醪中酵母在前期的过快生长,保持其在发酵后期的活性细胞计数,使其能持续发酵生成更多风味物质,提升低盐发酵原油的整体风味。

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.

食品中霉菌和酵母能力验证的方法比对与结果分析

检验实验室质量管理体系运行情况,总结实验过程中影响霉菌和酵母计数的因素,确保检测数据的准确性。参照能力验证参考指导书和GB 4789.15—2016,以不同的实验人员和不同的培养基进行比对,对能力验证样品进行检验和计数,把测试结果换算成对数形式,计算Z值,对实验室能力进行评价。结果表明,编号为22-C203样品上报的结果为4 200 CFU/mL,22-V106上报的结果为410 CFU/mL,取得的Z值分别为0.1和-0.4,能力验证结果为满意。表明实验室具备食品中霉菌和酵母计数的检测能力,提升了实验室内部体系管理水平,也为之后参加此项能力验证的实验室提供参考依据。

不同产香酵母接种方式对甜面酱品质的影响研究

通过向甜面酱保温发酵过程中添加不同种类(鲁氏酵母和异常威克汉姆酵母)和不同比例(1∶2、1∶1、2∶1)的耐盐产香酵母,对比不同产香酵母接种方式对甜面酱品质及风味的影响。结果表明,先添加鲁氏酵母后添加异常威克汉姆酵母,且二者比例为1∶1时,更有利于氨基酸态氮和有机酸的积累,保温发酵结束后,产品的还原糖含量为20.02 g/100 g,总酸含量为2.21 g/100 g,氨基酸态氮含量为0.49 g/100 g,有机酸含量为5.61 mg/g。产品中共检测出挥发性风味成分45种,含量为84.36×10^(-2)μg/g。综合理化指标及风味成分指标,甜面酱保温发酵过程中最优的酵母添加方式为先添加鲁氏酵母后添加异常威克汉姆酵母,且二者添加比例为1∶1时更有利于保温发酵甜面酱品质和风味的提升。

补氮方式对酿酒葡萄转色期生理调节及品质的影响

氮肥在促进酿酒葡萄营养生长的同时还能有效调节其生殖生长,补氮时期和补氮方式对于协调葡萄营养生理和品质构成的关系尤为重要,尤其在葡萄品质形成最为关键的转色期进行氮素调控对浆果品质组分的影响最为显著。以8年生酿酒葡萄品种“赤霞珠”为研究对象,在葡萄转色期内以清水为对照,设置叶面喷施、滴灌补氮、喷滴结合和减量补氮4种补氮方式,研究补氮方式对葡萄生理、品质和酵母可同化氮的影响。结果表明:与喷施清水相比,4种补氮方式均能提高葡萄果实的果形指数;滴灌补氮能有效增加叶片叶面积、水分利用率和叶绿素b含量,其中水分利用率显著提高81.53%;葡萄浆果的单宁、花色苷和总酚提升效果在叶面喷施氮素时分别显著提高11.69%、99.19%、21.60%;减量补氮可以显著促进叶绿素的合成与积累,同时可溶性固形物显著增加16.15%;喷滴结合提升叶片净光合速率、类胡萝卜素和酵母可同化氮含量的效果最佳,分别显著增加35.15%、38.46%和53.04%,同时百粒重显著提高44.88%。综合评价发现,在贺兰山东麓葡萄酒产区通过叶面喷施和滴灌追施的补氮方式是调节酿酒葡萄生理、改善葡萄浆果品质的有效途径。

6种益生菌及其添加方式对发酵米酒品质的影响

目的:探究不同益生菌及其添加方式对米酒制作效果的影响。方法:以糯米为原料,通过外源添加鼠李糖乳杆菌BV-77、发酵乳杆菌TSF-331、唾液乳杆菌AP-32、约氏乳杆菌MH-68、嗜酸乳杆菌TYCA-06、干酪乳杆菌CS-773酿制益生菌米酒,对比只添加糖化酶、同时添加糖化酶和酵母以及添加方式(益生菌麸曲和益生菌菌粉)对米酒酒精度、还原糖、总酸和感官质量的影响。结果:酵母对益生菌米酒的发酵有促进作用,可显著提高米酒酒精度和还原糖含量。利用发酵乳杆菌菌粉同时添加用量为200 U/g的糖化酶和0.04%的酵母,在30℃发酵3 d的米酒发酵效果最佳,酒精度和还原糖含量达到最高,分别为7.20%vol和270.00 mg/mL,总酸含量达到最低,为8.40 mL/L,酿造出的米酒口感较为浓郁。结论:发酵乳杆菌是6种益生菌中最适宜酿造米酒的益生菌。

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.

茅台镇不同海拔酒厂环境可培养酵母多样性分析

以中国酱香酒核心产区仁怀市茅台镇为研究区域,探索高温大曲和环境中可培养酵母的物种多样性,以及在不同海拔下的种群结构和相对丰度。结果显示,从高温大曲和环境样品中最终分离474株酵母,鉴定为21属32个种,其中从环境样品中共分离269株酵母,鉴定为19属28个种;从大曲中共分离205株酵母,鉴定为15属18个种。Wickerhamomyces anomalus在环境和大曲中均作为核心酵母菌。环境中分离出的酵母菌多样性指数高于高温大曲。在不同海拔梯度上,环境样品中酵母多样性和丰度差异明显,高温大曲中酵母多样性和丰度差异较小,主要的优势菌群均保持一定的相似性和稳定性。总的来说,海拔500 m以上的酵母菌群落结构较为相似;海拔500~600 m的酵母菌群相对丰度及多样性最高,海拔600 m以上酵母多样性及丰度要低于海拔500 m以下。

酵母种类和发酵方式对冻融前后冷冻熟制空心面品质的影响

为开发一款品质优良的冷冻熟制空心面(frozen cooked hollow noodles,FHN),考察活性即发干酵母(instant dry yeast,I)和半干酵母(semi-dry yeast,S)两种酵母种类,以及直接发酵法(F1)、液态预发酵法(F2)、面絮发酵法(F3)3种发酵方式对冷冻熟制空心面蒸煮品质、质构特性和感官评分等指标的影响。结果表明:酵母种类和发酵方式对冻融前后冷冻熟制空心面的蒸煮品质、质构特性均有显著影响(P<0.05)。冻融前,采用S-F2的FHN吸水率最高,为160.76%;采用I-F2的FHN蒸煮损失率最少,为3.55%;采用S-F2的FHN硬度最高,为3570.68 g。冻融10次后,采用I-F3的FHN吸水率最高,为259.10%,采用S-F3的FHN蒸煮损失率最小,为1.70%,且硬度最高,为2398.80 g。

食品中霉菌与酵母菌计数能力验证结果的分析探讨

本研究依据GB 4789.15—2016 《食品安全国家标准食品微生物学检验霉菌和酵母计数》以及ACAS—PT1588(2023)食品中霉菌和酵母计数能力验证(第一轮)参试指导书,对2份样品分别进行稀释、培养、计数。结果表明,营口市食品药品检验检测中心实验室(下文简称“本实验室”)具备食品中霉菌和酵母菌的检测能力。

贵州传统小曲酵母菌分子指纹图谱分析

采用26S rRNA基因D1/D2区域序列分析方法将59株分离自贵州5个不同地区传统小曲的酵母菌鉴定为6个酵母菌种:1株阿萨丝孢酵母(Trichosporon asahii)、32株扣囊覆膜孢酵母(Saccharomycopsis fibuligera)、8株Saccharomycopsis malanga、4株伯顿丝孢毕赤酵母(Hyphopichia burtonii)、6株异常威克汉姆酵母(Wickerhamomyces anomalus)和8株酿酒酵母(Saccharomyces cerevisiae)。本研究展示了6个酵母菌种的WL培养基菌落特征和5.8S rRNA基因ITS区限制性片段长度多态性酶切图谱特征,为这6个菌种的快速鉴定提供依据。在菌种鉴定的基础上,本研究进一步采用串联重复-tRNA指纹图谱法分析酵母菌的种内差异,共展示了17个基因型,H.burtonii、W.anomalus与S.fibuligera分别鉴定出4、3种和8种基因型,其中S.fibuligera基因型10、11、12、15、16与17之间的遗传关系较近,而其余菌种内部基因型之间的遗传关系相对较远。

不同发酵方式对骏枣果脯品质的影响

以阿克苏产新鲜骏枣为原料,分别采用接种乳酸菌、酵母菌、乳酸菌与酵母菌混合菌发酵及自然发酵4种方式进行发酵,以发酵过程中样品的pH值、总酸、总糖、还原糖、总酚含量的变化为评价指标,研究不同发酵方式对果脯品质的影响,以筛选出最佳发酵方式。结果表明:随着发酵时间的延长,4种发酵方式pH值变化不明显,总糖含量呈下降趋势,还原糖含量整体呈增加趋势。发酵结束时,自然发酵样品的总酸含量最低,为0.29%,总糖含量最高,为24.22%;混合菌发酵样品的总糖与还原糖均最低分别为19.69%、3.20 g/100 g,总酚含量最高,为47.83 mg/g,总酸含量为0.45%,低于乳酸菌发酵,高于其它2种发酵方式。综上,4种发酵方法中,混合菌发酵枣脯总糖含量最低、总酚含量最高、品质最优,是枣脯较为理想的发酵方式。

响应面法优化磁性酵母微球对废水中Cr的吸附

酵母菌被广泛用于重金属废水处理,为了提高酵母菌的吸附效率,合成制备了一种磁性酵母微球,并将其运用于含铬废水的处理。以铬的吸附率位响应值,首先通过单因素实验,发现投加量,pH和温度3种因素为主要影响磁性酵母微球对铅去除的因素,进而采用响应面分析法研究了这3种因素对磁性酵母微球对铬的吸附交互影响。结果表明磁性酵母微球对初始浓度10 mg/L的Cr废水的最佳去除工艺条件为初始投加量154.02颗/10 mL,pH=6.13,温度为33.43℃,该条件下磁性酵母微球对Cr的吸附率可达96.87%,使Cr废水达到国家排放标准。因此,磁性酵母微球可作为一种高效吸附剂用于低浓度Cr废水的治理。