Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.     

Repeated transfer of small RNA virus populations leading to balanced fitness with infrequent stochastic drift

The population dynamics of RNA viruses have an important influence on fitness variation and, in consequence, on the adaptative potential and virulence of this ubiquitous group of pathogens. Earlier work with vesicular stomatitis virus showed that large population transfers were reproducibly associated with fitness increases, whereas repeated transfers from plaque to plaque (genetic bottlenecks) lead to losses in fitness. We demonstrate here that repeated five-plaque to five-plaque passage series yield long-term fitness stability, except for occasional stochastic fitness jumps. Repeated five-plaque passages regularly alternating with two consecutive large population transmissions did not cause fitness losses, but did limit the size of fitness gains that would otherwise have occurred. These results underscore the profound effects of bottleneck transmissions in virus evolution.     

Genetic variability in two hydrophilous species ofPotamogeton,P. pectinatus andP. filiformis (Potamogetonaceae)

Genetic variability inPotamogeton pectinatus andP. filiformis was studied by means of isozymes. The overall levels of variability were similar to some other well studied hydrophilous species, and were shown to be distributed more between than within populations. This partitioning of variability was attributed to three main factors. (1) Clonal growth (as measured by the frequency of multi-enzyme phenotypes) was shown to be a major factor in both species, although more important inP. pectinatus. (2) Low levels of sexual reproduction were shown to be a likely contributor to the partitioning of variability inP. pectinatus; such reproduction is probably limited by seedling recruitment rather than by infrequent flowering. (3) Geographical isolation was indicated as a factor regulating gene flow at distances of more than about 1000 km in both species, with the data suggesting that dispersal between populations is mainly by seed rather than by vegetative means.     

可育的抗除草剂溴苯腈转基因小麦

报道了采用微粒轰击（Ｍｉｃｒｏｐｒｏｊｅｃｔｉｌｅ ｂｏｍ ｂａｒｄｍ ｅｎｔ） 幼胚将除草剂抗性基因导入小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ Ｌ．）的转化研究。实验共使用了１３ 个小麦品种, 从开花后１４～１８ ｄ 的籽粒中剥取幼胚, 植物表达质粒含有ＣａＭＶ ３５Ｓ启动子控制的除草剂溴苯腈抗性基因ｂｘｎ 以及筛选标记基因ＮＴＰⅡ。采用高压放电基因枪,用质粒ＤＮＡ 包被的钨粒轰击预培养３ ｄ 的幼胚。在含有卡那霉素类似物ｇｅｎｅｔｉｃｉｎ Ｇ４１８ｓｕｌｐｈａｔｅ 的ＭＳ培养基上, 经过多步骤筛选和分化, 从８００ 多个幼胚中获得了１６ 株转化苗。除草剂抗性鉴定和Ｓｏｕｔｈｅｒｎ 杂交分析证明, 其中４ 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为６ 个月, 因此, 该方法是一项快速有效的基因导入技术     

泡沙参同工酶基因位点的遗传分析

利用聚丙烯酰胺凝胶电泳技术 ,对来自天然群体 (居群 )的泡沙参 (Adenophora potaninii Korsh.)及其人工杂交子代进行了 8种同工酶的电泳检测和谱带遗传分析 ,以确定编码这些酶系统的基因位点和等位基因。选用 4种不同的凝胶缓冲系统 ,对下列不同酶系统进行了酶谱的遗传分析 :天冬氨酸转氨酶 (AAT)、酯酶 (EST)、甲酸脱氢酶 (FDH)、谷氨酸脱氢酶 (GDH)、异柠檬酸脱氢酶 (IDH)、乳酸脱氢酶(LDH)、苹果酸酶 (ME)和超氧化物歧化酶 (SOD)。结果表明 ,这 8种酶系统至少由 1 8个基因位点编码 ,其中 1 2个位点为遗传稳定的等位酶位点 ,是可靠的遗传标记。酶谱的分离式样表明 ,EST为单聚体结构 ,AAT、FDH、IDH、SOD为二聚体结构 ,GDH为六聚体结构。最后对同工酶的器官和发育特异性以及同工酶基因位点的遗传分析进行了讨论     

小麦与玉米及鸭茅状摩擦禾远缘杂交的胚发育过程中同工酶和蛋白质的变化

通过比较小麦与玉米及鸭茅状摩擦禾属间杂交获得的胚与小麦正常自交的胚之间在不同发育时期过氧化物酶和酯酶的同工酶谱,发现过氧化物酶同工酶表现出时空顺序的特异性变化。在同一发育时期,远缘杂交的具胚子房和无胚子房之间存在过氧化物酶同工酶谱的差异,这可能涉及到与胚发育相关的同工酶的出现。远缘杂交的具胚子房和正常自交的小麦子房之间也有一定的酶谱差异。同时,同一材料还表现出不同发育时期的过氧化物酶酶谱差别。在远缘杂交后的胚发育期间,酯酶同工酶的时空表达不如过氧化物酶显著。此外,对远缘杂交后的胚中的水溶性蛋白质进行了SD S-PAGE分析,初步的分析结果表明,可能存在与胚发育相关的蛋白质。     

甘蓝类无蜡粉亮叶性状遗传规律及其利用的研究

我们于1987年从普通结球甘蓝“迎春”品种自交二代群体中,发现了无蜡粉亮叶甘蓝突变株, 经过多年对其遗传规律进行的研究,认为这一无蜡粉亮叶性状是由一对隐性纯合基因控制。利用这一性状可培育结球甘蓝及其它甘蓝类具有这同一性状的新类型、新品种,提高其品质,更可作一代杂种利用的标记性状,充分发挥一代杂种的优势。     

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.     

A laboratory-based method to measure relative pesticide and spray oil efficacy against broad mite, Polyphagotarsonemus latus (Banks)(Acari: Tarsonemidae)

Six pesticides and two spray oils were tested against Polyphagotarsonemus latus. The chemicals were evaluated under laboratory conditions, requiring the development of a novel bioassay method, which is reported here. The pesticide toxicities fell into three distinct groups, namely abamectin, conventional pesticides and oils. The relative pesticide toxicities at the LC₅₀ level were abamectin 4.9×10^-8 g ai l^-1, endosulfan 1.1×10^-3 g ai l^-1, fenpyroximate 2.3×10^-3 g ai l^-1, pyridaben 4.1×10^-3 g ai l^-1, tebufenpyrad 4.4×10^-3 g ai l^-1, dicofol 4.5×10^-3 g ai l^-1, petroleum spray oil 3.4×10^-1 g ai l^-1 and canola oil 4.1×10^-1 g ai l^-1. The calculation of the LC_99.9 values allows for resistance monitoring in P. latus and the suggested discriminating concentrations are abamectin 1.0×10^-4 g ai l^-1; endosulfan, pyridaben and dicofol 1.0×10^-1 g ai l^-1 fenpyroximate and tebufenpyrad 5.0×10^-1 g ai l^-1.