Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.     

赤眼蜂分子鉴定研究进展

赤眼蜂是害虫生物防治中的重要天敌资源。该属种类繁多,已报道有200余种,其蜂种的正确鉴定与选择是影响其田间防效的重要因素。依赖雄成蜂外生殖器形态特征的传统赤眼蜂分类鉴定技术不仅对专业技术要求高、耗时费力,而且无法用于孤雌产雌品系的种类鉴定以及近缘种的区分。分子鉴定技术可以通过选择合适的分子标记,为赤眼蜂鉴定提供准确、便捷、高效的方法。本文对赤眼蜂分子鉴定中分子标记的选择及常用分子鉴定方法进行了综述,并提出了未来可能的发展方向。     

Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

三角酵母D—氨基酸氧化酶基因的克隆,测序及表达

利用跨越内含子的ＰＣＲ技术,从三角酵母（Ｔｒｉｇｏｎｏｐｓｉｓｖａｒｉａｂｉｌｉｓ）变种ＦＡ１１０中扩增得到Ｄ氨基酸氧化酶基因（ｄａａｏ）,并通过ＴＡ克隆的方法将其克隆至ｐＧＥＭＴ载体。序列测定结果表明,所得ｄａａｏ基因的５′端内含子已被删除,基因总长度为１０７１ｂｐ,它与Ｔｒｉｇｏｎｏｐｓｉｓｖａｒｉａｂｉｌｉｓ的Ｄ氨基酸氧化酶同源性达９８３％,与Ｆｕｓａｒｉｕｍｓｏｌａｎｉ和Ｒｈｏｄｏｔｏｒｕｌａｇｒａｃｉｌｉｓ的同源性分别是３８９％和３０８％。为提高表达水平,又将此基因转移至高表达载体ｐＥＴ２８ｂ上,在大肠杆菌ＢＬ２１（ＤＥ３）中进行诱导表达。经ＩＰＴＧ诱导,目的蛋白的产生量可占菌体总蛋白量的４６％,分子量约为３８ｋＤ。Ｄ氨基酸氧化酶的活力可达８０２ｕ／Ｌ。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

Epidemiology of apple proliferation (AP) in northwestern Italy: evaluation of the frequency of AP-positive psyllids in naturally infected populations of Cacopsylla melanoneura (Homoptera: Psyllidae)

Adults of Cacopsylla melanoneura, vector of the apple proliferation (AP) phytoplasma, were collected every 2 weeks from January until May in 2000 and 2001 by the beating tray method in eight apple orchards of the Aosta Valley (northwestern Italy). Total DNA was extracted from batches of five insects and amplified with the universal phytoplasma primers P1/P7 in direct PCR. A nested PCR assay was then performed on P1/P7 amplicons using the primers fO1/rO1, specific for the AP‐ phytoplasma group. The digestion of fO1/rO1 amplicons with Ssp I restriction endonuclease confirmed that C. melanoneura adults harboured the AP phytoplasma. The data obtained with PCR were used to estimate the proportion of AP‐positive insects in over wintered and offspring adults. Percentages of AP‐positive insects of 3.6% and 0.8% were estimated in 2000 among over wintered and offspring psyllids respectively. In 2001 only the over wintered insects were found infected, with an estimated proportion of 2.8%. The seasonal abundance of the vector was measured using yellow sticky traps. C. melanoneura was always present at a low population level, and the highest density was recorded from mid‐February until mid‐March in both years. The results show that the overwintered population is higher and spends a longer period in apple orchards, suggesting the crucial role of the overwintered adults in vectoring AP.     

PCR快速分析水稻cDNA文库构建质量方法的简化

对由水稻cDNA文库铺成平板的噬菌斑,采用tip吸头穿刺噬茵斑后在PCR反应混合液中吸打2次,直接作为PCR扩增模板进行PCR反应和电泳分析的方法,可以达到快速、简便地分析水稻cDNA文库构建的质量.此法无需提取噬茵斑DNA作为PCR扩增模板,操作简单、方便、快捷.