DNA recovery from agarose gels with a simple centrifuge-driven sephadex filtration

Conventional methods of DNA recovery from agarose gel generally require expensive equipment, extended elution times, or considerable handling of the sample after elution. We developed a simple protocol for a quick and effective recovery of DNA from agarose gels with good yield and quality. Using a Sephadex resin filled spin column, DNA fragments of 500 bp to 6 kb in an agarose gel slice were easily recovered by a 2 min centrifugation. The recovery efficiencies were over 40%-50% and the eluted DNA can be used directly for downstream application, such as polymerase chain reactions (PCR) and restriction enzyme digestion. This method could also be used to recover large DNA fragment (48 kb) without degradation. The use of Sephadex helps to remove small molecular impurities from agarose and it also reduces the chance of clogging the column filter caused by direct contact with agarose.     

双向电泳在植物蛋白质组中的应用及影响因素分析

双向电泳(two-dimensional gel electrophoresis,2-DE)是蛋白质组学研究中常用的技术之一,因具有较高的稳定性和重复性,在植物蛋白质组学中应用广泛。蛋白质双向电泳(2-DE)过程复杂、时间持久以及影响因素众多,本文立足蛋白质双向电泳实验过程中可能出现的各种现象,如：分离的蛋白质点过少、条纹过多导致凝胶背景模糊、高丰度蛋白点聚集沉淀及低丰度蛋白点被掩盖等,着重分析其产生的原因并提出具体的解决办法,为蛋白质双向电泳技术初学者提供参考。     

The origin of six-rowed ‘wild’ barley from the western Himalaya

Summary Plants were grown from seed of two-rowed wild barley, Hordeum spontaneum, and six-rowed brittlerachised barley. H. agriocrithon, collected in Ladakh, north-western India. Whereas the H. spontaneum remained true to type, segregation for morphological characters was observed in progeny rows grown from heads of H. agriocrithon plants. The H. agriocrithon heads also showed segregation for a biochemical character, the polypeptide pattern of the endosperm storage protein fraction (hordein). The H. agriocrithon seed therefore originated from natural hybridization between cultivated H. vulgare and weedy H. spontaneum. Crosses of H. vulgare and H. spontaneum gave progeny which resembled H. agriocrithon and showed similar hordein polypeptide segregation patterns. The results indicate that six-rowed brittle-rach ised barleys from the Himalayas have a similar origin to forms found in the Middle East, and that H. agriocrithon does not play a direct role in the evolution of barley.     

Comparison of the isozyme variation in tall fescue parents and their somaclones

Summary This study was conducted using the isozymes ACP-1, ADH-1, GOT-2, GOT-3, MDH, 6-PGD-1 and PGI-2 to: a) compare isozyme banding patterns of tall fescue somaclones with parents and b) correlate tissue culture-induced chromosome abnormalities with variant banding patterns. The 174 somaclones were grouped into seven categories based on their meiotic analyses and time of regeneration from culture. Differences in isozyme frequency between categories compared by chi-square tests were greatest for MDH, 6-PGD-1 and PGI-2, and least for ACP-1. The most significant differences in frequency were found between somaclones and parents. In comparisons of somaclone categories, the most different isozyme distributions were between the early vs. late regenerated somaclones. No significant differences in isozyme frequencies were found between all 42-chromosome somaclones vs. aneuploid somaclones and the three somaclone groups (42-normal, 42-abnormal, aneuploid) compared to each other. This study suggests that culture-induced isozyme variation alters the distribution of the isozyme phenotypes, but is not directly correlated with chromosome abnormalities.     

Relationship Between Gluten Strength and Glutenin Proteins in Durum Wheat Cultivars

An electrophoretic study of gliadin and glutenin proteins, mainly low-molecular-weight (LMW) Glutenin Subunits, was undertaken to investigate possible assoeiations between these proteins and gluten strength. Thirty-eight durum wheat cultivars having different origins and currently grown in Spam were analysed. Different electrophoretic methods were used to analyse the seed storage proteins. Protein grain content was estimated and gluten strength was measured by the SDS-sedimentation test. New patterns for LMW glutenins were observed. Besides the known patterns of LMW-1, associated with γ-gliadin 42, and LMW-2 associated with γ-gliadin 45, six cultivars had LMW-2^? associated with γ-gliadin 43, one cultivar showed LMW-2* associated with γ-gliadin 44, and another cultivar, null for γ-42 and γ-45, had LMW-1^?. Significant differences for gluten strength were found among groups of cultivars with different LMW patterns. High molecular weight glutenins were found in general to be poor indicators of viscoelastic properties, although band 20 showed a negative influence on quality. The results are discussed in relation to development ol cultivars with good gluten strength.     

Meiotic and Isozymic Analyses of Tall Fescue × Giant Fescue Hybrids and Amphiploids1

The meiotic behavior of three tall fescue (Festuca arundinacea, 2n = 6x = 42) genotypes, giant fescue (F. gigantea, 2n = 6x = 42), and their reciprocal F₁ hybrids and C₁, amphiploids was evaluated to determine the parental genomic relationships. Isozyme banding patterns were used to confirm the parental identity of the hybrids and amphiploids. At meta-phase I, the parents had predominantly bivalent pairing. The hybrids had an average of 9.51 I, 16.02 II, 0.12 III, 0.02 IV, and the amphiploids had 2.17 I, 38.82 II, 0.60 III, 0.58 IV, 0.01 V—VIII. The prevalence of bivalent pairing in both hybrids and amphiploids suggested a homoeologous relationship between the six genomes, with four of the six being more closely related. Bivalent pairing in the amphiploids indicated genetic regulation of chromosome pairing. Zymograms were obtained for acid phosphatase (ACPH), alcohol dehydrogenase (ADH), glutamate oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD) and phosphoglucoisomerase (PGI). The three tall fescue and giant fescue parents had different zymograms for ACPH, MDH, 6-PGD and PGI; thus, the tall fescue parents of the hybrids and amphiploids could be determined based on the banding patterns of these four enzymes. Phenotypes were determined for ACPH-1, PGI-2 and 6-PGD-1. ACPH-1 may be used to follow the introgression of giant fescue chromatin into a certain tall fescue genotype.     

Inheritance of peroxidase isozymes in mulberry (Morus spp.)

Summary Electrophoretic variants of peroxidase in mulberry (Morus spp.) were demonstrated by thin-layer gel isoelectric focusing. Of these variants, three isozyme band groups were found to be controlled by codominant alleles at a single locus. The gene symbol Px ₁ was given to this locus, with alleles Px ₁ ¹ and Px ₁ ² assigned to the A6-A7-A8 and A7-A8-A9 band groups, respectively. The A6-A7-A8-A9 band group proved to be controlled by the Px ₁ ¹ and Px ₁ ² heterozygote.Additional experiments showed that among the three banding types, there were no statistically significant differences in leaf blade length, leaf blade width, length-width ratio of leaf blade, internode length, phyllotaxis, leaf shape, tree vigor and resistance to powdery mildew, but there were significant differences in leafstalk length.     

Diet-related composition of the gut microbiota of Lumbricus rubellus as revealed by a molecular fingerprinting technique and cloning

Interactions between earthworms and microorganisms are essential for the functioning of soil ecosystems as they affect organic matter degradation and nutrient cycling. This is also true for the alpine region, where socio-economic changes lead to the increasing abandonment of pastures, which in turn, causes a considerable shift in the diet of saprotrophic invertebrates and thus impacts food web and decomposition processes. To enhance our understanding of how this diet shift influences earthworms and associated microorganisms, we studied the gut content and cast microbiota of Lumbricus rubellus (Lumbricidae, Oligochaeta), a key macrodecomposer on alpine pastureland in the Central Alps. A feeding experiment with L. rubellus and three different food sources that represent the vegetation shift from an alpine pasture to an abandoned site was set up. Earthworms were collected in the field, transferred to a climate chamber and fed with cow manure, dwarf shrub or grass litter for six weeks. PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) analysis of the DNA extracted from the substrates, the earthworms' gut contents and casts revealed that the gut and cast microbiota was strongly influenced by the food source ingested. Cloning of bacterial 16S rRNA gene fragments demonstrated that the intestinal content was dominated by Proteobacteria, especially from the Gamma-subclass, followed by members of the phyla Bacteroidetes, Actinobacteria and Firmicutes. In contrast, Actinobacteria were detected abundantly in all samples types when a cultivation approach was used. In conclusion, the gut microbiota of L. rubellus was shown to be substantially affected by the food source ingested, suggesting that this essential macrodecomposer is exposed to the diet shift resulting from a land-use change in the alpine area.     

复合磷酸盐对鲜切牛肉肌球蛋白凝胶保水性的影响

研究了焦磷酸钠(SAP)、三聚磷酸钠(STP)、六偏磷酸钠(HMP)及复合磷酸盐对牛肉肌球蛋白凝胶保水性的影响。结果显示,当复合磷酸盐中SAP,STP和HMP的质量比为2∶1∶1,添加量为0.2%时,可获得最佳的感官品质和较好的凝胶保水性,影响凝胶保水性因素的主次顺序为SAP>HMP>STP,且SAP和HMP对凝胶保水效果具有显著影响。     

植物表达载体pGMAR-BADH的构建

将甜菜碱醛脱氢酶基因BADH和植物表达载体pGMAR通过内切酶BamHI和KpnI双酶切,T4连接酶进行连接反应。重组质粒在大肠杆菌菌株DH5α内扩增,提取并纯化质粒。经鉴定,基因BADH已被完整、正确的插入到pGMAR载体中,并成功将BADH插入到载体pGMAR的两个MAR序列之间。