Nitrate Reduction by Roots of Soybean (Glycine max [L.] Merr.) Seedlings

Studies were conducted with 9 to 12 day-old soybean (Glycine max [L.] Merr. cv. Williams) seedlings to determine the contribution of roots to whole plant NO(3) (-) reduction. Using an in vivo -NO(3) (-) nitrate reductase (NR) assay (no exogenous NO(3) (-) added to incubation medium) developed for roots, the roots accounted for approximately 30% of whole plant nitrate reductase activity (NRA) of plants grown on 15 mm NO(3) (-).Nitrogen analyses of xylem exudate showed that 53 to 66% of the total-N was as reduced-N, depending on the time of day of exudate collection. These observations supported enzyme data that suggested roots were contributing significantly to whole plant NO(3) (-) reduction. In short-term feeding studies using (15)N-NO(3) (-) significant and increasing atom percent (15)N excess was found in the reduced-N fraction of xylem exudate at 1.5 and 3 hours after feeding, respectively, which verified that roots were capable of reducing NO(3) (-).Estimated reduced-N accumulation by plants based on in vivo -NO(3) (-) NR assays of all plant parts substantially over-estimated actual reduced-N accumulation by the plants. Thus, the in vivo NR assay cannot be used to accurately estimate reduced-N accumulation but still serves as a useful assay for relative differences in treatment conditions.     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

Quantitative Properties of Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA analysis (RAPD) is a methodology that has been used as a tool for monitoring microbial communities. To be useful in this application RAPD, and any other methodology, must show properties that allows for the detection of quantitative changes in composition of the microbiota. Therefore, the objective of this study was to establish whether RAPD possesses such properties. The strategy was to use genomic DNA, extracted from a set of tertiary bacterial mixtures defined according to an experimental mixture design, and containing varying proportions of Escherichia coli, Bacillus subtilis, and Pseudomonas CF600. RAPD-PCR was performed on the mixed DNA extracts and the amplified DNA fragments were separated on sequencing gels to produce genomic fingerprints that were digitized and modeled by Partial Least Squares regression (PLS). Significant predictions were obtained using an external test set for validation, with Root Mean Square Error of Predictions (RMSEP) of 0.21, 0.19 and 0.20 for the proportion of E. coli, B. subtilis and Pseudomonas CF600 respectively. Taken together, the results showed that RAPD patterns quantitatively represented the initial mixture proportions. Therefore, the view that RAPD could be useful for whole microbial community monitoring was strengthened.     

低磷对大豆主根伸长生长的影响

文章采用卷纸培养和分层琼脂培养的方法,研究磷对大豆主根伸长影响的结果表明:低磷[0.2 μmol(KH2PO4)·L-1]显著促进大豆主根伸长,特别是延长大豆主根根尖至最新侧根间的距离;组织切片表明,低磷对主根伸长的促进主要是通过延迟主根伸长区的分化实现的,并且低磷对主根的促进作用不受亚磷酸盐的影响.琼脂分层培养的结果表明,在磷分布不均匀的条件下,低磷影响主根的仲长生长,上层或下层不施磷的大豆主根伸长均有增加.     

Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress

Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from −0.6 megapascal to −1.7 megapascals after 4 days of stress; then to −3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-¹⁴C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids.     

Protein Synthesis during Natural and Precocious Soybean Seed (Glycine max [L.] Merr.) Maturation

Protein synthesis was studied during precocious and natural soybean seed (Glycine max [L.] Merr.) maturation. Developing seeds harvested 35 days after flowering were precociously matured through controlled dehydration. Total soluble proteins and proteins labeled with [³⁵S]methionine were extracted from control, developing seeds and from precociously and naturally matured seeds and were analyzed by one-dimensional PAGE and fluorography. The results demonstrated that several polypeptides which were designated “mature polypeptides,” were synthesized de novo during precocious and natural seed maturation. Two of these polypeptides, 31 and 128 kilodalton in mass, also stained intensely with Coomassie blue, suggesting their abundant accumulation during seed maturation. Results from in vitro translation experiments showed that the mRNAs corresponding to these “maturation polypeptides” accumulated during precocious maturation and in naturally matured seeds, but not in seeds freshly harvested 35 days after flowering (control). The role of the “maturation polypeptides” is currently unknown; however, their presence and that of their corresponding mRNAs was coincident with the ability of matured seeds to establish seedling growth. This study has demonstrated that precocious seed maturation treatments may be extremely useful for investigations of metabolic events and molecular control mechanisms affecting soybean seed maturation.     

利用RAPD技术分析实验用比格犬的遗传背景

目的　运用RAPD技术对实验用比格犬 (Beagle)进行遗传背景分析。方法　筛选的 16个RAPD引物对 4 0个样品的分析 ,共得到 93个扩增条带。结果　所有样品的相似系数 80 4 %到 10 0 %间变动 ,平均相似系数为93 2 8% ,聚类分析得到了这些个体的同源树资料。结论　本遗传背景资料可以为以后的比格犬繁育生产和生物医药学的研究应用提供技术指导     

Sequence Level Analysis of Recently Duplicated Regions in Soybean [Glycine max (L.) Merr.] Genome

A single recessive gene, rxp, on linkage group (LG) D2 controlsbacterial leaf-pustule resistance in soybean. We identifiedtwo homoeologous contigs (GmA and GmA') composed of five bacterialartificial chromosomes (BACs) during the selection of BAC clonesaround Rxp region. With the recombinant inbred line populationfrom the cross of Pureunkong and Jinpumkong 2, single-nucleotidepolymorphism and simple sequence repeat marker genotyping wereable to locate GmA' on LG A1. On the basis of information inthe Soybean Breeders Toolbox and our results, parts of LG A1and LG D2 share duplicated regions. Alignment and annotationrevealed that many homoeologous regions contained kinases andproteins related to signal transduction pathway. Interestingly,inserted sequences from GmA and GmA' had homology with transposaseand integrase. Estimation of evolutionary events revealed thatspeciation of soybean from Medicago and the recent divergenceof two soybean homoeologous regions occurred at 60 and 12 millionyears ago, respectively. Distribution of synonymous substitutionpatterns, K_s, yielded a first secondary peak (mode K_s = 0.10–0.15)followed by two smaller bulges were displayed between soybeanhomologous regions. Thus, diploidized paleopolyploidy of soybeangenome was again supported by our study.     

用RAPD技术对罗非鱼遗传变异的研究及应用(英文)

应用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术检测了一个奥利亚罗非鱼（ａｕ）和湘湖（ｎｘ）、美国（ｎｍ）、沙市（ｎｐ）三个尼罗罗非鱼养殖群体（Ｔａｂｌｅ１）。在２０个引物（Ｔａｂｌｅ ２）中筛选到１２个引物,它们的扩增产物显示了罗非鱼和尼罗罗非鱼二者在群体内或群体间存在遗传差异。其中（Ｆｉｇ．１）, ＯＰＺ０６、 ＯＰＺ１６、 ＯＰＺ１２和 ＯＰＺ１９四个引物分别有一个扩增片段具有种的特异性。它们的大小分别是９００、１５００、１７００和７３０ｂｐ。可以作为鉴别罗非鱼和尼罗罗非鱼二者的分子遗传标记。湘湖（ｎｘ）、美国（ｎｍ）和沙市（ｎｐ）三个尼罗罗非鱼群体内遗传变异相似性指数Ｓ分别为 ０．７９８、 ０．７９５和０．８２４（Ｔａｂｌｅ ３）。表明：这三个尼罗罗非鱼群体都保留了较高水平的遗传变异。而奥利亚罗非鱼（ａｕ）的群体内遗传变异最小。奥利亚罗非鱼（ａｕ）与湘湖（ｎｘ）、美国（ｕｍ）、沙市（ｕｐ）三个尼罗罗非鱼群体之间的遗传距离分别是０．２８５、０．２６２和０．３４４（Ｔａｂｌｅ ３）,说明奥利亚罗非鱼（ａｕ）和沙市尼罗罗非鱼（ｎｐ）杂交将可能产生较强的杂种优势。     

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.     

A Pod Leakage Technique for Phloem Translocation Studies in Soybean (Glycine max [L.] Merr.)

Radioactive photosynthetic assimilates, translocated to a soybean (Glycine max [L.] Merr. `Fiskeby V') pod can be measured directly by excising the stylar tip of the pod under 20 mm ethylenediaminetetraacetate solution (pH 7.0) and allowing the material to leak into the solution. Pods at the source node received approximately 50% of the ¹⁴C exported from the source leaf to the pod and leaked approximately 1 to 3% of this into the solution. More than 90% of the ¹⁴C that leaked from the pods was found in the neutral fraction and, of this, about 93% was in sucrose. Fifteen amino acids were identified in the leakage including: alanine, arginine, asparagine, γ-aminobutyric acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine. The majority of the ¹⁴C in the basic fraction was found in serine (30%) and asparagine (23%). The inorganic ions K, Ca, P, Mg, Zn, and Fe were found in the leakage component. Nitrate was not detectable in the collected leakage solution. The absence of NO₃⁻ and the large proportion of the label in sucrose suggest a possible phloem origin for most of the material. The technique provides an uncomplicated, reproducible means of analyzing the material translocated into and through the soybean pod, as well as following the time course of label arrival at the pod.     

牛血清白蛋白在植物RAPD分析中的作用

以水杉、七子花DNA为模板,添加牛血清白蛋白（BSA）,观察其对植物RAPD扩增效果的改善情况。研究显示,在水杉及七子花RAPD扩增体系中,改善RAPD扩增反应的最佳的BSA浓度是不同的,分别为06μg/μl与1μg/μl。另外,BSA还可以封闭乙酰BSA对RAPD扩增反应的抑制作用,降低RAPD反应系统中Taq酶的用量。 Abstract:Using Metasequoia glyptostroboides and Heptacodium miconioides DNA as templates,the effect of bovine serum albumin(BSA) on RAPD in plants was studied.The results showed that suitable concentrations of BSA used in Metasequoia glyptostroboides and Heptacodium miconioides RAPD were different,which were 06μg/μl and 1μg/μl,respectively.The inhibition of acetylated BSA on the amplification of plant RAPD could be relieved by BSA.BSA could reduce the dosage of Taq DNA polymerase.     

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N₂ per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N₂ per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Cloning, mapping, and analyses of expression of the Em-like gene family in soybean [Glycine max (L). Merr.]

 The entire Em-like Group-1 late embryogenesis abundant (Lea) gene family from soybean was cloned and characterized. The five Group-1 Lea genes (Sle1-5) were divided into two classes based on sequence identity. Sle1-4 were genetically mapped to four different linkage groups. Nucleotide sequencing indicated that Sle1, Sle2, Sle3, and Sle5 encode polypeptides differing primarily by the presence of a repeated 20-amino acid motif. Sle1 and Sle5 were shown by Northern analysis to be expressed in developing embryos weeks earlier than Sle2 and Sle3. Sle4 was shown to be a pseudogene. Maximal levels of mRNA for all functional Sle genes accumulated in maturation-phase seeds, before significant desiccation had occurred, and declined rapidly upon seed imbibition. Desiccation did not induce Sle expression in seeds or vegetative tissue. Sle expression was confined to embryo tissues and Sle mRNA accumulated at similar levels in both the embryo axis and in the cotyledons. Received: 25 September 1996 / Accepted: 27 September 1996     

RAPD技术及其在哺乳动物生态学研究中的应用

1　引　言现代生态学的不断发展 ,改变了过去主要采用宏观的方法来研究生物与生物及环境的关系。在种群生态学研究中 ,越来越多的学者采用微观的分子生物学技术来研究种群的生存问题 ,新学科分子生态学得到快速的发展。目前 ,从分子水平来研究种群生态 ,较多采用限制性酶切图谱 (ＲＦＬＰ)和以ＰＣＲ为基础的ＲＡＰＤ技术以及ＤＮＡ序列分析技术。在使用这些技术时 ,很多实验的条件往往会影响实验的结果。本文根据作者在实验中的体会 ,结合一些研究者已有的工作 ,对ＲＡＰＤ的原理、影响因素以及在哺乳动物生态学研究中的应用作简单介绍。2 …     

Light Regulation of beta-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.)

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of β-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)⁺RNA with a probe derived from the coding region of a previously characterized soybean β-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of β-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of β-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of β-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.     

大豆遗传转化研究进展

综述了大豆遗传转化体系及其优缺点,转基因大豆的研究成果、生产状况和生物安全性评价,分析了大豆遗传转化中存在的一些问题及其解决办法,展望了未来大豆遗传转化的发展前景。