利用RAPD技术快速鉴定番茄体细胞无性系变异

从以镰刀菌酸为选择剂筛选的番茄再生植株和未经筛选的植株叶片中提取ＤＮＡ,建立了适合番茄ＲＡＰＤ分析的ＰＣＲ条件,进一步在６０个随机引物中找到了４个可用于鉴定四个番茄品种体细胞无性系变异的引物。利用该法鉴定体细胞无性系变异不仅简单、迅速、可靠,而且因ＤＮＡ的用量少,不影响被检植株的后期生长,可尽早淘汰那些生理适应性而非分子水平发生变异的再生植株。     

八种昆虫离体细胞系对灭多威农药的敏感性研究

采用ＭＴＴ法,研究了灭多威农药对八昆虫离体细胞系敏感性的差异。结果显示,同一浓度处理下,不同细胞系细胞病变的程度不同,敏感性差异很大。ＬＣ５０值在１０＾２－１０＾５μｇ／ｍｌ范围内变化,其中,白纹伊蚊细胞系是最敏感的,而小菜蛾细胞系几乎不敏感。     

昆虫离体细胞总膜蛋白的提取及双向电泳分析

探讨适用于双向电泳的昆虫离体细胞总膜蛋白提取技术及适于双向电泳染色的高灵敏度蛋白质银染技术。以粉纹夜蛾细胞系BT1-TN-5B1-4为材料,比较了传统的Kwa法和Sigma公司的ProteoProp^TMMEMBRANE EXTRACTION KIT提取BT1-TN-5B1。离体细胞总膜蛋白,SDS—PAGE及双向电泳结果表明Sigma公司试剂盒提取的总膜蛋白效果较好,并且适用于双向电泳分析;比较了两种蛋白银染方法,确定并优化了一种灵敏度较高适于双向电泳的银染方法,得到了理想的膜蛋白2-DE图谱。     

RAPD鉴定栽培稻与野生稻体细胞杂种

利用随机引物扩增DNA(RAPD)技术,对栽培稻和野生稻原生质体融合获得的体 细胞杂种进行了鉴定。证实了它们包含有双亲的基因组成分。但来自双亲的基因组成分并不是对等的。一些体细胞杂种含有一个亲本更多的基因组成分,而另一些相反。利用RAPD数据和聚类图讨论了体细胞杂种和双亲的亲缘关系。     

5S rDNA间隔序列分析和RAPD技术用于鉴定体细胞杂种

主要对狭叶柴胡(Bupleurum scoronerifolium)与川西獐芽菜(Swerita musstii Franch) 的科间体细胞杂种愈伤组织、小麦(Triticum aestivum)与燕麦(Avena sativa)的族间体细胞杂种愈伤组织及再生植株进行5S rDNA 间隔序列及RAPD 分析鉴定, 确证了它们为体细胞杂种。     

RAPD分析─鉴定柑桔体细胞杂种的快速方法

本文利用改进的DNA提取方法,从Volkamer柠檬(Citrus volkameriana Ten. and Pasq.)和酸橙(C. aurantium L.)及其原生质体杂种植株的叶片中抽提总DNA,进行RAPD(Random Amplified Polymorphic DNA)分析。结果表明: 在随机选取的15种引物中,有10种可单独或与其它引物一道鉴定这一组合的体细胞杂种。与形态学性状观察、同工酶及ONA杂交分析等方法比较,RAPD分析是一种可在试管苗期即可直接、准确、快速鉴定柑桔体细胞杂种的方法。     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

利用RAPD分析鉴定花椰菜杂种纯度

应用随机扩增多态性DNA(RAPD)分析方法,鉴定花椰菜F1代杂种纯度。筛选出20个10bp随机引物对杂种F1代和父母本基因组DNA进行RAPD分析,共获得扩增片段124条,分子量在0.3-3kb之间,其中2个引物S120和S174可用来鉴定杂种纯度。RAPD分析结果与田间形态鉴定结果基本一致,表明RAPD分析方法适用于花椰菜杂种的纯度鉴定。     

应用RAPD标记法鉴定甘蓝型油菜杂种纯度

近年 ,华中农大作物遗传改良国家重点实验室国家油菜改良武汉分中心的在职研究生段院和刘平武等 ,对用RAPD标记鉴定甘蓝型油菜杂种H990 9的纯度作了研究和报道。他们对杂交组合H990 9两亲本的指纹图谱作了分析研究 ,选出S374、SI334、SI36 5、UBC5 2 34个引物作杂种的纯度鉴定     

用RAPD技术鉴定小麦属间不对称体细胞杂种

用随机引物扩增多态DNA(RAPD)技术对三种不同组合:小麦(Triticum aestivum)( )簇毛麦(Haynaldia villosa);小麦( )羊草(Leymus chinensis)和小麦( )高冰草(Agropyron elongatum)的属间不对称杂种进行分子鉴定,不同杂种植株的基因组经随机引物扩增后,均出现双亲的多态特异产物,证实它们含有双亲的基因组。将引物OPJ-12扩增的高冰草多态特异产物(分子量为0.77bp的DNA片段)分离纯化并标记作探针,用Southern杂交证明了小麦( )高冰草杂种经OPJ-12扩增的0.77kbp特异片段与高冰草这一片段具有同源性。本文结果证明,RAPD技术可作为小麦属间不对称体细胞杂种的一种快速、简便、有效的分子鉴定方法。     

Mechanism of Action of Bacillus thuringiensis Insecticidal Delta-endotoxin on Insect Cells in Vitro

Cultured insect cells, TN-368 from the cabbage looper, swelled and burst upon treatment with the enzyme-digested delta-endotoxin of Bacillus thuringiensis var. aizawai. The cytotoxic sweelling was depended upon the amount of the delta-endotoxin added and the concentration of NaCl or KC1 in the isotonic solution. The concentration of Na⁺ in the swollen cells approximately doubled in isotonic NaCl, while that of K⁺ decreased to 10% of the original cellular concentration. The cell swelling was inhibited by tetrodotoxin and also by ouabain in only KC1 isotonic solution. On the other hand, 4-aminopyridine stimulated the swelling in the isotonic KC1 solution, These results indicate that the delta-endotoxin induces the stimulation of Na+ influx and K+ efflux in the isotonic NaCl solution, and also stimulates the Na⁺, K⁺-ATPase in the isotonic KC1 solution. The cytotoxic swelling was also blocked by cAMP, AMP, ATP, GTP, and NAD, but not by adenosine and GMP. These results suggest the participation of nucleotide derivatives in the action of delta-endotoxin.     

Applying Multiphoton Imaging to the Study of Membrane Dynamics in Living Cells

The endomembrane system of a cell is a highly dynamic, ephemeral structure that is difficult to visualize. Reconstructions from sections of fixed material can provide high-resolution information on intercellular membrane architecture, but such techniques are fraught with artifacts and are of little help in understanding the dynamics of intracellular membrane traffic. Recently, the availability of fluorescent membrane probes and the development of techniques for optically sectioning intact specimens have allowed glimpses of membrane dynamics to be visualized in living tissue. In this review we discuss the potential of a new optical sectioning technique, multiphoton imaging, for visualizing membrane dynamics in living cells. Multiphoton microscopy offers an unparalleled ability to obtain images from deep within specimens while minimizing the effects of phototoxicity.     

烟草初生胚乳细胞在微室培养中的发育

烟草初生胚乳细胞在微室培养中的发育 李师弢 房克凤 杨弘远*     

大鼠髓核细胞原代培养及表型鉴定

目的:探讨Ⅱ型胶原酶联合透明质酸酶消化分离培养髓核细胞及免疫细胞化学表型鉴定的可行性。方法:无菌条件下分离SD大鼠凝胶状髓核,采用Ⅱ型胶原酶联合透明质酸酶消化分离髓核细胞并连续培养,倒置相差显微镜下观察,随后进行免疫细胞化学染色检测不同代次髓核细胞HIF-1、Ⅰ、Ⅱ型胶原、MMP2及蛋白聚糖的表达情况,并给予MTT法测定髓核细胞生长曲线。结果:Ⅱ型胶原酶联合透明质酸酶分离培养原代髓核细胞需要12 d左右贴壁,达95%融合需要34 d,而传代髓核细胞贴壁速率明显增快至10 h,且其倍增时间约为2.5 d;免疫细胞化学显示髓核细胞均表达HIF-1、Ⅰ、Ⅱ型胶原、MMP2和蛋白聚糖,且随着髓核细胞传代其HIF-1α、HIF-1β、Ⅰ型胶原及MMP2表达均增加,但Ⅱ型胶原表达降低,而蛋白聚糖表达无明显差异;MTT法显示随着髓核细胞传代其增殖有所减缓。结论:Ⅱ型胶原酶联合透明质酸酶可成功分离髓核细胞,提高培养效率,且HIF-1α、HIF-1β、Ⅰ、Ⅱ型胶原及MMP2可作为髓核细胞表型分子用于髓核细胞的鉴定。     

昆虫细胞无血清培养

血清是细胞培养基常用的添加剂。目前应用最广泛的动物血清是胎牛血清。随着现代细胞生物学在细胞和组织培养方面的进步以及细胞培养方法的标准化,人们更多的注意到了胎牛血清收集中的伦理道德问题。按照3Rs的原则,科学家希望通过减少血清用量和开发使用血清替代物的方法来减少每年对血清的需求;另外由于血清成分并不明确,考虑到改进细胞和组织培养方法的要求,很多无血清细胞培养基陆续开发成功,成为替代胎牛血清的一个比较科学的方法。     

Ultrastructural Study of Proplastid Ontogeny in Tobacco Mesophyll Cells in Vitro

Since the discovery of plastid DNA the continuity of plastids has well been established. It is known that in plant cultures a form of plastid can differentiate into others. However, only a little has been made in studing chloroplast dedifferentiation in vitro. In the work present here, we reported on ultrastructural changes of chloroplasts dedifferentiation and the proplastid origin in the mesophyll cells of cultured tobacco leaf explant. Fully expanded leaves of haploid tobacco (cv. Ge Xin No. 1) were cut into pieces of 5–6 mm width. These were inoculated on MS medium supplemented with 1 mg/L 2,4-D and 1 mg/l kinetin. The cultures were maintained at (30±2) ℃ and illuminatied by a bank of fluorescent lamps. For electronmicroseopic investigation, after 0, 1, 2, 3, 6 days of culture small leaf fragments were cut off along the cut edges of the explants. The samples were fixed and processed in the manner as described earlier. The sections were examined with a Hitachi HU-11A or a JEM-100CX electronmicroscope. Electronmicroscopic observation shows that the uncultured mesophyll cells are highly vacuolete, with a thin peripheral layer of cytoplasm in which a nucleus and some chloroplasts and other organelles are found in it. But these cells do not contain proplastids (Fig. l). In the explants cultured for 1 day there are no obviously changes in mesophyll cells, except a few cytoplasmic strands extend from periphery to central vacuole. At 2 days of culture quite obvious changes can be detected. A increase in the amount of cytoplasm becomes apparent and transvacuolar cytoplasmic strands grow up. Following cytoplasmic growth, the nucleus and chloroplasts move away from the peripheral cytoplasm and enter the central vacuolate zone (Fig. 2). At this stage some of mesophyll cells have completed the first cell division. After 3 days of culture numerous mesophyll cells have undergone several divisions and formed multicellular masses. In those subdivided cells a more important change of the chloroplasts is the occurrence of protrusions which we call proplastid buds. This phenomenon has also been named as chloroplast budding. According to observations on a large amount of sections chloroplast budding is a common phenomenon in the dedifferentiating mesophyll cells of tobacco leaf explants. Fig ure 3 exhibits a typical profile of a chloroplast with a proplastid bud. The proplastid buds observed are generally long-oval in shape and 1.0–2.5 μm long and about 0.5–0.7 μm thick. These dimensions agree with those of proplastids in meristematie cells. Inside of proplastids ribosomes and electron opaque areas containing DNA fibrils can be seen (Fig. 3). Near the proplastid buds proplastids can often be found (Fig.5). According to above observations we can conclude that the proplastids in dedifferentiating mesophyll cells originate from the proplastid buds by chloroplast budding. The newly formed proplastids usually surround the nucleus and sometimes undergo equal division to increase their number (Figs.5, 6). There are no inner membranes in the newly formed proplastids except vesicles connected with inner membrane of the envelope (Fig.7). While the proplastids are continuously produced, the chloroplasts themselves are filled with starch and gradually turned to large amyloplasts (Fig.5). On the other hand, a few of chloroplasts can divide into equal parts following the chloroplast budding (Fig.4). Israel and Steward (1967) suggested that when cultured carrot cells developed into plantlets the chloroplasts turned into leucoplastids, chromoplastids or proplastids. However, they did not describe how chloroplast became a proplastid. Several investigators reported that the chloroplasts in the dedifferentiating cells gradually lost their grana and intergranal lamellae and then became eueoplasts or proplastids. But according to our observation in tobacco explants, the initiation of proplastids is due to unequal division of chloroplasts, i.e. “budding fission” as described by Malzan and Miihlethaler in Splachnum ampullaceum. Since the proplastid is an organelle characteristic of meristematie cells, the ontogeny of proplastids and its control mechanism should be very important in studing cell dedifferentiation.     

A Novel Technique for Identifying the Instar of Field-Collected Insect Larvae

Many field studies of insects have focused on the adult stage alone, likely because immature stages are unknown in most insect species. Molecular species identification (e.g., DNA barcoding) has helped ascertain the immature stages of many insects, but larval developmental stages (instars) cannot be identified. The identification of the growth stages of collected individuals is indispensable from both ecological and taxonomic perspectives. Using a larval–adult body size relationship across species, I present a novel technique for identifying the instar of field-collected insect larvae that are identified by molecular species identification technique. This method is based on the assumption that classification functions derived from discriminant analyses, performed with larval instar as a response variable and adult and larval body sizes as explanatory variables, can be used to determine the instar of a given larval specimen that was not included in the original data set, even at the species level. This size relationship has been demonstrated in larval instars for many insects (Dyar’s rule), but no attempt has been made to include the adult stage. Analysis of a test data set derived from the beetle family Carabidae (Coleoptera) showed that classification functions obtained from data sets derived from related species had a correct classification rate of 81–100%. Given that no reliable method has been established to identify the instar of field-collected insect larvae, these values may have sufficient accuracy as an analytical method for field-collected samples. The chief advantage of this technique is that the instar can be identified even when only one specimen is available per species if classification functions are determined for groups to which the focal species belongs. Similar classification functions should be created for other insect groups. By using those functions together with molecular species identification, future studies could include larval stages as well as adults.