Production of ramoplanin analogues by genetic engineering of Actinoplanes sp.

Ramoplanins are lipopeptides effective against a wide range of Gram-positive pathogens. Ramoplanin A2 is in Phase III clinical trials. The structure–activity relationship of the unique 2Z,4E-fatty acid side-chain of ramoplanins indicates a significant contribution to the antimicrobial activities but ramoplanin derivatives with longer 2Z,4E-fatty acid side-chains are not easy to obtain by semi-synthetic approaches. To construct a strain that produces such analogues, an acyl-CoA ligase gene in a ramoplanin-producing Actinoplanes was inactivated and a heterologous gene from an enduracidin producer (Streptomyces fungicidicus) was introduced into the mutant. The resulting strain produced three ramoplanin analogues with longer alkyl chains, in which X1 was purified. The MIC value of X1 was ~0.12 μg/ml against Entrococcus sp. and was also active against vancomycin-resistant Staphylococcus aureus (MIC = 2 μg/ml).     

番鸭呼肠孤病毒YB株σB蛋白的基因序列分析及其原核表达

番鸭呼肠孤病毒(Muscovy duck reovirus,DRV)是造成雏番鸭高死亡率的重要病原体,深入其检测与免疫研究对于防控DRV感染意义重大。利用RT-PCR和测序技术,对3株福建DRV分离株的S3基因进行序列分析,发现DRV-YH、YJL株与禽呼肠孤病毒(Avian reovirus,ARV)遗传距离较近,同源性高达94.6%～98.9%,而DRV-YB株与ARV同源性仅为60.6%～61.7%。构建和鉴定DRV YB株重组原核表达质粒pET-30a-S3,并转化大肠杆菌BL21,SDS-PAGE表明,表达的目的蛋白分子量约为42ku,IPTG最适诱导浓度为0.1mM,最适诱导时间为5h,最适诱导温度为37℃,以包涵体形式存在。薄层扫描显示重组DRVσB蛋白占菌体总量的67.7%。以Ni 2+柱亲和层析纯化蛋白,纯化后的目的蛋白纯度为93%,质量浓度为0.86g/L。Western blot分析该融合蛋白能与抗DRV阳性血清发生特异性反应,表明重组DRVσB蛋白具有良好的免疫反应性。     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

北京地区油菜软腐病病原菌的鉴定

摘要:【目的】确定引发北京地区油菜［Brassica campestris L．ssp．chinensis (L.) Makino var． communis Tsen et Lee］软腐病的病原。【方法】结合病原菌形态、BIOLOG 及生理生化、16S rＲNA 基因序列及亚种IGS区特征分析,对从北京大兴和通州区油菜软腐病样中病原菌进行生物学鉴定。【结果】分离的40 个菌株均能引发 油菜软腐病,但分别为胡萝卜果胶杆菌(Pectobacterium carotovorum) 的2个不同亚种,其中13株为P．carotovorum subsp．carotovorum(Pcc),另27株为P．arotovorum subsp． brasiliensis(Pcb)。接种白菜(Brassica campestris L．ssp．pekinensis)致病力测定分析表明,亚种内、来源相同与16S rRNA基因序列相同的菌株间均存在明显的致病力分化。【结论】Pectobacterium carotovorum subsp．carotovorum和Pectobacterium carotovorum subsp. brasiliensis是引发北京地区油菜软腐病的致病菌,后者为首次报道能引起白菜类蔬菜软腐病的常见致病菌。     

Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺ and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺ condition. The cell wall‐derived pectin homogalacturonan (GalA)₃ partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺ stress.     

Estimation of genetic parameters for disease‐resistance traits in Cynoglossus semilaevis (Günther, 1873)

The objective of this study was to estimate genetic parameters for three traits based on 28 Cynoglossus semilaevis families approximately 6 months of age (at least 5 cm total length), including trait_1 (survival of 26 families, 3434 individuals in total subjected to challenge tests with Edwardsiella tarda), trait_2 (survival of 20 families, 2016 individuals in total subjected to challenge tests with Vibrio anguillarum) and trait_3 (survival of 27 families, 9340 individuals tagged at circa 180 days of age and reared in indoor ponds for circa another 5 months). The result showed that there were large differences in the survival of the families after challenge (11.11–65.31% for E. tarda and 9.18–70.54% for V. anguillarum). Additionally, the survival of families reared in indoor ponds was also different, varying from 21.00% to 73.67%. Heritabilities of the three traits varied from 0.14 to 0.26, as estimated by the linear model (LM) and the threshold model (TM). The trait_1 heritabilities (0.26 and 0.19 estimated by LM and TM) were higher than those of the others (0.20 and 0.23 estimated by LM, 0.14 and 0.19 estimated by TM). The estimates of heritabilities using LM were consistently higher than those of TM in this study. There were significant medium genetic correlations of 0.44 and 0.42 between trait_1 and trait_2 obtained from LM and TM (P < 0.05). However, very low and non‐significant genetic correlations of trait_1 and trait_3 (?0.10 for LM, ?0.05 for TM), as well as those of trait_2 and trait_3 (0.05 for LM, 0.04 for TM) were obtained. Therefore, indirect selection for trait_1 and trait_2 was effective, but almost ineffectual for trait_1 and trait_3 as well as trait_2 and trait_3. Otherwise, there was no significant difference in the predictive abilities of LM and TM. Two families resistant to both Edwardsiella tarda and Vibrio anguillarum were selected plus one family resistant to both Vibrio anguillarum and naturally infected by unknown pathogens through family selection. As there was very low and non‐significant genetic correlation of trait_3 and trait_1 as well as trait_2, superior strains are anticipated with the ability to resist two or more kinds of diseases, through the crossing of families selected for the three traits described above. The results support the hypothesis that genetic variation exists for disease survival, which could be used to design a breeding program for selecting strains of Cynoglossus semilaevis with high disease resistance.