2型猪链球菌Fbps基因敲除突变体的构建及毒力分析

目的:敲除2型猪链球菌(SS2)强毒株05ZYH33中的Fbps基因,研究该基因敲除对菌株生物活性及毒力的影响,为深入探讨猪链球菌致病机制提供实验基础。方法:从05ZYH33基因组中扩增Fbps基因上、下游同源臂,从pSET1质粒中扩增氯霉素抗性基因Cm,通过重叠PCR的方法将3个片段整合后连接到温敏自杀载体pSET4s上,电转入05ZYH33感受态细胞,通过改变培养温度实现双交换和质粒丢失,最后经抗性筛选获得敲除株05ZYH33ΔFBPS,分析敲除株的生物学性状,以CD1小鼠作为体外感染模型对突变株和野生株进行毒力比较。结果:PCR分析和测序结果均显示Fbps基因敲除成功,动物实验结果显示Fbps基因敲除后05ZYH33的毒力有所下降。结论:与野生株相比,突变株对小鼠的毒力有所降低。     

Ⅱ型猪链球菌二元信号转导系统2148hk/rr基因敲除突变体的构建

构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH33二元信号转导系统2148hk/rr基因敲除突变体.构建中间为壮观霉素抗性基因,两侧为2148hk/rr编码基因上、下游同源序列的基因敲除质粒,通过同源重组筛选2148hk/rr编码基因敲除突变体.PCR分析和Southern杂交结果均显示2148hk/rr编码基因完全被壮观霉素抗性基因替代,基因敲除突变体构建成功.筛选获得05ZYH33二元信号转导系统2148hk/rr基因敲除突变体,为阐明该调控系统在猪链球菌致病过程中的作用奠定了基础.     

Ⅱ型猪链球菌二元信号转导系统2148hk/rr基因敲除突变体的构建

构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH33二元信号转导系统2148hk/rr基因敲除突变体。构建中间为壮观霉素抗性基因, 两侧为2148hk/rr编码基因上、下游同源序列的基因敲除质粒, 通过同源重组筛选2148hk/rr编码基因敲除突变体。PCR分析和Southern杂交结果均显示2148hk/rr编码基因完全被壮观霉素抗性基因替代, 基因敲除突变体构建成功。筛选获得05ZYH33二元信号转导系统2148hk/rr基因敲除突变体, 为阐明该调控系统在猪链球菌致病过程中的作用奠定了基础。     

2型猪链球菌MocR家族转录调控因子SSU0562基因敲除突变体的构建及毒力分析

目的:构建2型猪链球菌强毒株05ZYH33中MocR家族转录调控因子SSU0562基因敲除的突变株,探索SSU0562基因缺失对细菌基本生物学特性和毒力的影响。方法:构建左右两侧为SSU0562基因上下游的同源序列,中间部分为壮观霉素抗性基因(Spcr)的基因敲除质粒,通过同源重组的方法筛选SSU0562基因敲除突变株Δ0562。对突变株与野生株的基本生物学特性进行系统的比较分析,并且将小鼠作为动物感染的模型来研究突变株的毒力。结果:组合PCR的分析及基因测序结果均表明Spcr完全取代了S.suis2中SSU0562基因位点,表明基因敲除突变体Δ0562构建成功,反转录PCR(RT-PCR)证实了突变株Δ0562中SSU0562基因在转录水平的缺失;在溶血活性、生长速率及对小鼠的致病力方面,突变株Δ0562与野生株05ZYH33相比均无显著差别,然而革兰染色实验显示突变株Δ0562的成链能力明显减弱。结论:猪链球菌强毒株05ZYH33的毒力并未因SSU0562基因的缺失而发生显著性改变,表明SSU0562基因并非猪链球菌的毒力决定因子,但很有可能参与猪链球菌成链能力的调控。     

猪链球菌2型ABC转运蛋白gene0910敲除突变体的构建及活性分析

目的:构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH3389K毒力岛上的ABC转运蛋白gene0910敲除突变体,并初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以猪链球菌2型05ZYH33基因组为模板,扩增gene0910两侧各约500bp左右的片段为上下游同源臂,以pSET1质粒为模板,扩增氯霉素抗性基因Cm为中间片段,采用重叠PCR方法搭建三个片段,并克隆到自杀载体pSET4S上,构建基因敲除的载体。电转化05ZYH33感受态细胞,经30℃双交换和40℃质粒丢失,最后点板法筛选出基因敲除突变体△0910。对突变株和野生株的生物学活性及小鼠的致病性进行了初步比较。结果:PCR分析和测序结果均显示gene0910完全被氯霉素抗性基因Cm所替代,基因敲除突变体构建成功。结论:突变株的生物学活性和对小鼠的致病性与野生株相比差异不显著。     

猪链球菌2型菌毛骨架蛋白编码基因SSU2101敲除突变株的构建及其生物学功能

利用同源重组基因敲除方法构建猪链球菌2型(Streptococcus suis serotype2,S.suis2)中国强毒株05ZYH33菌毛骨架蛋白(Backbone protein,BP)编码基因SSU2101敲除突变株。采用引物特异性PCR分析、Southern杂交及RT-PCR等方法鉴定,证实成功构建了BP基因缺失突变株。生物学特性显示,突变株的菌落形态、溶血活性以及染色特性方面与野生株之间均无明显差异。小鼠致病性试验结果显示,突变株的毒力比野生株显著减弱。研究结果提示菌毛在S.suis2感染致病过程中起重要作用,为系统研究S.suis2菌毛分子装配机制及其生物学功能奠定了基础。     

猪链球菌cAMP结合蛋白基因敲除株的构建及生物学特性

目的:构建猪链球菌2型强毒株05ZYH33 c AMP结合蛋白(CRP)编码基因敲除突变株及基因回复互补株,并探究CRP基因的缺失对细菌生物学特性及毒力的影响。方法:构建中间为壮观霉素抗性基因(Spcr)、两侧为CRP编码基因上下游同源序列的基因敲除质粒,通过同源重组筛选CRP编码基因敲除突变株ΔCRP;构建CRP编码基因的互补质粒,通过电转化敲除株ΔCRP,筛选CRP的基因回复互补株CΔCRP;比较分析突变株、野生株和回复互补株的基本生物学特征的差异,并以小鼠作为动物感染模型对突变株、互补株及野生株的毒力进行评估分析。结果:应用组合PCR和基因测序分析,证实构建了CRP的突变株ΔCRP,并筛选出CRP的回复互补株CΔCRP;逆转录PCR证实在突变株ΔCRP中CRP在转录水平缺失,而在回复互补株CΔCRP中其转录回复;在丰富营养情况下,突变株ΔCRP与野生株的溶血活性、生长速率及对小鼠的致病力均无显著性差异,但突变株的成链能力减弱。结论:CRP编码基因的缺失并未显著改变野毒株05ZYH33的基本生物学特性和毒力,提示CRP可能不是猪链球菌的关键毒力决定因子,其参与碳源代谢等功能有待进一步研究。     

猪链球菌2型05Z33强毒株转录调控因子Rgg基因敲除突变体的构建及毒力分析

目的:构建猪链球菌2型强毒株05ZYH33转录调控因子Rgg的基因敲除突变体,观察其生物学性状,并在动物感染实验中比较敲除株与野生株的毒力差异,为进一步研究猪链球菌转录调控因子在致病中的作用提供实验基础。方法:分别以猪链球菌2型05ZYH33基因组和pSET1质粒为模板,扩增基因SSU05_1997两侧各约500 bp的片段为上下游同源臂,氯霉素(Cm)抗性基因为中间片段,采用重叠PCR方法连接3个片段;连接产物先克隆到T载体上,再经过酶切克隆到温度敏感自杀载体pSET4S上;将构建的基因敲除载体pSET4S-1997电转化入05ZYH33感受态细胞,通过改变培养温度筛选出基因敲除突变体05Z33△rgg;对敲除株和野生株的生物学性状及小鼠和猪的致病性进行了初步比较。结果:PCR分析和测序结果均显示基因SSU05_1997完全被Cm抗性基因所替代,基因敲除突变体构建成功;05ZYH33△rgg对小鼠和猪的致病性与野生株相比无明显差异。结论:转录调控因子Rgg可能和猪链球菌2型的毒力无关。     

猪链球菌2型Ⅳ型分泌系统组分VirD4与毒力相关性分析

摘要:【目的】构建猪链球菌2型强毒株05ZYH33毒力岛89 K上的Ⅳ型分泌系统组分VirD4敲除突变株,初步分析其活性和毒力,为进一步研究猪链球菌2 型在逃避宿主天然免疫杀伤中的作用提供基础。【方法】以05ZYH33基因组为模板,PCR扩增VirD4基因上下游同源臂,以穿梭质粒pSET1为模板,PCR扩增氯霉素抗性基因Cm,通过重叠PCR技术搭建上述3个片段并连接至温敏载体pSET4s,构建基因敲除载体pSET4s∷VirD4;通过同源重组构建基因敲除突变株ΔVirD4;通过体外全血杀伤实验、CD1小鼠竞争感染及攻毒实验 对突变株和野生株的毒力进行比较分析。【结果】获得了基因敲除突变株ΔVirD4,通过对比发现其毒力与野生株相比有所降低。【结论】猪链球菌2型Ⅳ型分泌系统组分VirD 与其毒力相关,并在早期抵抗天然免疫细胞杀伤中发挥一定作用。     

2型猪链球菌SSU0448基因敲除突变株的构建及其毒力分析

【目的】 通过构建2型猪链球菌(SS2)强毒株05ZYH33的SSU0448基因缺失突变株Δ0448和互补株CΔ0448,探索SSU0448基因缺失对细菌基本生物学特性和细菌毒力的影响。【方法】用同源重组基因敲除方法构建筛选强毒株05ZYH33中N-乙酰半乳糖胺和半乳糖胺代谢途径相关转录调节因子SSU0448基因的缺失突变株,比较分析突变株Δ0448与野生株05ZYH33、互补株CΔ0448的基本生物学特性,小鼠毒力实验分析SSU0448基因缺失对细菌毒力的影响。【结果】PCR检测分析显示,SSU0448基因在转化重组体中被壮观霉素抗性基因所替代,表明基因敲除突变株构建成功;同时构建了基因功能互补株CΔ0448。生物学特性实验表明突变株Δ0448在成链能力上较野生株明显减弱,对数生长期稍短,快速到达平台期;而菌落形态、革兰氏染色和溶血活性方面无明显差异;小鼠毒力实验发现,突变株毒力并无显著改变。【结论】SSU0448基因的敲除能够改变2型猪链球菌的成链能力;不影响其侵袭致病能力,可能延缓2型猪链球菌的发病过程,此研究为2型猪链球菌致病感染奠定了基础。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Scales of spatial patterns of distribution of intertidal invertebrates

Few comparative studies of spatial patterns at different scales have examined several species in the same habitat or the same species over a range of habitats. Therefore, variability in patterns among species or among habitats has seldom been documented. This study quantifies spatial patterns of a suite of intertidal snails and a species of barnacle using a range of statistical techniques. Variability in densities was quantified from the scale of adjacent quadrats (over a distance of centimeters) to tens of kilometers. Significant differences in abundances occurred primarily at two spatial scales. Small-scale differences were found at the scales of centimeters or 1–2 m and, for many species on many shores, these accounted for most of the variability in abundances from place to place. These are likely to be determined by behavioural responses to small-scale patches of microhabitat. Large-scale differences in abundance were also found in most species at the scale of hundreds of meters alongshore. These are likely to be due to variation in recruitment (and/or mortality) because of limited dispersal by adults of these species. There was little or no additional variation among shores, separated by tens of kilometers, than was shown among patches of shore separated by hundreds of meters. Identification of the scale(s) at which significant differences in abundance are found focus attention on the processes (and the scales at which these processes operate) that influence patterns of distribution and abundance. Some of the advantages and disadvantages of various procedures are discussed.     

Identification of the site of glycation of human insulin

This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1–5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH₂-terminal Phe¹ residue. This was confirmed by automated Edman degradation with glycated human insulin.