Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Probiotic properties of lactic acid bacteria isolated from stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China

A total of 567 lactic acid bacteria (LAB) strains were isolated from the stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China. In order to reduce the number of strains for further examinations, 36 isolates were screened out for further examination whilst the other strains, which had lower probiotic properties, were not suitable for yogurt production due to the absence of growth in pH 3.5 MRS medium and no curding during fermentation, and so were excluded. The result of identification by API, sequence analysis of 16S rRNA and random amplified polymorphic DNA (RAPD) analysis showed that there were three strains of Lactobacillus acidophilus, 10 strains of Lactobacillus rhamnosus, three strains of Lactobacillus casein, three strains of Lactobacillus brevis, two strains of Enterococcus faecium, two strains of Enterococcus faecalis, four strains of Bifdibacterium infantis, three strains of Bifdibacterium brevise, three strains of Bifdibacterium bifidium, two strains of Bifdibacterium adolecentis and one strain of Bifdibacterium longam among the 36 isolates. These strains were evaluated by in vitro methods including survival upon exposure to pH 2.0, 3.0 and/or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. The results presented here show that L. rhamnosus LV108, L. rhamnosus F, B. brevise R39 and B. infantis R42 are acid and bile tolerant, adhere to the cultured human intestinal Caco-2 cell line, antagonistic activity against potential pathogenic bacteria infection in vitro, and so are potential strains for probiotic use.     

成都地区四种食尸性蝇类 mtDNA中 COⅠ基因序列检测

通过检测食尸性苍蝇线粒体DNA(mtDNA)上细胞色素氧化酶亚基Ⅰ(COⅠ)中278bp 基因序列,鉴定食尸性苍蝇的种类,解决依据形态学方法不能鉴定苍蝇卵的种类、很难鉴定幼虫种类的难题 ,作为法医鉴别食尸性苍蝇及其幼虫、卵种类依据。随机采集放置在成都地区室外草地兔尸体 上的4 种15个食尸性苍蝇。利用改进的小型昆虫DNA匀浆方法提取上述苍蝇mtDNA;通过Perkin Elmer 9600扩增仪进行PCR扩增;聚丙烯酰胺非变性凝胶连续缓冲体系垂直电泳和银染显色技 术进行扩增结果检测;PCR胶回收试剂盒纯化;ABI 377测序仪测序;MEGA2.1软件包进行序列 分析和构建系统发育树。在双翅目食尸性苍蝇的种内进化分歧均数小于1%,种间进化分歧均数 大于7%。mtDNA上COⅠ序列分析能有效地对主要的食尸性苍蝇进行种类鉴定。该检测方法快速、 简便和精确,能作为法医鉴别食尸性苍蝇种类的可靠依据。     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd∶YAG激光微束将处于有丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收。将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增。Southern杂交证明4个特定区域的DNA确实来自于小麦基因组。用一系列(42对引物)位于6B染色体和其他染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证。结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体。将6B染色体4个片段的第二轮PCR产物克隆到pGEMT-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×10~5、2.74×10~5、2.45×10~5和2.93×10~5个重组子克隆。每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证。结果显示:插入片段大小在300～1800 bp之间,平均大小为820～870 bp,其中43％～48％的克隆为低/单拷贝序列,42％～47％为中/高拷贝序列。本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础。     

植物与病原真菌互作的形态变化及其生理 生化机制和基因调控

本文从生理、生化、酶学及基因调控等方面对植物与病原真菌互作过程中的形态变化进行了综述,以期为植物抗病方面的研究提供参考。     

大肠杆菌TdcC、SstT和LIV-1系统缺失对胞外L-苏氨酸积累的影响

【目的】比较分析苏氨酸吸收系统TdcC、SstT和LIV-1缺失对大肠杆菌吸收和积累胞外苏氨酸的影响。【方法】从菌株E.coli W3110出发,敲除tdcC、sst T和liv J基因,构建Tdc C、SstT和LIV-1系统单缺失和多缺失菌株,将过量表达苏氨酸操纵子基因的重组质粒pKKthr AC1034TBC分别转入原始菌和重组菌,考察各菌株吸收和积累胞外苏氨酸的能力。【结果】敲除tdc C和sst T基因的重组菌T04的苏氨酸吸收能力比原始菌W3110降低了43.28%,T04(pKKthr AC1034TBC)胞外苏氨酸积累量最高达到1.09 g/L,比对照菌W3110(pKKthr AC1034TBC)高出172.5%。敲除tdcC、sstT和livJ基因的重组菌T07的苏氨酸吸收能力比T04降低了12.97%,然而T07(pKKthr AC1034TBC)胞外苏氨酸积累量最大为0.63 g/L,与T04(pKKthr AC1034TBC)相比降低了42.2%。【结论】阻断Tdc C和Sst T系统,能有效降低大肠杆菌吸收苏氨酸的能力,提高苏氨酸的胞外积累量。阻断LIV-1系统,虽然能减少大肠杆菌对苏氨酸的吸收,却不利于菌株积累胞外苏氨酸。     

浙江古田山自然保护区野含笑群落特征研究

野含笑为我国省级珍稀濒危植物。通过对野含笑群落的特征进行分析和研究,结果表明。野含笑群落植物种类丰富,科属组成分散,区系成分复杂。从属的地理成分来看,热带分布的属明显多于温带分布的属。群落的生活型以高位芽植物为主,地面芽植物次之。该群落叶的性质以小型叶、单叶、革质、非全缘为主。群落垂直结构复杂,地上成层明显,可分为乔木层、灌木层和草本层,并有一定数量的层间植物。灌木层一乔木层一草本层物种多样性依次递减。野含笑种群的年龄结构属稳定型。     

Changes of zeatin riboside content in rice plants due to infestation by Nilaparvata lugens (Homoptera: Delphacidae)

The effect of Nilaparvata lugens (St?l) (Homoptera: Delphacidae), infestation on the content of zeatin ribosides (ZR) in rice plants was investigated with enzyme-linked immunosorbent assay. Hydroponics experiments were conducted on 'Zhendao 2' rice, in which plants were subjected to N. lugens infestation at three nonhopperburn-causing densities (15, 30, and 60 nymphs per hill) for 2, 4, 6, and 8 d and at one hopperburn-causing density (240 nymphs per hill) for 2, 4, and 6 d, respectively. When rice plants were infested at the nonhopperburn-causing densities, ZR content in leaves varied significantly with the infestation density. Compared with the control plants, ZR content in rice leaves decreased significantly after infestation by 60 nymphs per hill for 2 d, but it tended to increase due to prolonged infestation at all the nonhopperburn-causing densities. In contrast, ZR content in rice roots significantly reduced after the plants being infested at the density of 15 nymphs for 2 d and at all densities for prolonged duration, except for the plants infested by 60 nymphs for 6 and 8 d, in which the ZR content increased or did not change significantly. However, infestation at the hopperburn-causing density caused significant reduction in ZR content in rice roots, regardless of infestation duration, and in rice leaves from the plants subjected to 2-d infestation. These results are discussed in relation to the possible physiological reaction of rice plants to N. lugens infestation and the resultant severe damage or hopperburn.     

Molecular and biochemical characterization of three WD-repeat-domain-containing inositol polyphosphate 5-phosphatases in Arabidopsis thaliana

Type II inositol polyphosphate 5-phosphatases (5PTases) in animals and yeast have been known to be important for regulating inositol and phospholipid signaling by hydrolyzing phosphate from both inositol polyphosphates and phosphoinositides. However, the molecular and biochemical properties of type II 5PTases in plants have not yet been studied. In this report, we show that three Arabidopsis genes, At5PTase12, At5PTase13 and At5PTase14, encode proteins with a 5PTase domain and a WD-repeat domain, a novel combination present only in plant 5PTases. We demonstrate that these genes are differentially expressed in Arabidopsis organs and At5PTase13 is induced in response to ABA and wounding treatments. Our biochemical studies reveal that although both At5PTase12 and At5PTase13 exhibit phosphatase activity toward only Ins(1,4,5)P3, At5PTase14 hydrolyzes phosphate from PI(4,5)P2, PI(3,4,5)P3 and Ins(1,4,5)P3 with the highest substrate affinity toward PI(4,5)P2. All three At5PTases require Mg2+ for their phosphatase activities. Our molecular and biochemical characterization of three WD-repeat-domain-containing At5PTases provides a foundation for further elucidation of their cellular functions in Arabidopsis.     

Amphivasal vascular bundle 1, a gain-of-function mutation of the IFL1/REV gene, is associated with alterations in the polarity of leaves, stems and carpels

In Arabidopsis stems, the vascular bundles in the stele are arranged in a ring-like pattern and the vascular tissues in each bundle are organized in a collateral pattern. We have shown previously that the semidominant amphivasal vascular bundle 1 (avb1) mutation transforms the collateral vascular bundles into amphivasal bundles and disrupts the ring-like arrangement of vascular bundles in the stele. In this study, we show that the avb1 mutation occurred in the putative microRNA 165 target sequence in the IFL1/REV gene and caused an amino acid substitution in the putative sterol/lipid-binding START domain. We present direct evidence that the wild-type IFL1/REV mRNA was cleaved within the microRNA 165 target sequence and the avb1 mutation resulted in an inhibition of cleavage and a higher level accumulation of full-length mRNA, suggesting a role of microRNA 165 in the regulation of IFL1/REV gene expression. In addition to an alteration in vascular patterning, the avb1 mutation also caused dramatic changes in fiber cell wall thickening and organ polarity, including aberrant formation and proliferation of cauline leaves and branches, production of trumpet-shaped leaves with reversed adaxial-abaxial identity, ectopic growth of carpel-like structures on the outer surface of carpels, and fasciation of inflorescence. Ectopic overexpression of the avb1 mutant cDNA not only phenocopied most of the avb1 mutant phenotypes but also led to additional novel phenotypes such as formation of leaves with extremely narrow blades and ectopic production of branches in the axil of siliques. Taken together, these results suggest that the avb1 gain-of-function mutation of the IFL1/REV gene alters the positional information that determines vascular patterning and organ polarity.