两种HMW-GS基因启动子片段的克隆及分析

利用聚合酶链反应(PCR)技术从小偃6号中获得400bp左右的扩增产物,将其与pGEM-T Easy载体连接后转入大肠杆菌,经过筛选获得HMW-8-P和HMW-38-P两种类型克隆。序列分析表明：HMW-38-P包括了HMW-GS14基因上游启动子及信号肽对应编码区,而另一段(HMW-8-P)为一未知HMW-GS基因启动子区及信号肽对应的编码区。将两序列和GenBank中已知的35种HWM-GS基因启动子区序列进行多序列比对,最后获得HMW-GS启动子的系统发生树。通过系统发生树可以清晰地看出位于不同染色体上的不同亚基类型的HMW-GS基因的进化关系,并可确定HMW-8-P为Glu-D-1类型HMW-GS的启动子区,小偃6号中Glu-D-1类型的亚基为2亚基,所以HMW-6-P为2亚基启动子区序列。     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

中国特有小麦Gli-1、Gli-2和Glu-1位点的遗传多样性(英文)

运用APAGE和SDS_PAGE方法 ,研究了 32份中国特有小麦Gli_1、Gli_2和Glu_1位点的遗传多样性。在 1 4份云南铁壳麦 (Triticumaestivumssp .yunnaneseKing)中 ,共出现 8种醇溶蛋白带型和 3种高分子谷蛋白带型。在 9份西藏半野生小麦 (T .aestivumssp .tibetanumShao )中 ,发现 9种醇溶蛋白带型和 4种高分子谷蛋白带型。在 9份新疆稻麦 (T .petropavlovskyiUdacz.etMigusch .)中 ,观察到 9种醇溶蛋白带型和 5种高分子谷蛋白带型 ,其中 1份新疆稻麦 (稻麦 2 )具有Glu_D1编码的新亚基 2 .1 1 0 .1。在这 3种中国特有小麦群体中 ,Gli_1位点分别检测出 1 0、1 4和1 1个等位基因 ;Gli_2位点各具有 1 1、1 4和 1 2个等位基因 ;Glu_1位点也分别出现 5、6和 8个等位基因。云南铁壳麦、西藏半野生小麦和新疆稻麦群体内的Nei’s遗传变异系数分别为 0 .3798、0 .56 2 5和 0 .56 93。这些结果说明 ,与云南铁壳麦相比 ,西藏半野生小麦和新疆稻麦群体内的遗传变异相对较大。     

Thermal properties of corn gluten meal and its proteic components

Thermal properties of corn gluten meal (CGM) and of its extracted proteic components (zein and glutelin) at 0% moisture content, is studied by dynamic mechanical thermal analysis (DMTA) and modulated differential scanning calorimetry (MDSC). The glass transition temperature (T_g) on first heating, is measured at 176 and 174°C, respectively, for hot-air-dried and native CGM. For zein and glutelin isolated fractions, the measured T_g values are 164 and 209°C, respectively. The calculated T_g from using Matveev’s method (Matveev YI. Spec Publ R Soc Chem 1995;156;552) is in good agreement with experimental data for zein, a well defined protein. MDSC allows the measurement of change in heat capacity at T_g (ΔCp) with a single heating scan, avoiding sample alteration, and ΔCp values are 0.365 J/g per K for zein and 0.184 J/g per K for glutelin. The differences observed in T_g, relaxation temperatures, ΔCp and tan δ peak height are related to differences in the structure of the proteins, through the cross-linkages and hydrogen or van der Waals interactions. Experimental data from DMTA and MDSC, and the Couchman–Karasz thermodynamic approach indicate that CGM behaves as a miscible blend of its components, with high non-polar interactions between zein and glutelin proteins.     

Immunochemical Assay for Quality and Immunological Homology of Storage Proteins in Different Closely Related Genera and Species of Wheat

An immunochemical assay using polyclonal and monoclonal antibodies of high-molecular-weight glutenin subunit (HMW-GS) in Triticum aestivum L. was carried out to determine the quality of wheat and to investigate the immunological homology of the storage proteins in cereal endosperms in different closely related wheat genera and species. The results showed that correlation between the antigen-antibody reaction and the wheat quality varied with the type of antibodies used and the quality. The correlation coeffecient was slightly higher when the polyclonal antibodies were used than monoclonal antibodies were used. The correlation coeffecient was high between the antibody binding and the protein content, and wet/dry gluten coment, with Zeleny sedimentation value, while that between antibody binding and bread characters was lower. The highest correlation coeffecient between the polyclonal antibody binding and the protein content in grains, wet and dry gluten contents, bread volume and bread ratio volume was 0. 762 0, 0. 894 2, 0. 887 3, 0.610 3, 0.459 8 and 0.474 4 respectively, while the highest correlation coeffecient between the monoclonal antibody binding and the above parameters was 0. 783 7, 0. 774 5, 0.782 2, 0. 684 1, 0. 687 3 and 0. 598 2 respectively. The immunological homologies between I-IMW-GS 1Dyl0 in common wheat and endosperm storage protein in wheat grains of different genera and species were noticed. The cross-reac-tion among Triticum aestivum L., Secale cereale, T. spelta L., Aegilops squearrosa L. and T. Turgidtan L. was stronger than that among other cereals.     

谷蛋白聚合体大小分布与面粉揉面特性的初步研究

用单向一步SDS-PAGE方法分析表明小麦品种Suneca和Cook在麦谷蛋白5个亚基位点（Glu-B1,Glu-D1,Glu-A3,Glu-B3和Glu-D3)均含不同等位基因。选用Suneca×Cook的F4代群体中麦谷蛋白亚基位点均为纯合基因的60个系,研究麦谷蛋白基因型不同的株系间谷蛋白聚合体粒度大小分布（用SE-HPLC测定）和面粉揉面特性的变异。结果表明,不同的谷蛋白基因型,其谷蛋白聚合体粒度大小相对分布（用不溶谷蛋白聚合体占总谷蛋白聚合体含量的百分数表示,即UPP%）和面团形成时间（即揉面仪曲线图峰值的和面时间,简写PTM）均有显差异;面粉的揉面曲线形状与其UPP%值密切相关;UPP%与PTM呈极显正相关,与揉面仪曲线图峰高（PHM）呈显负相关;与面粉蛋白质含量（FP%）相比,UPP%对PTM和PHM的影响更大些,可作为育种早代品质性状选择一个指标。     

BiP, HSP70, NDK and PDI in wheat endosperm. I. Accumulation of mRNA and protein during grain development

Biosynthesis and accumulation of seed storage proteins such as the wheat glutens depend on the activity of a variety of other proteins, including chaperones and foldases. cDNA probes and antibodies to two chaperone proteins and a foldase were used to follow mRNA and protein accumulation in developing grains of wheat ( Triticum aestivum , cvs Cheyenne and Butte). Endosperm was separated from other grain components and protein accumulation was analyzed on a per mg fresh weight basis. The ER resident chaperone BiP (binding protein) and foldase PDI (protein disulfide isomerase) accumulated to maximal levels in the middle stage of endosperm development, a period of rapid cell expansion and storage protein accumulation, whereas levels of a cytosolic chaperone, HSP70, remained relatively constant throughout grain development. In contrast, nucleoside diphosphate kinase (NDK), a cytosolic enzyme needed for synthesis of nucleoside triphosphates, accumulated early in endosperm development during the period of nuclear division and cell formation. When analyzed as a fraction of total protein the relative abundance of all four proteins peaked early in grain development and then declined. Accumulation of mRNA for the four proteins also peaked early in grain development. Although BiP and PDI formed a declining percentage of total protein as storage protein accumulated, their pattern of accumulation was compatible with a proposed role as catalysts for storage protein folding and accumulation in the ER.     

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.     

Gamma-crystallin, a major cytoplasmic polypeptide disulfide linked to membrane proteins in human cataract

Examination of human cataract has revealed the presence of a number of unique complexes containing cytosol and membrane components: the high molecular weight disulfide linked aggregates and membrane preparations containing disulfide linked cytosol polypeptides. It is now shown that a major cytosol species associated with these complexes is gamma-crystallin. This conclusion is based upon investigation of polypeptides released by reduction and comparisons based on amino acid, immunochemical and sequence analyses. It is suggested that two types of complexes may be closely related.