利用培养和免培养方法研究红壤的真菌多样性

利用培养和免培养方法研究了一个云南红壤的真菌多样性。扩增的18S rRNA基因片段经过RsaⅠ、HinfⅠ、HaeⅢ三种限制性内切酶消化后,培养方法共获得16种限制性酶切长度多态类型（Restriction Fragment Length Polymorphism,RFLP）,而免培养方法仅获得了12种RFLP类型。每个代表类型经DNA测序及系统发育分析表明,免培养方法得到的真菌全部属于子囊菌门。优势物种与Aspergillus niger类似,占免培养真菌的40．9％。其次是Penicillium属和Paecilomyces属真菌,分别占免培养真菌的18％和17％。此外,有13个克隆仅和环境克隆具有较近的亲缘关系。培养方法获取的真菌包括Ascomycota门（11．5％）、Zygomycota门（86．5％）以及Basidiomycota门（1．9％）真菌,优势克隆来自Mortierella属,占全部培养克隆的55．8％,有21．2％的序列与Absidia glauca相似。两种方法观察到的真菌物种完全不同。     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

用富集文库克隆人胰岛素基因组基因

通过构建可富集人胰岛素基因的λ噬菌体文库,克隆了人胰岛素基因组基因．首先从中国人血液白细胞中提取到人基因组ＤＮＡ,用ＥｃｏＲⅠ和ＢｇｌⅡ对基因组ＤＮＡ进行全酶切,经０．４％琼脂糖凝胶电泳,特异回收９．５ｋｂ左右的ＤＮＡ片段．将该片段与λＥＭＢＬ３／ＢａｍＨⅠ臂连接,构建成一个特殊的人基因组λ噬菌体文库（富集文库）,效价为２×１０４．同时采用ＰＣＲ方法及用引物Ⅰ：５′ＧＧＡＣＡＧＧＣＴＡＣＡＴＣＡＧＧＡＡＧＡＧＧ３′,引物Ⅱ：５′ＣＴＧＣＧＴＣＴＡＡＴＴＧＣＡＧＴＡＧＴＴＣ３′,从人基因组ＤＮＡ中扩增出一段含胰岛素基因的１．３６ｋｂＤＮＡ片段,做为放射性标记探针,对文库进行了噬菌斑原位杂交筛选,从１×１０４个噬菌斑中筛选到一个含人胰岛素基因组基因的阳性克隆,并进一步完成了亚克隆和该基因１７３２ｂｐＤＮＡ序列的测定．结果该基因的１７３２ｂｐＤＮＡ序列包括部分５′端和３′端与国外发表的人胰岛素α型等位基因的序列相同     

一种改良的启动子序列克隆的染色体步查法

利用染色体步行法,从已知DNA序列克隆侧翼未知序列是非常有效的方法之一,但由于所选用的特定限制性内切酶对目标基因组不能酶解成合适大小的片段,因而受PCR扩增能力的局限,往往扩增不出有效产物. 针对这一点,这里我们介绍一种简单有效的改良方法,它包括以下步骤：首先用不同的限制性内切酶(包括平末端和粘性末端) 酶解目标基因组DNA,接着,选择能将基因组酶切成弥散、分布均匀的限制性内切酶,如DraⅠ和HindⅢ,合成相对应的接头;然后,选择弥散的、分布均匀的限制性内切酶的酶解产物,构建成含相应接头的基因组DNA文库,用作PCR的模板;最后,用接头引物和特异引物,通过巢式PCR扩增目的片段,获得了理想的扩增效果.采用改进后的染色体步查法,有效地从较复杂的棉花核DNA中克隆出6个棉花启动子序列.     

pBR322DNA与8-甲氧补骨脂素光化学反应部位的碱基顺序特异性

用限制性核酸内切酶酶切试验研究了质粒pBR322 DNA经8-MOP及近紫外线作用后损伤部位的碱基顺序特异性。实验研究发现PUVA损伤的DNA在HindⅢ及RsaⅠ识别位置上酶切反应受到严重抑制,而在SphⅠ,EcoRⅠ,PvuⅡ,BamHI,PstⅠ识到位置上抑制轻微。通过对不同识别位置上碱基顺序及其光化学反应敏感性的分析,推断出DNA的TpA顺序可能是最易接受8-MOP光化学反应的部位。     

Abundance and polymorphism of simple repetitive DNA sequences in Bmssica napus L.

Summary The Brassica napus genome has been investigated by DNA fingerprinting with six synthetic oligonucleotide probes complementary to simple repetitive sequences, namely (GATA)₄, (GACA)₄, (GGAT)₄, (CA)₈, (CT)₈ and (GTG)₅. While all sequence motifs were found to be present in the B. napus genome, their organization and abundance varied considerably. Among the investigated probes, (GATA)₄ revealed the highest level of intraspecific polymorphism and distinguishes not only between cultivars but even between different individuals belonging to the same cultivar. In contrast, (GTG)₅, (GACA)₄ and (GGAT)₄ produced relatively homogeneous fingerprint patterns throughout different cultivars, while hybridization to (CT)₈ and (CA)₈ resulted in only a few weak bands superimposed on a smear. The isoschizomeric pair Hpa II and Msp I revealed the presence of methylated cytosines in the vicinity of (GATA)_m repeats. The applicability of simple repetitive sequence polymorphisms as molecular markers for Brassica species is discussed.     

鲤鱼mtDNA酶切图谱的构建

采用密度梯度离心法及ＲＮａｓｅ消化法制备并纯化了鲤（ＧｙｐｒｉｎｕｓｃａｒｐｉｏＬｉｎｎａｅｕｓ）肝脏线粒体ＤＮＡ（ｍｔＤＮＡ）,用１０种限制性内切酶对ｍｔＤＮＡ进行了分析,鲤鱼ｍｔＤＮＡ分子量约１０．１２×１０￣６,约１６．４９ｋｂ．ＳａｌⅠ、ＰｓｔⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＢｇｌⅠ、ＰｖｕⅡ、ＸｈｏⅠ、ＥｃｏＲⅠ、ＤｒａⅠ和ＨｉｎｄⅢ分别为１、１、３、３、３、４、１、４、４、和６个切点。根据单酶解及双酶解结果,构建了鲤ｍｔＤＮＡ１０种具酶３０个切点的限制性酶切图谱。     

Restriction Endonuclease Digestion of Amplification Products Generated by RAPD Technique in a Population of Glycine soja

n a population of Glycine soja L., the polymorphic loci could be hardly detected by RAPD markers, using several primers. These non-polymorphic amplification products were cleaved by some restriction endonuclease, such as Msp Ⅰ , Hinf Ⅰ , Taq Ⅰ , EcoR Ⅰ , Sal Ⅰ , Dra Ⅰ and Hae Ⅲ. After cleaving, the digested amplification products were detected on polyacrylamide gel electrophoresis with silver staining. It was found that: 1 ) some restriction endonucleases could not, and some others could effectively digest the random amplication products of the DNAs of G. soja; 2) some endonucleases could produce polymorphic DNA fragments after digestion of the non-poly-morphic products, but others could not even after digestion; 3) non-polymorphic amplification products amplified by some primers could produce polymorphic DNA fragments after digestion, while those by other primers could not. It could be concluded that the restriction endonuclease digestion of amplification products could increase significantly detectability of polymorphic DNA by RAPDs technique.     

水稻OsNCED3基因的RNAi载体构建

水稻OsNCED3基因是水稻抗逆过程中重要的基因之一.以水稻中花10号幼苗为材料,提取基因组DNA.设计引物扩增区段cDNA并引入相应的酶切位点,以基因组DNA作为模板,进行RNAi-OsNCED3顺式和反式目的片段的PCR扩增.将PCR产物连接到pMD19-T载体上,经酶切和PCR检测后进行测序.测序结果表明:RNAi-OsNCED3顺式和反式目的片段均已正确的连接到pMD19-T载体上.然后将RNAi-OsNCED3顺式和反式目的片段通过酶切和连接,连接到含有发夹结构的质粒pFGC5941上.PCR及双酶切结果显示,构建的pFGC5941-OsNCED3即RNAi-OsNCED3载体结构完整.     

The organization of the evolutionarily conserved GATA/GACA repeats in the mouse genome

Simple repeated GATA and GACA sequences which were originally isolated from sex-specific snake satellite DNA have been found subsequently in all eukaryotes studied. The organization of these sequences within the mouse genome was investigated here by using synthetic oligonucleotide probes as a novel tool in comparison with conventional hybridization probes. Southern blot hybridization showed sex-specific patterns with both the (GATA)₄ and (GACA)₄ oligonucleotide probes, as previously described with conventional probes. The quantitative analysis of two mouse DNA phage libraries and of 25 isolated GATA-positive phage clones revealed intensive interspersion of GATA sequences with GACA, and with other repetitive and single-copy sequences. Ubiquitous interspersion and homogeneous genomic distribution of GATA and GACA sequences were confirmed by hybridization in situ of the oligonucleotide probes to metaphase chromosomes. The lengths of the GATA and GACA stretches were found to vary considerably in the individual phage clones. DNA inserts from 20 phages were assigned to autosomes and sex chromosomes and three genomic fragments were found to be confined to the Y chromosome. The organization of GATA and GACA sequences is discussed in the context of their evolutionary potential and possible conservation mechanisms.     

The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Ase I fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dra I map was obtained by positioning the Dra I sites relative to the Ase I map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Ase I and Dra I fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The twoterminal Ase I fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.     

Transgenic Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>) plants obtained by biolistic transformation of embryogenic suspension cells

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric -glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.     

六株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学分析

对 6株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学进行了分析 .6株菌与nifHDK基因有杂交 .菌株总DNA经BamHⅠ酶切后与pEA9 DNA进行Southern杂交 ,只有 2株菌具有完整的质粒DNA ,其余菌株质粒DNA发生了 15 3～ 137 7kb不同程度的缺失 .用切割位点较少的限制性内切酶XbaⅠ酶切 6株菌的总DNA ,经脉冲场凝胶电泳 (PFGE)后用pEA9 DNA为探针进行Southern杂交 ,每株菌的pEA9 DNA明显大于用BamHⅠ酶切后的杂交结果 ,表明质粒与染色体发生了整合 .转座子Tn5或插入序列IS 12 2 2和IS 12 71可能参与质粒与染色体的整合过程 .     

用衔接头PCR克隆新的胡萝卜Ⅱ型转化酶基因启动子

 为克隆新的胡萝卜 型转化酶基因启动子 ,将胡萝卜基因组 DNA分别用 Pvu 、Eco R 、Dra 和 Sma 酶切 ,酶切片段与一特殊的衔接头连接 .取连接产物作模板 ,以衔接头引物和基因特异引物做 PCR,得到的主要 PCR产物分别为 3.4kb、1 .3kb、0 .4kb和 0 .6kb.将 Eco R -衔接头体系的 PCR产物克隆和测序 ,并将其序列与 Gen Bank中的已知序列进行比较分析 ,发现了一个新的胡萝卜 型转化酶启动子序列 ,它含有类似于 TATA box和 CAAT box的元件 ,在启动子的远上游区域还含有多个 AT富含区 .该启动子的发现对于研究植物中糖代谢具有重要意义 .     

Construction of Chloroplast Genomic Library of Amaranthus cruentus R104 and Cloning of the Gene for the Large Subunit of Ribulose-1, 5-disphosphate Carboxylase/Oxygenase

Grain amaranth is an annual food and forage crop and C4-plant with characteristics of rich nutrients, high photosynthesis efficiency and strong stress-resistance. Chloroplast DNA (ctDNA) was prepared from Amaranthus cruentus R104. The ctDNA was digested with restriction enzymes Barn HⅠ, EcoRⅠ, BgⅢ, PstⅠ and SaⅡ, and the fragments thus obtained were electrophoresed in 0.7% agarose gel. The Length of the doublestranded ctDNA was measured to be 140 kb or so. BamHI-digested fragments of R104 ctDNA were inserted into pBR322 and a chloroplast genomic library has been constructed. The recombinant clone containing rbcL gene has been identified and selected out from the library. The restriction analysis of the clone showed that the rbcL gene of A. cruentus R104 has the structure similar to that of C4-plant corn. Moreover the reason of unsuccessful cloning of 5.9 kb BamHI fragment containing psbA gene is discussed.     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

非缺失型α地中海贫血基因的克隆化分离

从一例基因型为αα~T/—的血红蛋白H病患者的手术切除脾制备了染色体DNA,用BamHⅠ水解后,回收14±1kb的DNA片段,作为插入体。以λEMBL_4噬菌体经非超离心法制备基因组DNA作为替换载体。经BamHⅠ水解回收左右臂,与α地贫脾DNA 14kb片段连接,包装、传染后,得10~4—10~5重组噬菌体/μg。以人α珠蛋白基因特异的探针作Benton原位杂交,M4×10~3克隆中筛出两个含α珠蛋白基因序列的克隆株。其中一株扩增后制备的重组DNA经酶解图谱及Southern印迹杂交鉴定,中间可替换部分含有14kb的人α珠蛋白基因组DNA。从而在我国首次从中国地贫病人中,以基因工程的方法,分离了克隆化的非缺失型α地贫基因,为研究其结构与功能打下了重要基础。     

勒氏笛鲷微卫星位点的筛选及特征分析

采用PCR法快速筛选勒氏笛鲷(Lutjanus russelli)基因组文库, 以获得(CA)_n微卫星位点。勒氏笛鲷基因组DNA经限制性内切酶HaeⅢ+ DraⅠ双酶切后, 连接T-载体克隆, 构建基因组文库。以通用引物M13+/-与重复序列引物(CA)15对基因组文库进行筛选, 二次筛选后得到121个可能含有微卫星位点的阳性克隆。进行序列测定, 共获得53个CA(n≥7)重复序列, 重复次数主要分布于7~15(80.77%)。在所得微卫星序列中, 重复单元除CA外, 还观察到单碱基、三碱基、四碱基、五碱基重复单元。根据侧翼序列设计48对引物, 通过优化PCR反应条件, 可获得清晰可重复的目的条带。研究旨在为勒氏笛鲷遗传多样性研究及遗传图谱的构建等奠定基础, 为勒氏笛鲷资源的合理开发利用提供参考。