带芒草属物种新型高分子量谷蛋白亚基的鉴定

采用SDSPAGE方法对牧草带芒草属3个种8份材料的高分子量谷蛋白进行了检测和鉴定。结果显示,带芒草物种具有的高分子量谷蛋白亚基与普通小麦中发现的不一样,其迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率大。8份材料中共发现了4种x型亚基新类型(Tax1,Tax2,Tax3和Tax4),5种y型亚基新类型(Tay1,Tay2,Tay3,Tay4和Tay5)和6种亚基组合类型(Tax1+Tay3,Tax3+Tay2,Tax4+Tay1,Tax1+Tay1,Tax2+Tay5,Tax4+Tay2),该项研究结果揭示了带芒草属植物可能具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。     

应用SDS-PAGE分析和PCR鉴定长发带芒草的高分子量谷蛋白亚基

利用SDS-PAGE分析、PCR扩增和序列测定与分析研究了长发带芒草(Taeniatherumcrinitum)的高分子量谷蛋白亚基及其基因。结果显示,长发带芒草中发现的高分子量谷蛋白亚基与普通小麦中的类似,但迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率更快。本研究结果揭示了带芒草属具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。     

小麦新品种“川麦42”低分子量谷蛋白亚基新基因的分子克隆

采用PCR方法从小麦（Triticum aestivum L.）新品种“川麦42”中克隆得到一个低分子量谷蛋白亚基（LMW-GS）新基因,暂命名为LMWCM42-1。该基因编码区全长846 bp,编码281个氨基酸,具有LMW-GS基因的典型结构特征。推导氨基酸序列比较显示,尽管LMWCM42-1与已知LMW-GS高度相似,但在N-末端重复区部分重复单元和C-末端区中仍存在明显差异。聚类分析表明,LMWCM42-1可能是由Glu-D3位点编码的。     

滨麦低分子量谷蛋白亚基（LMW-GS）基因的分离与序列分析

采用PCR方法,从滨麦(Leymus mollis)基因组中分离出8条LMW-GS基因序列.核苷酸序列分析表明,序列GQ169791在起始密码子上游包含318 bp的启动子序列,该序列包含-300元件、GCN4 motif、种子贮藏蛋白盒等基因特异表达的顺式或反式作用调控元件.推导的氨基酸序列分析表明,8条序列的编码区依次有信号肽,N-末端区,中部重复区和C-末端Ⅰ、Ⅱ、Ⅲ区等典型LMW-GS多肽一级结构特征;序列HQ416909、HQ416914和HQ416915具有单一完整的开放阅读框(ORF);序列GQ169791、HQ416910、HQ416911、HQ416912和HQ416913在中部重复区和C-末端区出现了4个或5个提前终止密码子,推断其为假基因.8条序列都含有8个或9个半胱氨酸残基(C),N-末端区起始氨基酸序列为METSRIPG-或METTRIPG-,推断其为LMW-m型LMW-GS基因.系统进化分析表明,8条序列与华山新麦草(Psathyrostachys huashanica)LMW-GS基因(HM475146,GQ223386)和野大麦(Hordeum brevisubulatum)的B-hordein基因(AY695368)具有相对较近的同源关系.该研究为挖掘利用滨麦LMW-GS的基因提供了理论依据,对小麦品质改良具有一定参考价值.     

小麦Glu-B3位点LMW-GS基因的克隆及序列分析

以小麦特殊遗传材料———六倍体普通小麦阿勃二体、1A缺体、1B缺体和1D缺体,四倍体硬粒小麦墨西粒卡以及二倍体节节麦的总基因组DNA为模板,对D Ovidio等曾报道的硬粒小麦Glu-B3位点LMW-GS基因特异引物对P1(5-′tcctgagaagtgcatgacatg-3′)和P2(5-′gtaggcaccaactccggtgc-3′)进行了PCR扩增验证.结果表明,该引物对同样能特异扩增普通小麦Glu-B3位点LMW-GS基因.利用这对引物通过AS-PCR方法克隆得到优良小麦品种小偃6号1B染色体1个LMW-GS基因片段.该基因全长为1 089 bp,包含了完整的编码区和其上游318 bp的胚乳特异表达启动子区.该基因被命名为XY-Glu-B3-LMW2(GenBank登录号为DQ630442).XY-Glu-B3-LMW2的推测蛋白含256个氨基酸(包括N-端20个氨基酸的信号肽),其成熟蛋白有8个保守的Cys残基,均分布在C-末端区.XY-Glu-B3-LMW2是从小偃6号克隆到的第2个LMW-GS基因.     

四川小麦地方品种AS1643中低分子量谷蛋白基因的克隆及其蛋白质的二级结构预测

根据小麦低分子量谷蛋白基因保守区序列设计引物P1/P2, 采用PCR法对四川小麦地方品种AS1643的基因组DNA进行扩增, 获得1条约900 bp的片段, 分离、纯化后连接到载体pMD18-T上, 对筛选阳性克隆测序, 获得1个低分子量谷蛋白基因LMW-AS1643(GenBank登录号: EF190322), 其编码区长度为909 bp, 可编码302个氨基酸残基组成的成熟蛋白。序列分析结果表明, LMW-AS1643具有典型的低分子量谷蛋白基因的基本结构, 其推导氨基酸序列与其它已知的LMW-GS相比, 最高相似性为93.40%。生物信息学分析表明, 在LMW-AS1643低分子量谷蛋白中, 无规则卷曲含量最高, 为67.90 %, 其次是a-螺旋, 占30.46 %, b-折叠含量最少, 为1.64 %。     

部分小麦育种材料低分子量谷蛋白亚基等位基因变异

改进小麦LMW-G S分离技术,利用改良一步单向连续梯度SDS-PAGE技术分析40份具有较好品质、产量特性的小麦育种亲本材料的LMW-G S类型和组成形式。结果表明:采用适当的方法提取谷蛋白亚基,在4.8%浓缩胶(pH 6.8,C=2.6%),12.0%~14.0%连续梯度分离胶浓度(pH 8.8,C=1.3%),电流35 mA,电泳330 m in后可以获得良好的LMW-G S分离效果。G lu-3位点出现11种变异类型、19种亚基组合形式。G lu-A 3位点出现G lu-A 3a、G lu-A 3b、G lu-A 3c、G lu-A 3d 4种类型,频率分别为40.0%、12.5%、10.0%、37.5%;G lu-B 3位点出现7种亚基类型,其中G lu-B 3d、G lu-B 3b、G lu-B 3 j为主要类型,频率分别为40.0%、20.0%、12.5%。亚基组合形式G lu-A 3a G lu-B 3d组合频率最高,频率为22.5%;9个亚基组合分别只在一个亲本出现,占分析材料总数22.5%。G lu-A 3位点的遗传变异指数为0.673 8,G lu-B 3位点的遗传变异指数为0.800 8,表明G lu-3位点遗传变异丰富。     

小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

水稻富硫10kD醇溶谷蛋白基因的克隆和序列分析比较

用ＰＣＲ技术从我国水稻品种“广陆矮”（Ｏｒｙｚａ ｓａｔｉｖａ ｖａｒ． ｉｎｄｉｃａ）和“中花８ 号”（Ｏ． ｓａｔｉｖａｖａｒ． ｊａｐｏｎｉｃａ）中特异地扩增并克隆测序了富硫１０ ｋＤ醇溶谷蛋白基因,它共有５２５ 个核苷酸,编码１３４ 个氨基酸。经分析,克隆的基因与水稻属其它种或品种的同类基因同源率为９６．６％ 到１００％ ,与玉米１０ ｋＤ醇溶蛋白基因同源率为３４．２％ ;与某些双子叶植物的贮藏蛋白也有一定的同源性,例如和巴西豆富硫水溶蛋白同源率达３１．２％ 。水稻１０ ｋＤ醇溶谷蛋白Ｎ 端有一段信号肽含２４ 个氨基酸,经分析,发现这段信号肽与禾谷类玉米、高粱和燕麦的贮藏蛋白信号肽同源率很高,分别为６５．０％ 、６５．０％ 和６２．５％ ,而在双子叶植物的贮藏蛋白中未发现有相似序列。所测定的“广陆矮”和“中华８ 号”１０ ｋＤ醇溶谷蛋白基因序列已被ＥＭＢＬ数据库接受,收录号分别为Ｌ３６６０４ 和Ｌ３６６０５     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.     

Isolation and partial characterization of a low molecular weight trypsin inhibitor from human urine

A low molecular weight glycoprotein which completely inhibited trypsin at a 1 : 1 molar ratio was isolated from human urine. It was generated from a precursor molecule which in turn derived from plasma inter-alpha-trypsin inhibitor. It had one polypeptide chain with a molecular weight of about 20 000 and a high content of half-cystine residues. Its amino-terminal amino-acid sequence was Val-Thr-Glu-Val-Thr-X-Leu-Glu-Asp-.     

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica

Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M_r polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.     

Isolation and partial characterization of a novel form of low molecular weight kininogen from guinea pig plasma

Two kinds of low molecular weight kininogen (termed LK1 and LK2) were isolated from pooled plasma of guinea pigs. When polyclonal antisera raised against the individual proteins were used, immunological cross-reactions were observed between LK1 and high molecular weight kininogen (HK), but not either between LK1 and LK2 or between LK2 and HK. After tissue injury, plasma level of LK1 doubled while those of LK2 and HK remained relatively unchanged.     

Isolation and partial characterization of high and low molecular weight acid phosphatases from chicken liver

Two acid phosphatase forms were isolated from chicken liver by gel filtration on Sephadex G-100. These enzymes, termed I and II, have similar Km- and Vmax-values, but differ in molecular weight, optimum pH, sensitivity to various inhibitors and substrate specificity. The results were compared with the numerous literature reports of mammalian acid phosphatases.