A smoke-derived butenolide improves early growth of tomato seedlings

3-Methyl-2H-furo[2,3-c]pyran-2-one is an active compound isolated from plant-derived smoke water. It has a stimulatory role during seed germination similar to that of smoke or aqueous extracts of smoke. The present study was undertaken to gain insight into the physiological events involved in seed germination and seedling development and which are affected by butenolide using tomato (Lycopersicon esculentum Mill.) cultivar “Heinz 1370” seeds. No stimulatory role on the seed germination of tomato was recorded following the use of the butenolide, however, post-germinative growth of tomato seedlings was significantly improved over the control (P ≤ 0.05). The emergence of the radicle and elongation of the hypocotyls and radicles were accelerated in seeds imbibed with butenolide at 10⁻⁷ M. Flow cytometry studies showed that in butenolide-treated seeds the ratio of cells with replicated DNA was increased. Seedling vigour and weight were significantly increased by the butenolide (P ≤ 0.05). An inverse correlation was observed between the weight of cotyledons and the weight of the hypocotyls and radicle during seedling development. This is an indication that the butenolide is implicated in mobilization and utilization of stored reserve materials in developing tomato seedlings.     

A butenolide, isolated from smoke, can overcome the detrimental effects of extreme temperatures during tomato seed germination

The butenolide, 3-methyl-2H-furo[2, 3-c]pyran-2-one, is an highly active compound isolated from plant-derived smoke. This compound is known to stimulate seed germination in a wide range of plants akin to smoke or aqueous extracts of smoke. The present study attempted to elucidate the role of the butenolide in overcoming detrimental effects of low and high temperatures on tomato seed germination and seedling growth. The germination percentage followed a parabolic curve for temperatures ranging from 10 to 40°C, with 25°C being the optimum for all treatments. Control seeds showed radicle emergence at two extreme temperatures (10 and 40°C) and seedlings failed to develop further, even upon prolonged incubation. By comparison the butenolide-treated seeds grew into phenotypically normal seedlings at these non-optimum temperatures. The smoke–water-treated seeds had an intermediate response as only a fraction of germinated seed developed into normal seedlings. Seedling vigour indices as well as seedling weight were significantly higher (p ≤ 0.05) for butenolide-treated seeds at all temperatures. Furthermore, seedlings developed in the presence of the butenolide had about a 1:1 correspondence between root and shoot length. Butenolide-treated seeds grew better than the control seeds in the temperature shift experiments. A gradual decline in the vigour index values was recorded with an increased duration of incubation at the extreme temperatures. Results of the present study are very important from an horticultural point of view as they indicate the potential use of the butenolide compound in restoring normal seed germination and seedling establishment in tomato below and above optimum temperatures.     

Isolation of Resistance Gene Analogs in Pepper Using Modified AFLPs

An efficient technique for isolation of resistant gene analogs (RGAs) in pepper from silver stained denaturing polyacrylamide gel was developed using a modified amplified fragment length polymorphism (AFLP) strategy. Pepper DNA was digested, ligated and pre-amplified as in a normal AFLP method. The selective amplification was made by using combinations with oligonucleotide primers based on conserved motifs in and around nucleotide binding site (NBS) of known NBS-leucine-rich repeats resistance proteins from known resistant genes. The amplified products were separated by using denaturing polyacrylamide gels and silver staining instead of radioactive labelling. We isolated specific polymorphic AFLP bands directly from the gels with one round of polymerase chain reaction amplification, in order to confirm, after sequencing, that these bands have homologies with products of resistance genes described so far. Two bands (R2: 250 bp and R6: 150 bp) are particularly highlighted because they could be considered as RGAs related to resistance to Phytophthora capsici in pepper, because their sequences have a very high homology with other resistant gene analogs that have already been described. Besides, they were only detected in the resistant parent and in the bulked resistant segregants but not in the susceptible parent or susceptible F₂ segregants. We can conclude that the technique used is clean, quick and efficient for the isolation of RGAs in pepper. This revised version was published online in July 2006 with corrections to the Cover Date.     

蓝狐-卫星DNA的克隆及序列分析

利用限制性内切酶酶切蓝狐基因组, 经琼脂糖凝胶电泳, 对特异性亮带进行克隆、测序及序列分析。结果获得42个卫星DNA序列, 该卫星DNA单体大小为737 bp, G+C含量为51.9%, 单体之间同源性为91%~97%; 每个单体由3个约245 bp的亚重复串联构成, 亚重复之间的同源性为49%~55%; 在物种进化过程中, 该卫星DNA有G+C含量逐渐降低而A+T含量逐渐上升的趋势; 该卫星DNA为犬科动物种属所特有, 与犬着丝粒相关卫星DNA为同类卫星DNA, 同源性为74%, 命名为α-卫星DNA。     

Butenolide from plant-derived smoke enhances germination and seedling growth of arable weed species

We tested the applicability of the recently identified major germination cue from smoke (a butenolide 3-methyl-2Hfuro[2,3-c]pyran-2-one) on 18 weed species from non-fire prone environments. For the study species we compared the relative effectiveness of alternating temperatures, KNO₃, GA₃, smoke water and the butenolide on germination percentage, germination rate and seedling mass. We found that while smoke stimulated germination in a number of species it also had negative impacts on other species. In addition, the butenolide was effective on the widest range of species in terms of enhancing germination percentage, rate and seedling mass. However, none of the treatments, including butenolide were effective on all species. Our data demonstrate that butenolide may have wide applicability as a germination and seedling growth stimulant irrespective of whether the species come from fire-prone habitats.     

苎麻细胞质雄性不育“三系”ISSR 特异片段克隆和序列分析

利用ISSR 分子标记技术对苎麻细胞质雄性不育“三系”mtDNA 进行多态性分析; 在选用的38 个ISSR引物中, 有6 个引物的扩增产物在不育系、保持系和恢复系之间存在差异。对这些特异性片段进行克隆和序列测定, 结果表明: 片段21-MS 全长956 bp , 包含一个525 bp 的完整编码区, 共编码174 个氨基酸。片段31-M􊄯R 全长778 bp , 包含一个404 bp 的不完整编码区, 共编码134 个氨基酸; 其核苷酸和氨基酸序列与已报道的多种植物中的番茄红素β-环化酶基因分别存在71%～76%和73%～77%的同源性。     

<Emphasis Type="Italic">Ptcorp</Emphasis> gene induced by cold stress was identified by proteomic analysis in leaves of <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.     

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR<Subscript>1</Subscript>

Background  

Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR₁), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR₁ action.     

苎麻细胞质雄性不育"三系"ISSR特异片段克隆和序列分析

利用ISSR分子标记技术对苎麻细胞质雄性不育"三系"mtDNA进行多态性分析;在选用的38个IS-SR引物中,有6个引物的扩增产物在不育系、保持系和恢复系之间存在差异。对这些特异性片段进行克隆和序列测定,结果表明:片段21-MS全长956bp,包含一个525bp的完整编码区,共编码174个氨基酸。片段31-M/R全长778bp,包含一个404bp的不完整编码区,共编码134个氨基酸;其核苷酸和氨基酸序列与已报道的多种植物中的番茄红素β-环化酶基因分别存在71～76和73～77的同源性。     

Fluorescent detection of differentially expressed cDNA using SYBR gold nucleic acid gel stain

We describe herein a modified differential gene display (DGD) technique that can be rapidly and simply performed and that eliminates the need for radioactivity by fluorescent visualization of complementary deoxyribonucleic acid (cDNA) bands with SYBR gold nucleic acid gel stain. To streamline the DGD procedure, a number of modifications were employed. Ribonucleic acid isolated from differentially treated populations of human trabecular bone-derived mesenchymal progenitor cells was reverse-transcribed into cDNA using oligo-dT primer, and subsequent amplification of differentially expressed cDNAs was done using arbitrary 25-mer primers and oligo-dT₉ 30-mer primers. Moderate-sized nondenaturing 6% polyacrylamide gels (30 × 20 cm) of 1.5-mm thickness were used for easier handling and increased sample loading capacity. Gels were subjected to electrophoresis overnight, stained with SYBR gold, and visualized and photographed using a commercially available gel imager. DNA bands ranging in size from 100 to 400 bp were visualized directly on an ultraviolet transilluminator, excised from the gel, and reamplified. The cDNA amplicons were subcloned, sequenced, and gene sequences were identified by a Basic Local Alignment Search Tool of genomic databases. Overall, this rapid and functional method proved quite effective for identification of novel genes that may be of interest in studies of cartilage and bone differentiation.     

A micronucleus-limited sequence family in Tetrahymena thermophila: Organization and sequence conservation

During mocronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronu-cleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited. © 1992 Wiley-Liss, Inc.     

Analysis of integration sites of T-DNA insertions in transgenic tobacco plants

DNA fragments containing T-DNA/plant DNA junctions isolated from 17 transgenic tobacco plants were amplified using inverse PCR. Analysis of the nucleotide sequences of 34 cloned DNA fragments revealed 100% homology with vector sequences outside T-DNA in 10 cases. Nine nucleotide sequences had homology with the repeats in the tobacco genome. The percentage of homology varied from 70 to 90%, with the identified repeats belonging to different types. In most clones no homology was revealed with the GENEBANK sequences. Alignment of the sequences truncated during the integration of the left and the right borders of the T-DNA insertions demonstrated significant clusterization (10 bp region) of truncation sites for the left border. Five sequences had identical truncation sites (+23 T) that showed the perferable use of this nucleotide. The AT content varied from 51 to 72% which was close to the total percentage of AT pairs in the tobacco genome.     

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

家蝇幼虫差异表达基因的克隆筛选与分析

目的是利用mRNA差异显示(DDRT-PCR)技术对诱导后家蝇幼虫差异表达基因进行克隆分析。取诱导和未诱导家蝇(Musca domestica vicina)三龄幼虫总RNA进行mRNA差异显示反应,产物经6％非变性聚丙烯酰胺凝胶电泳展开,银染分析后,回收差异显示条带。随机选取4条诱导家蝇三龄幼虫中上调表达的基因条带,进行Northern杂交验证,有2条被验证为真实带,命名为YD1和YD2。对这2条基因片段进行T-A克隆和测序,长度分别为495bp和265bp,登录NCBI运用Blastn程序进行同源性比较,发现两者与任何已知基因同源性都低于70％,提示为两个未报道的新基因,在家蝇免疫反应中可能起着一定的作用。     

Differentially methylated genomic fragments related with sexual dimorphism of rice pests,Sogatella furcifera

Sogatella furcifera (Hovarth) is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation‐sensitive representational difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tramtrack genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S. furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.     

燃烧植物产生烟与热的生理生态效应

燃烧植物产生的烟与热对植物的生理生态功能有重要的影响,相关研究已成为生态学研究的热点之一。植物源烟对一些植物种子的萌发和幼苗生长有促进作用,这种促进作用与GA和细胞分裂素的作用相似。在植物烟水溶液中分离得到了具有促进植物种子萌发作用的化合物丁烯羟酸内酯,该物质具有热稳定性、挥发性和有效浓度范围广等特点。丁烯羟酸内酯可以通过纤维素加热产生,因而几乎所有的植物燃烧产生的烟中都可以产生丁烯羟酸内酯。热因子对植物种子萌发有利作用表现为打破种子休眠、清除限制种子萌发的物理、化学因素和减轻种子病原体等方面。大量研究显示,不同植物对烟与热的响应机理存在显著的差异,这是植物群落过火后物种组成改变的重要原因之一。烟与热因子对植物生理生态作用的研究我国开展较少．这与我国是一个森林、草原火灾频繁的国家是不相称的,加强这方面的研究很有必要。另外,今后我国可以在烟与热因子对植物作用的机理,揭示传统用烟火处理土壤促进农林业植物生长的物理和化学本质,以及这些机理在发展有机农业中运用等方面开展深入的研究。     

大鳞副泥鳅3个Dmrt基因DM保守区的序列分析

摘要: 利用简并PCR克隆技术, 扩增和克隆了大鳞副泥鳅(Paramisgurnus dabryanus)Dmrt基因的DM结构域, 获得了3个具有不同DM序列的克隆。结果表明, 在大鳞副泥鳅基因组中存在Dmrt基因家族的多个成员。同源性比较和系统进化分析显示不同进化地位脊椎动物的Dmrt基因存在高度的进化保守性