The genetical control and tissue-specificity of esterase isozymes in hexaploid wheat

A comparative genetic analysis of esterase (E.C.3.1.1.1) isozymes of wheat cultivar Chinese Spring in endosperm, embryo, coleoptile, leaf and root tissues revealed eight sets of isozymes characterised by different tissue specificities, pI ranges and the chromosomal locations of their controlling genes. This data was considered together with previously published work, resulting in a proposed rationalization of nine sets of wheat esterase isozymes. Although this classification included two sets of isozymes controlled by genes on the short arms of homoeologous group 3 chromosomes and three sets on the long arms of the same chromosomes, for which no recombination evidence of genetic distinctness has been obtained among either group, it is argued that the different characteristics of the various sets warrant retention of separate set nomenclatures. Previously unreported esterase genes includeEst-9, a low pI, monomeric, embryo-specific group with controlling genes on chromosomes 3BS and 3DS and two further members ofEs-1,Est-H1 inHordeum vulgare andEst-S ^l1 inAegilops longissima.     

抗白粉病6R(6B)染色体异代换系的选育

以中７９０２（６Ｂ）单体为母本,Ｈｏｌｄｆａｓｔ－ＫｉｎｇⅡ６Ｒ二体异附加系为父本配制杂交组合,经白粉病抗性追踪和细胞学鉴定,选育出抗白粉病６Ｒ（６Ｂ）二体异代换系。其与普通小麦中８６０１以及与“中国春”６Ｂ重双端体的Ｆ１植株,在减数分裂中期Ⅰ染色体构型分别为２０″＋２′和２０″＋１′＋２ｔ′ ,从而,证实６Ｂ染色体被 ６Ｒ染色体代换。同时,对６Ｒ染色体上抗白粉病基因的进一步利用进行了讨论。     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

The genetic control of grain esterases in hexaploid wheat

Summary Analysis of grain esterase isozymes in Chinese Spring aneuploid genotypes by IEF confirmed that genes on the long arms of chromosomes 3A, 3B and 3D (Est-5) control the production of 19 isozymes. Allelic variants have been found for the isozyme pattern controlled by each chromosome. Segregational data involving null alleles and complex phenotypic differences indicate that the wheat grain esterases are encoded by three compound and probably homoeoallelic loci, each capable of producing at least six different isozymes. In a sample of 138 hexaploid genotypes, seven alleles were distinguished.     

水稻半矮秆基因sd－g的染色体定位研究

以标志基因系和ＩＲ３６三体为工具材料,通过杂交,研究了籼稻矮秆材料双矮所携半矮秆基因ｓｄ－ｇ在染色体上的位置。结果表明：半矮秆基因ｓｄ－ｇ与标志基因系Ｍ４所携隐性主基因ｇｈ－１和Ｍ２７所携隐性主基因ｎ１表现连锁。ｓｄ－ｇ与ｇｈ－１之间的交换值为２４．３３％±３．９６％,ｓｄ－ｇ与ｎ１之间的交换值为２９．４４％±４．８１％。由于ｇｈ－１和ｎ１均位于第５染色体,因而推定ｓｄ－ｇ位于第５染色体上。     

Preliminary analysis of root exudates of in vitro-micropropagated Alnus glutinosa clones

Biochemical analysis at Alnus glutinosa (L.) Gaertn. root exudates was carried out in order to investigate the probable role of these exudates in the establishment of the Alnus-Frankia symbiosis. Sufficient amounts of sterile root exudates were collected from plants originating from five in vitro-micropropagated A. glutinosa clones (AG1, AG2, AG3, AG4, AG8) grown under aerobic conditions in nutrient solution with Ballotini glass beads. Cytological controls showed good preservation of roots at the end of the collecting period. The total protein and sugar contents of crude root exudates were determined and related to the dry root weight. These results showed a provenance effect. Soluble high molecular-weight fractions (i.e. the mucilage) were isolated following rigorous purification of the crude root exudates and within this fraction proteins were characterized by isoelectric focusing. The protein pattern of the five clones tested showed major bands in common and some minor specific ones. These experimental data will serve as a useful basis for future physiological investigations.     

双向凝胶电泳的实验操作及进展

双向聚丙烯酰胺凝胶电泳是研究蛋白质组的核心技术,本文重点介绍了双向凝胶电泳的原理、实验操作过程中的具体步骤以及近年来在实验过程中发展的一些新方法和进展。     

The semidwarf gene,sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection

To establish the location of the semidwarf gene, sd-1, the anthocyanin activator (A), purple node (Pn), purple auricle (Pau), and the isozyme locus, EstI-2, in relation to DNA markers on the molecular linkage map of rice, 20 RFLP markers, previously mapped to the central region of chromosome 1 (McCouch et al. 1988), were mapped onto an F₂ population derived from the cross Taichung 65 (A,Pn,Pau)/Taichung 65 (sd-1). sd-1 and EstI-2 were determined to be linked most tightly to RFLP markers RG 109 and RG 220, which cosegregated with each other. The distance between these RFLP markers and sd-1 was estimated to be 0.8 cM, based on an observed recombination value of 0.8%. The order of genes and markers in this region of chromosome 1 was determined to be sd-1 — (EstI-2 — RG220 — RG109) — RG381 — A — Pn — Pau. To test the efficacy of selection for sd-1 based on these linked markers, 50-day-old F₂ seedlings derived from another cross, Milyang 23/Gihobyeo, were analyzed for marker genotype. At this age, the semidwarf character could not be clearly detected based on phenotype. In addition, plant height was normally distributed in this population, making it difficult to unambiguously identify plants carrying sd-1. Thirteen seedlings homozygous for the sd-1-associated allele at EstI-2, RG220 and RG109, and 13 seedlings homozygous for the Sd-1-associated allele at all three marker loci were selected for further genetic analysis. At 20 days after heading, the culm lengths of these 26 plants were measured and the expected phenotype was confirmed in every case. These 26 plants were then selfed for four generations and F₆ lines were again evaluated to determine whether any recombination among the three molecular markers, or between these markers and the sd-1 gene, could be detected. No recombinants were identified, confirming the tight linkage of these loci and the usefulness of genotypic selection for this recessive semidwarf character prior to the time when it can be evaluated based on phenotype.     

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electrosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The ∼90 fl injection volume for individual cells is 7×10⁴ times lower than those previously reported. Adult (normal and elevated A₁), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A₀, A_1c, S and F determined. The pH gradient for cIEF was linear (r² = 0.9984), which allowed tentative identification of Hb F_ac. Variants differing by as little as 0.025 pI units were resolved.     

北京鸭LDH同工酶的研究——Ⅲ.LDH座位在北京鸭发生过程中的表现方式

采用电泳结合光密度扫描的方法,对北京鸭肝脏、胸肌、心肌和肾脏4种组织的LDH同工酶进行了发生遗传学分析。发现LDH座位在北京鸭发生过程中有三种表现方式:肝脏表现出B型酶到A型酶的典型转换;胸肌则表现出AB型酶到A型酶的特殊转换;心肌和肾脏始终保持B型酶为主,只表现出量上的增加趋势,同时也发现LDH1同工酶各亚带也具有组织的和发育阶段的特异性。显然这都是由于LDH座位即ldh a和ldh b两个基因的差别表达所造成的。     

中国几种特殊普通小麦酯酶同工酶研究

采用聚丙烯酰胺凝胶电泳法对中国几种特殊普通小麦的幼芽进行了酯酶同工酶分析。结果表明:同种不同来源的麦酶谱差异不大,较整齐一致,只有个别材料有酶带的增减及沾性强弱有别;对于不同的物种云南铁壳麦与西藏半野生小麦的酶谱很相近似,属同一类群,新疆稻麦慢带较模糊,少第3条酶带归属别一类群。     

OKP-B-13型β-内酰胺酶的克隆与表达

目的构建OKP-B-13型β-内酰胺酶的表达载体。方法抽提菌株的质粒,应用PCR扩增OKP-B-13基因全长编码序列,扩增产物经Nde I、Xho I酶切后连接至pET-26b(+)表达载体,重组质粒经酶切及DNA测序确证后,转入大肠埃希菌BL21(DE3),IPTG诱导表达。超声破碎法提取表达蛋白产物,检测其活性,等电聚焦电泳检测蛋白的等电点(pI)。结果PCR扩增获得879 bp的产物,重组表达载体经Nde I、Xho I酶切及DNA测序后表明,目的基因已成功接入表达载体,重组菌的粗提物经头孢硝噻吩检测显示具有β-内酰胺酶活性,显示载体[pET-26b(+)/OKP-B-13]构建成功。目的等电点为7.1。结论β-内酰胺酶OKP-B-13在原核表达细胞中实验了基因重组表达,为进一步分析酶的特性提供条件。     

Spirocamallanus cricotus (Nematoda): isoelectric focusing and spectrophotometric characterization of its hemoglobin and that of its piscine host, Micropogonias undulatus

The hemoglobins of Spirocamallanus cricotus, a reddish-colored, camallanid nematode, and its Atlantic croacker fish host, Micropogonias undulatus, were characterized with spectrophotometry and isoelectric focusing. Hemoglobin from female parasites' perienteric fluid and homogenized male parasites gave Spectrophotometric peaks at 412, 539, and 575 nm, whereas female worms drained of perienteric fluid and homogenized differed by having a Soret peak of 408 nm. Changing the ionic strength of the buffer from 0.1 to 0.01 M shifted the Soret peak to 406 nm for the female parasites' perienteric fluid and ground male parasites and 404 nm for homogenized female parasites. In all cases, the β band had a higher absorption than the α band suggesting a high O₂ affinity for the parasite hemoglobin. Host hemoglobin had peaks of 406, 437, and 577 nm. Isoelectric focusing not only confirmed the Spectrophotometric evidence that host and parasite hemoglobins differed, but also showed that the parasite's analyzed hemoglobin fractions differed from one another by having different isoelectric points.     

SHV-59型β-内酰胺酶的克隆与表达

目的构建SHV-59型β-内酰胺酶的表达载体。方法抽提菌株的质粒,应用PCR扩增SHV-59基因全长编码序列,扩增产物经NdeI、XhoI酶切后连接至pET-26b（＋）表达载体,重组质粒经酶切及DNA测序确证后,转入大肠埃希菌BL21（DE3）,IPTG诱导表达。超声破碎法提取表达蛋白产物,检测其活性,等电聚焦电泳检测蛋白的等电点（PI）。结果PCR扩增获得879bp的产物,重组表达载体经NdeI、XhoI酶切及DNA测序后表明,目的基因已成功接入表达载体,重组菌的粗提物经头孢硝噻吩检测显示具有β-内酰胺酶活性,表明载体[pET-26b（＋）／SHV-59]构建成功。目的等电点为7．6。结论β-内酰胺酶SHV-59在原核表达细胞中实验了基因重组表达,为进一步分析酶的特性提供条件。     

菠菜中的乙醇酸氧化酶是一个同工酶

乙醇酸氧化酶（ＥＣ．１．１．３．１５．ＧＯ）是光呼吸途径的关键酶,降低其活性可提高Ｃ３植物如水稻的产量,在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下,对ＧＯ的研究具有重要的理论意义和实际应用价值．在光呼吸途径被提出后的几十年间,人们对如...     

滇池高背鲫和方正银鲫酯酶、乳酸脱氢酶同工酶的比较研究

本研究以方正银鲫(Carassius auratus gibelio Bloch)普通鲫(Carassius auratus)和滇池高背鲫(Carassius sp.)的各种组织器官为材料,进行酯酶(Esterase)和乳酸脱氢酶(LDH)同工酶电泳图谱的分析比较。结果表明:9种不同组织中酯酶同工酶谱带各不相同,有明显的组织特异性。滇池高背鲫的酯酶谱图有3种表型。方正银鲫和滇池高背鲫同一组织的LDH同工酶酶谱也有明显差异。等电聚焦凝胶电泳(T=7.5％,C=5％)的结果又表明这二种鱼的肝脏、脑、卵的酯酶同工酶酶谱及电泳扫描图亦有差异。这些结果揭示滇池高背鲫与方正银鲫至少在生化水平上已有明显的分化,很可能起源于不同的地区,由不同的祖先,独立演化而形成。滇池高背鲫与云南普通鲫的LDH酶谱较为接近,这说明滇池高背鲫最可能起源于云南本地的普通鲫。     

Localization to chromosomes of structural genes for the major protease inhibitors of barley grains

Summary Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous -amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3.     

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.