水稻线粒体DNA酶切带型研究

水稻IR36线粒体DNA经6种限制酶酶切,用脉冲电泳和长距离琼脂糖凝胶电泳分离酶切片段,获得高分辨率的清晰带型。每组酶切片段加和测得水稻IR36线粒体基因组大小分别为227kb(HindⅢ)、253kb(EcoRⅠ)、253kb(XhoⅠ)、294kb(BamHⅠ)、239kb(SalⅠ)和283kb(xbal)采用9个来自水稻和玉米线粒体基因组的基因探针与酶切条带杂交发现,水稻线粒体基因组含有包括编码基因在内的重复顺序。     

细胞质雄性不育高粱叶绿体 ndh D 基因的序列变异

片段SAAU－02 700特异地扩增自7种具可育细胞质的高粱材料的总DNA,含有叶绿体psa C(88bp)和ndh D(192bp)基因的部分序列。该片段与Eco Ri HindⅢ酶切的总DNA,线粒体DNA和叶绿体DNA杂交,在总DNA中获得了0.74kb的杂交带,而在叶绿体中获得0.74kb和0．45kb两条杂交带。与线粒体DNA无杂交;与经Hae Ⅲ酶切的总DNA杂交,在不育系中获得4．9kb的杂交带,而保持系的杂交带为4．45kb。参考GenBank中高粱的近缘物种玉米叶绿体基因组的序列,构建了ndh D基因区的酶切位点图谱,借此分析得出高粱不育系的叶绿体ndh D基因序列已发生改变。这种变异与高粱细胞质雄性不育反生的关系正在探讨中。     

五种常见嗜尸性蝇类的分子鉴定

为了解决法医学人员对于嗜尸性蝇类的鉴定难题,对中山市、广州市及西安市5种常见嗜尸性蝇类共17个样本,对其线粒体COⅠ基因的348 bp大小片段进行了RFLP和DNA序列分析,采用ABI377测序仪测序,DNASTAR软件预测限制性位点,DdeⅠ, DraⅠ和HinfⅠ3种限制性内切酶消化,非变性聚丙烯酰胺凝胶电泳检测酶切结果,MEGA3.0软件包进行序列分析和构建系统发育树。结果表明： 采用mtDNA扩增结合银染技术和DNA序列分析,均可以方便快捷地进行上述三地5种常见嗜尸性蝇类的种类鉴定。本文结果为法医昆虫学中嗜尸性昆虫DNA鉴定数据库的建立提供了数据资料。     

黑曲霉糖化酶高产菌株的糖化酶结构基因的分离及其序列分析

从糖化酶高产菌株(Aspergills niger)T21中分离出染色体DNA.Southern印跡分析表明糖化酶结构基因位于约2.5kb的EcoRⅠ-EcoRV片段中.该染色体DNA经EcorⅠ、EcoRⅤ完全酶切后,用琼脂糖凝胶电泳分离,回收2.0—3.0kb的片段,与载体pBR322连接后转化宿主菌大肠杆菌DH5,获得转化子.通过原位杂交,从转化子中筛选出4个阳性克隆.阳性克隆的进一步酶切鉴定及序列分析表明,黑曲霉T21糖化酶结构基因大小为2.3kb,含有4个内含子.     

水稻线粒体atpA基因的克隆及其与细胞质雄性不育的关系

本研究以水稻BT型细胞质雄性不育系秋光和相应的保持系秋光为材料,提取线粒体DNA,用限制性内切酶完全酶解,以玉米线粒体atpA基因和波菜叶绿体atpA基因作为探针,进行分子杂交,将保持系线粒体atpA基因定位在3.5kb的Bam HI酶切片段上,并且以pBR322为载体,克隆了这一片段,另外,在Bam HI完全酶解普带的杂交结果中,不育系线粒体基因组中有两条阳性杂交带,分别是3.5kb和2.9kb,而保持系线粒体基因组中只有3.5kb一条阳性杂交带,因而认为水稻不育系线粒体基因组中可能有两个atpA基因拷贝,而相应的保持系线粒体基因组中只有一个atpA基因拷贝。     

水稻线粒体atpA基因的克隆及其与细胞质雄性不育的关系

本研究以水稻BT型细胞质雄性不育系秋光和相应的保持系秋光为材料，提取线粒体DNA，用限制性内切酶完全酶解，以玉米线粒体atpA基因和波菜叶绿体atpA基因作为探针，进行分子杂交，将保持系线粒体atpA基因定位在3.5kb的Bam HI酶切片段上，并且以pBR322为载体，克隆了这一片段，另外，在Bam HI完全酶解普带的杂交结果中，不育系线粒体基因组中有两条阳性杂交带，分别是3.5kb和2.9kb，而保持系线粒体基因组中只有3.5kb一条阳性杂交带，因而认为水稻不育系线粒体基因组中可能有两个atpA基因拷贝，而相应的保持系线粒体基因组中只有一个atpA基因拷贝。     

中国人β类珠蛋白基因多态性研究Ⅱ.ψβ_1基因中及其3′侧序列中的多态性HincII酶切位点

本文首次报告在中国人的ψβ_1基因中及其3′侧序列中存在两个HincⅡ的多态性酶切位点。甩含(?)β_1序列的重组质粒pp 3.9的BglⅡ和XbaⅠ双酶切片段(1.8kb)作为探针,与正常染色体DNA的HincⅡ酶解片段行Southern印迹杂交,所得杂交自显影图谱上可呈现7.6kb、6.0kb和3.0kb等条带,表明存在酶切位点的多态性。结果显示,在β_1基因之中及其3′侧序列中出现多态性HincⅡ酶切位点的频率分别为30.6％和39.0％。同时观察到,在所检测的18个个体中,按杂交片段所表示的基因型有4种,即纯合子7.6kb/7.6kb型和3.0kb3.0kb型,杂合子7.6kb/3.0kb型和7.6kb/6.0kb型。     

北京鸭肝线粒体DNA的克隆

 本文采用限制内切酶HindⅢ切割经Sepharose 4 B柱纯化的北京鸭肝线粒体DNA,得到五个片段,其大小分别为:A——6.56kb,B——3.12kb,C——3.12kb,D——2.40kb,E——1.40kb。以质粒pWR33为载体,在HindⅢ切点处插入线粒体DNA的HindⅢ酶切片段,将体外重组质粒转化到大肠杆菌HB101内,经筛选、分析:如菌落原位杂交,限制性内切分析和Southern吸印法分析,首次得到了线粒体DNA的HindⅢB、C、D、E四个片段的克隆子。另外还得到了一个与片段A同源的1.60kb大小的片段。     

雨生红球藻基因组文库的构建及鉴定

本研究以雨生红球藻34-1n为材料,提取其基因组DNA,利用限制性内切酶Sau3AⅠ对基因组DNA进行酶解,回收6~8kb的基因组DNA片段,并浓缩至200ng/μL。该片段与经BamH Ⅰ酶切和去磷酸化处理后的pUC18载体连接,然后电击转化到受体菌Escherichia.coli DH5α中,获得雨生红球藻34-1n的基因组文库。该文库的平均插入片段长度约为6.5kb,获得6×105个克隆数。通过PCR筛选,由雨生红球藻基因组文库中获得含bkt1序列的单克隆菌,与β-胡萝卜素氧化酶序列(GenBank:DQ086233.1)进行比对,结果表明bkt1基因组序列含有6个外显子。本研究为进一步鉴定雨生红球藻相关基因提供了一个文库平台。     

水稻线粒体DNA的提取和纯化

继玉米细胞质雄性不育在生产上利用以 来，我国杂交稻的大面积推广种植对我国粮食 增产起了巨大的作用，并推动了对细胞质雄性 不育的深人研究〔11。玉米和高粱雄性不育的研 究表明，细胞质雄性不育是由线粒体基因组编 码的。Levings等人发现玉米正常的和细胞质 不育的S, T, C型线粒体DNA的限制性内切 酶切割片段是不相同的。限制性内切酶片段的 分析可以是鉴定各种玉米雄性不育细胞质的有 效手段1410     

Transfer of a male-sterility-inducing cytoplasm from onion to leek ( Allium ampeloprasum)

Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [ Allium cepa (2 n=2 x=16, CC)] and leek [ A. ampeloprasum (2 n=4 x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC(1) plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30-33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC(2) progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.     

Analytical study of phenotypic and biochemical attributes of onion cultivars in relation to infestation of onion thrips,Thrips tabaci

Onion thrips is a major threat to onion crop throughout the world. It is a potential vector of Iris yellow spot virus and causes significant economic damage to bulb production. Phenotypic and biochemical traits of onion cultivars were assessed against Thrips tabaci. Onion Gawran LR-241 (OG) cultivar was tolerant against the infestation of T. tabaci whereas Onion White (OW) was susceptible. Number and size of stomata, cuticle thickness, cell wall thickness and surface wax of onion leaves were studied with the help of scanning electron microscope and quantitative and qualitative analysis was carried out to estimate epicuticular wax and other bio-chemical components through GC/MS. Onion Gawran has thick cell wall, sharp and dense wax crystals, wider central angle and small sized stomata compared to other cultivars. Epicuticular wax components of OG cultivar were heptacosane (5.2%), octacosanol-1 (9.2%), 2-methyl octacosane (4.2%), heptadecanol-1 (5.2%), hexacosanol-1 (4.2%), azulene, 1,4-dimethyl-7-(1-methylethyl) (36.9%), hexadecanoic acid (1.95%), heptadecane (4.2%), triacontanol-1 (5.8%) and hentriacontanone-16 (23.40%). Azulene, 1, 4-dimethy-l-7-(1-methy-l-ethyl) was only found 36.9% in OG but absent in other three cultivars. 2-methyl octacosane was absent in Poona Red Desi and OW cultivars. Hentriacontanone-16, 2-methyl octacosane, fatty alcohols (Octacosanol-1 and Triacontanol-1) and azulene, 1, 4-dimethy-l-7-(1-methy-l-ethyl) were effective in the formation of epicuticular wax in onion cultivars. It implies that phenotypic and biochemical characteristics of OG cultivar proved as resisting features to T. tabaci.     

Molecular taxonomy and phylogeny of entomopathogenic nematode species (Rhabditida: Steinernematidae) by RFLP analysis of the ITS region of the ribosomal DNA repeat unit

The ITS region of 19 isolates of the genus Steinernema Travassos, 1927 belonging to 17 species was PCR amplified. The resulting products were then digested with 17 different restriction endonucleases and the fragments generated were separated by agarose gel electrophoresis. Some enzymes yielded patterns which were highly diagnostic (e.g. Alu I, Dde I, Hha I and Hinf I) while others showed similar RFLP patterns for the majority of species (e.g. Hpa II). All of the species could be unequivocally identified using this method. A tree was constructed based on band sharing which resulted in clusters of species which exhibited similar morphological features.     

RNA-Seq Reveals Leaf Cuticular Wax-Related Genes in Welsh Onion

The waxy cuticle plays a very important role in plant resistance to various biotic and abiotic stresses and is an important characteristic of Welsh onions. Two different types of biangan Welsh onions (BG) were selected for this study: BG, a wild-type covered by wax, which forms a continuous lipid membrane on its epidermal cells, and GLBG, a glossy mutant of BG whose epidermal cells are not covered by wax. To elucidate the waxy cuticle-related gene expression changes, we used RNA-Seq to compare these two Welsh onion varieties with distinct differences in cuticular wax. The de novo assembly yielded 42,881 putative unigenes, 25.41% of which are longer than 1,000 bp. Among the high-quality unique sequences, 22,289 (52.0%) had at least one significant match to an existing gene model. A total of 798 genes, representing 1.86% of the total putative unigenes, were differentially expressed between these two Welsh onion varieties. The expression patterns of four important unigenes that are related to waxy cuticle biosynthesis were confirmed by RT-qPCR and COG class annotation, which demonstrated that these genes play an important role in defense mechanisms and lipid transport and metabolism. To our knowledge, this study is the first exploration of the Welsh onion waxy cuticle. These results may help to reveal the molecular mechanisms underlying the waxy cuticle and will be useful for waxy gene cloning, genetics and breeding as well as phylogenetic and evolutionary studies of the Welsh onion.     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

葱小孢子的染色体和核型研究

 本文用胰酶-尿素双重处理法和SSG法,分别进行了葱小孢子染色体螺旋的诱导和染色体C-带的显带研究,首次成功地获得了葱小孢子染色体螺旋和染色体C-带,螺旋图象清晰;染色体C-带与体细胞的基本一致,所有染色体上均显示出明显的末端带,但未出现次缢痕带。另外,研究了葱小孢子的核型,其结果与体细胞的核型基本一致,但有差异。作者认为小孢子的核型比体细胞的核型准确,更能反映出葱的核型特征。     

Direct Species Identification of Common Pathogenic Dermatophyte Fungi in Clinical Specimens by Semi-nested PCR and Restriction Fragment Length Polymorphism

OBJECTIVE: To seek a rapid and reliable molecular biology method to identify the common pathogenic dermatophyte fungi from clinical samples. METHOD: The genome DNA was extracted from cultured strains of seven common dermatophyte fungi species and part of each positive clinical specimen by microscopy. Intergenic spacer regions of ribosomal DNA (ITS) were amplified by semi-nested PCR (snPCR) with three universal primers (NS5, ITS1, and ITS4) for fungi. The amplified products were digested with two restriction endonucleases (BciT130 I, Dde I), the Restriction Fragment Length Polymorphism(RFLP). The rest of each clinical specimen was cultured in Sabouraud's Agar medium. Then the results of RFLP were compared with the traditional culture results. RESULTS: The digestion of seven common dermatophyte fungi produced seven different restriction profiles. Restriction profiles of 17 clinical specimens matched, respectively, to that of the cultured strains, and 14 profiles of the 17 ones matched the culture result completely. The coincidence was 100.0%. CONCLUSIONS: snPCR-RFLP analysis of intergenic spacer regions of ribosomal DNA is a valuable method of exactness and clarity for species identification of common dermatophyte fungi from clinical specimens.     

Co-Planting Cd Contaminated Field Using Hyperaccumulator Solanum Nigrum L. Through Interplant with Low Accumulation Welsh Onion

Monoculture and intercrop of hyperaccumulator Solanum nigrum L. with low accumulation Welsh onion Renbentieganchongwang were conducted. The results showed that the remove ratio of S. nigrum to Cd was about 7% in intercrop plot when top soil (0–20 cm) Cd concentration was 0.45–0.62 mg kg^?1, which did not significantly impact the yield of low accumulation Welsh onion compared to the monoculture. The consistency of remove ratio in practice and theory indicated the remediation of S. nigrum to Cd was significant. The Cd concentration and yield of Welsh onion were not affected by the growth of S. nigrum either in intercrop plot. The Cd concentration in edible parts of Welsh onion was available either. In short, inter-planting hyperaccumulator with low accumulation crop could normally remediate contaminated soil and produce crop (obtain economic benefit), which may be one practical pathway of phytoremediating heavy metal contaminated soil in the future.     

Hepatoprotective Potentials of Onion and Garlic Extracts on Cadmium-Induced Oxidative Damage in Rats

The hepatoprotective effect of onion and garlic extracts on cadmium (Cd)-induced oxidative damage in rats is reported. Control group received double-distilled water alone. Cd group was challenged with 3CdSO₄·8H₂O (as Cd; 1.5 mg/kg bw per day per oral) alone, while extract-treated groups were pretreated with varied doses of onion and/or garlic extract (0.5 and 1.0 ml/100 g bw per day per oral) for a week and thereafter co-treated with Cd (1.5 mg/kg bw per day per oral) for 3 weeks. Cd caused a marked (p?<?0.001) increase in the levels of lipid peroxidation and glutathione S-transferase, whereas glutathione, superoxide dismutase, and catalase levels were decreased in the liver. We also observed a decrease in hepatic activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase and a concomitant increase in the plasma activities of ALT and AST. Onion and garlic extracts significantly attenuated these adverse effects of Cd. Onion extract proffered a dose-dependent hepatoprotection. Our study showed that Cd-induced oxidative damage in rat liver is amenable to attenuation by high dose of onion and moderate dose of garlic extracts possibly via reduced lipid peroxidation and enhanced antioxidant defense system that is insufficient to prevent and protect Cd-induced hepatotoxicity.