No evidence of inhibition of horizontal gene transfer by CRISPR–Cas on evolutionary timescales

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)–Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR–Cas maintenance and one of the causes of the patchy distribution of CRISPR–Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR–Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR–Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.     

Thermal tolerance in widespread and tropical Drosophila species: does phenotypic plasticity increase with latitude?

The distribution of insects can often be related to variation in their response to thermal extremes, which in turn may reflect differences in plastic responses or innate variation in resistance. Species with widespread distributions are expected to have evolved higher levels of plasticity than those from restricted tropical areas. This study compares adult thermal limits across five widespread species and five restricted tropical species of Drosophila from eastern Australia and investigates how these limits are affected by developmental acclimation and hardening after controlling for environmental variation and phylogeny. Irrespective of acclimation, cold resistance was higher in the widespread species. Developmental cold acclimation simulating temperate conditions extended cold limits by 2°-4°C, whereas developmental heat acclimation under simulated tropical conditions increased upper thermal limits by <1°C. The response to adult heat-hardening was weak, whereas widespread species tended to have a larger cold-hardening response that increased cold tolerance by 2°-5°C. These patterns persisted after phylogenetic correction and when flies were reared under high and low constant temperatures. The results do not support the hypothesis that widely distributed species have larger phenotypic plasticity for thermal tolerance limits, and Drosophila species distributions are therefore more closely linked to differences in innate thermal tolerance limits.     

The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.     

荧光标记复合PCR扩增微卫星位点的构建与优化

本研究采用毛细管电泳技术,构建并优化了荧光标记复合PCR同时扩增多个微卫星位点。主要过程为：首先根据设计所扩增微卫星位点的期望长度,将9个微卫星位点分成两组,5个位点用FAM（蓝色）标记,4个位点用HEX（绿色）标记;两种荧光类型分组优化,用琼脂糖胶电泳检测。其次,荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点,采用ABI3730xl毛细管电泳检测,以ROX500（红色）为长度标准物,结果经Genemapper3.5软件 分析,检测结果表明毛细管电泳检测荧光标记复合PCR产物不仅精确读取微卫星位点的长度（分辨率高达1bp）,还能区分微卫星位点复制时滑链所引起的“回声斑”;调整各微卫星位点引物比列使所有位点扩增强弱均匀。最后,逐一检测复合PCR基本参数（dNTP浓度、 PCR程序和模版DNA用量）对复合PCR产物的影响,优化PCR。结果表明通过毛细管电泳检测荧光标记复合PCR产物来读取微卫星位点的基因型具有精确性、高效性和稳定性。     

中国平脉树螽属五新种记述：直翅目：螽斯科：树螽亚科

本文报道中国平脉树螽属5个新种。每个新种皆有详细的形态描述和形态特征图。所有模式标本存于北京农业大学昆虫标本室。     

Phylogenetic and populational study of the Tuber indicum complex

When examined using SEM, Chinese samples of Tuber indicum and T. sinense displayed the same ascospore ornamentation as that of T. pseudohimalayense, T. indicum, collected in India by Duthie in 1899, and samples renamed T. himalayense in 1988. The different authors who named the four taxa (T. indicum, T. himalayense, T. sinense, T. pseudohimalyense) described differences in the surface of the peridium which could be considered as usual variations within a single species. We consider T. indicum, T. himalayense, T. sinense and T. pseudohimalayense as one species, T. indicum. Within this T. indicum complex, according to ITS and β-tubulin sequences, there are two groups in China, which could be considered as geographical ecotypes. This study is the first to identify a genetic and phylogeographical structure within the Chinese Tuber species.     

Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae

An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.     

Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.     

Systemic administration of epidermal growth factor increases UCP3 mRNA levels in skeletal muscle and adipose tissue in rats

We have previously reported that systemic epidermal growth factor (EGF) treatment in rats reduces the amount of adipose tissue despite an unaltered food intake. The mitochondrial uncoupling proteins (UCP2 and UCP3) are thought to uncouple the respiratory chain and thus to increase energy expenditure. In order to find out whether the UCP system was involved in the EGF-induced weight loss, the effects of EGF on UCP2 and UCP3 in adipose tissue and skeletal muscle were investigated in the present study. Eight rats were treated with placebo or EGF (150 microg/kg/day) for seven days via mini-osmotic pumps. The EGF-treated rats gained significantly less body weight during the study period than the placebo-treated animals and had significantly less adipose tissue despite a similar food intake. The placebo group and the EGF group had similar UCP2 mRNA expression (in both adipose tissue and skeletal muscle), whereas the EGF-treated group compared to the placebo group had significantly higher UCP3 mRNA expression in both skeletal muscle (3.76 +/- 0.90 vs 8.41 +/- 0.87, P < 0.05) and in adipose tissue (6.38 +/- 0.71 vs 12.48 +/- 1.79, P < 0.05). In vitro studies with adipose tissue fragments indicated that the EGF effect probably is mediated indirectly as incubations with EGF (10 microM) were unable to affect adipose tissue UCP expression, whereas incubations with bromopalmitate stimulated both UCP2 and UCP3 mRNA expression twofold. Thus, EGF treatment in vivo was found to enhance UCP3 mRNA expression in both adipose tissue and skeletal muscle, which may indicate that the EGF effect on body composition might involve up-regulation of UCP3 in skeletal muscle and adipose tissue.