Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

Physical characterization of a glucose-dehydrogenase-bearing plasmid from ketoacid-producingErwinia herbicola

Erwinia herbicola (ATCC 21998), a facultative anaerobe, has two plasmids: pVQ1 and pVQ2. Curing with mitomycin C indicated that pVQ2 was cryptic but pVQ1, a 7.4-kb plasmid, bears a 4.3SacI fragment which strongly hybridized to the C-terminal region of the glucose dehydrogenase gene ofAcinetobacter calcoaceticus. A restriction map of plasmid pVQ1 is presented.The authors are with the Department of Biotechnology, Regional Research Laboratory, Canal Road, Jammu Tawi-180 001, India;     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针在丁型肝炎病毒核酸杂交中的应用

应用异羟基洋地黄毒甙元(Digoxigenin)标记的RNA探针,检测了人和黑猩猩血清及肝脏中丁型肝炎病毒核酸,并与~(32)P标记同一探针做了比较。结果表明,异羟基洋地黄毒甙元标记的RNA探针的杂交效果与同位索探针一致(同源cDNA0.2pg),可用于人和动物血清及肝脏标本内HDV核酸的检测。     

Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis

Improvements to the sensitivity, speed, and reproducibility of digoxigenin (DIG)-labeled probes and chemiluminescent substrates makes these compounds increasingly popular to detect nucleic acids. High sensitivity and low background are essential in Southern blot analysis, particularly with plant DNA. This article describes a nonradioactive system to detect single-copy genes in transgenic plants. Labeling using the polymerase chain reaction (PCR) was employed to produce highly sensitive and reusable DIG-labeled probes. The background was reduced by immobilizing the DNA onto nylon filters by alkaline transfer and by minimized gel handling; the signal-to-noise ratio was improved by modification of the detection procedure.