Screening of proteins interacting with activation domain of macrophage migration-inhibitory factor

AIM: To screen the proteins that interact with the catalytic thiol-protein oxidoreductase (TPOR) domain of macrophage migration-inhibitory factor (MIF). METHODS: Yeast two-hybrid system was used to study the proteins that interacted with MIF. The bait vector pBTM116-MIF was constructed and transfected into L40 yeast strain. L40 competent cells expressing the TPOR domain were prepared and used to screen the proteins interacting with the TPOR domain of MIF in a human osteosarcoma cDNA library. The proteins interacting with TPOR domain were identified by HIS3 reporter gene and β-galactosidase assay. The techniques of co-immunoprecipitation and immunofluorescence were also used to verify the proteins interacting with TPOR domain. RESULTS: Wild-type pBTM116-MIF and pACT2-TXNL2 (thioredoxin-like 2 protein) were constructed and co-transfected into L40 yeast strain. The activation of HIS3 reporter gene and the positive result of β-galactosidase assay indicated that TXNL2 was the candidate protein that interacted with the catalytic TPOR domain of MIF. The vector of pcDNA3.1-Myc-TXNL2 was constructed and transfected into MCF-7 cell line. The result of co-immunoprecipitation assay showed that the catalytic TPOR domain of MIF co-immunoprecipitated with Myc-TXNL2 protein. The result of immunofluorescence assay demonstrated that the catalytic TPOR domain of MIF and TXNL2 co-localized in the cytoplasm of the cells. CONCLUSION: TXNL2 is the protein that interacts with the catalyzed TPOR domain of MIF.     

Interaction of NR2D subunit of NMDA receptor with MOCA

AIM: To identify the potential proteins interacting with NR2D subunit of NMDA receptor by yeast two-hybrid screening and to investigate the role of NR2D in excitotoxicity of the retina.METHODS: The Clontech GAL4 yeast two-hybrid system was used to screen the mouse brain cDNA library, and the bait plasmid containing C-terminus of NR2D was constructed. Physical interaction between 2 proteins was verified by co-immunoprecipitation assay. The subcellular localization of 2 proteins in the mouse retina was observed under microscope with immunofluorescence.RESULTS: Modifier of cell adhesion (MOCA) was identified as a new protein interacting with NR2D. MOCA and NR2D were co-expressed in the mouse retina. CONCLUSION: MOCA specifically interacts with NR2D, which provides the experimental basis for identifying the role of glutamate excitotoxicity in the retina neurodegeneration.     

酵母铬对热应激蛋用种公鸡精液品质的影响及作用机制研究

选用72只35周龄罗曼种公鸡随机平均分成6组,对照组饲喂基础日粮,分别在基础日粮中添加0.2、0.4、0.6、0.8、1.0 mg/kg铬量的酵母铬,作为试验I、II、III、IV、V组来探讨酵母铬对热应激蛋用种公鸡精液品质及内分泌机能的影响.结果表明:与对照组相比,日粮中添加0.4、0.6、0.8、1.0 mg/kg铬量的酵母铬均不同程度提高公鸡的精液量、精子活力和精子密度,降低公鸡精液的精子畸形率,而对精液的pH值影响不明显;试验各组公鸡血浆三碘甲腺原氨酸(T3)水平均有不同程度升高;皮质醇、甲状腺素 (T4)水平降低.上述结果表明:在热应激条件下,日粮中添加一定量的酵母铬可有效缓解热应激,显著提高夏季蛋用种公鸡的繁殖性能并影响其内分泌机能.     

Puroindoline-a and puroindoline-b interact with the Saccharomyces cerevisiae plasma membrane through different amino acids present in their tryptophan-rich domain

Puroindolines are two small, basic cysteine-rich proteins isolated from Triticum aestivum seeds and characterized by a tryptophan-rich domain. They form the molecular basis of wheat grain hardness and display antimicrobial activity that may contribute to plant defence. Their antimicrobial activity is presumed to be due to their hydrophobic tryptophan-rich domain. However, little is known about their mode of action and there is no in vivo evidence that the binding of puroindolines to membranes is mediated by their tryptophan-rich domain. In this study, using a yeast complementation assay, we showed that puroindolines interact with the Saccharomyces cerevisiae plasma membrane. By site-directed mutagenesis of their tryptophan-rich domain, we determined that two tryptophan residues (W41 and W44) are mandatory for interaction of puroindoline-a with the yeast membrane whereas interaction of puroindoline-b depends on lysine residues. These results highlight that other residues than tryptophan play a critical role in the interaction of puroindolines with membranes, and probably their affinity for lipids and antimicrobial activities.     

核蛋白筛选系统(NTT)的建立及鉴定

为了研究植物核蛋白的组成及其在各种环境条件下的动态变化,利用核蛋白的核定位信号(nuclear localization signals,NLS)筛选编码核蛋白的基因,建立了一套快速、省力、低成本的核蛋白筛选系统(nuclear transportation trap,NTT).通过鉴定发现,将合成的SV40蛋白大T抗原的核定位信号序列插入筛选载体的多克隆位点,转化酵母EGY48,插入核定位信号的筛选载体转化酵母后可以在选择性培养基(SD/His-/Leu-)上生长,而没有插入核定位信号的筛选载体转化酵母后不能在选择性培养基上生长,从而证明此核蛋白筛选系统有效.比较实验表明,本研究构建的核蛋白筛选系统与已报道的系统相比筛选效率更高.利用此系统从cDNA文库中分离编码核蛋白的基因,对于研究核蛋白的动态组成、了解核蛋白基因在调控细胞发育及其环境胁迫条件下的作用机制具有重要的意义.     

巴西橡胶树乳管细胞HblMYC3互作蛋白筛选与功能分析

MYC是b HLH转录因子家族的亚家族成员,在植物茉莉酸信号转导过程中发挥着重要的调节作用。Hbl MYC3是从巴西橡胶树的乳管细胞中分离鉴定到的MYC类转录因子,其基因表达受割胶和茉莉酸上调。采用酵母双杂交方法初步筛选Hbl MYC3蛋白的互作蛋白,旨在进一步了解Hbl MYC3的功能。结果表明:Hbl MYC1、Hbl MYC2、DNAJ蛋白、谷氧还蛋白2、含A20和AN1锌指结构域的胁迫相关蛋白5、28 ku热和酸稳定的磷蛋白以及25 ku泛素连接酶E2等7种蛋白不同程度地与Hbl MYC3蛋白互作。基于这些互作蛋白的功能,推测Hbl MYC1或Hbl MYC2通过与Hbl MYC3形成二聚体对小橡胶粒子膜蛋白基因表达进行调控,其他蛋白参与胁迫条件下维持二聚体的稳定性和胁迫反应后降解该二聚体。     

小麦转录因子TaMYB5 like转录自激活能力及其参与生理型花粉败育过程表达模式研究

化学杂交剂SQ-1诱导的小麦花粉败育是一个复杂的生理代谢过程,TaMYB5-like被发现参与此过程。为了解TaMYB5-like基因在SQ-1诱导小麦生理型花粉败育过程中的作用,以SQ-1诱导的小麦生理型雄性不育系PHYMS-1376和对照可育系CK-1376四分体、单核早期、单核晚期、二核期和三核期花药为试验材料,克隆TaMYB5-like基因,并对其进行序列结构、生物信息学、表达模式、亚细胞定位及转录自激活效应分析。结果发现,TaMYB5-like基因序列长5 207 bp,其cDNA长为1 133 bp,其中CDS序列长819 bp,编码272个氨基酸,含有2个SANT结构域,分子量为29.36 kDa,无信号肽,无跨膜结构,亚细胞定位在细胞核内;TaMYB5-like基因启动子涉及光响应、逆境胁迫响应、茉莉酸甲酯响应等顺式作用元件。自激活效应发现,TaMYB5-like蛋白的自激活区段位于N端,合适浓度的AbA和3-AT可以抑制BD-TaMYB5-like和BD-TaMYB5-like-N-SANT融合蛋白的自激活性;随着蛋白质氨基酸序列的截断,自激活性呈降低趋势。与CK-1376相比,PHYMS-1376四分体和单核早期花药中TaMYB5-like表达量差异不显著;花药发育到单核晚期、二核期和三核期时,TaMYB5-like表达量显著增加;CK-1376和PHYMS-1376三核期时花药内TaMYB5-like表达量均最高。     

利用大豆浓缩蛋白乳清制备水苏糖的发酵条件

利用酵母对大豆浓缩蛋白乳清进行发酵处理制备水苏糖.在确定最佳起始发酵液的糖度为31.8Brix后,对温度、pH、接种量、装液量等工艺条件进行了单因素试验及正交试验,结果表明最佳发酵条件为:温度32°C,pH5.5,接种量8%,在此发酵条件下利用酵母对大豆浓缩蛋白乳清进行发酵处理48 h,水苏糖的纯度达到了90%,保留率为68%.     

橙汁酿酒酵母菌株的分离筛选和发酵性能的测定

江西是中国著名的无公害柑橘生产基地之一,以南丰蜜橘为原料生产柑橘果酒,可以提高柑橘的附加价值,是柑橘深加工研究的方向之一。采用现代微生物实验技术,通过富集培养和分离技术,在橙汁平板培养基上分离出酿酒酵母的单菌落。通过进一步发酵鉴定经发酵性能测定和产品的感官评定,分离出1株菌种酵母菌(Yeast),编号为Y68。并利用该菌株对柑桔类果汁进行了一定的发酵性能实验,初步发酵证明酵母菌株Y68适合酿造柑橘果酒。