A simple and efficient method for isolating small RNAs from different plant species

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.     

Spatial structure of shell polychromatism in <Emphasis Type="Italic">Cepaea hortensis</Emphasis> in relation to a gradient of landscape fragmentation in Western France

Because of their highly polymorphic shell patterns, Cepaea land snails have been the subject of numerous studies in ecological genetics. Here, we investigated the spatial structure of polychromatism in Cepaea hortensis in agricultural landscapes with zones from low to high hedgerow densities. Our main purpose was to search for a relationship between landscape composition and spatial structuring of chromatism. Despite significant spatial heterogeneity in the three landscapes sampled, only the high hedgerow density landscape showed a significant spatial structuring of shell polymorphism. In order to understand this result, an investigation of daily movement patterns in relation to habitat form was carried out on a mark-release experiment under semi-artificial conditions. This experiment revealed a strong influence of a linear corridor on snail dispersal. In the field, spatial heterogeneity of shell polymorphism, related to the effects of genetic drift, was shaped by restricted dispersal in narrow corridors. In the more enclosed one, i.e. where hedgerow density was the highest, the significant spatial structure we detected involved a balance between local genetic drift and environmentally mediated gene flow. This isolation-by-distance pattern resulted from direct gene exchange through fields between neighbouring populations. When applying landscape distances based on hedgerow length, no significant spatial correlation with polychromatism was found. In the more fragmented sites, random genetic drift seemed to be the prevailing force and, at the scale of the whole sampled area, selective pressures potentially interfere with these genetic drift-dispersal events.     

Pharmacokinetic disposition of triclabendazole in cattle and sheep; discrimination of the order and the rate of the absorption process of its active metabolite triclabendazole sulfoxide

A comparative pharmacokinetic study was conducted to determine the order and the rate of absorption of triclabendazole (TCBZ) in cattle and sheep. A commercial suspension of TCBZ (Biofasiolex, Biogénesis S.A., Argentina) was administered at a dose rate of 10 mg/kg by the oral route to six Holstein female calves and six Corriedale female sheep. The plasma concentration profiles of the metabolites triclabendazole sulfoxide (TCBZ-SO) and triclabendazole sulfone (TCBZ-SO₂) were analysed by means of the non-compartmental method. The order of the absorption process of the active metabolite, TCBZ-SO, was determined by construction of curves of cumulative absorbed fraction of the drug by means of the Wagner-Nelson method. The appearance of TCBZ-SO in plasma of cattle and sheep resembles the entry of a constant quantity of drug into the organism per unit time. This is explained by the reservoir effect of the rumen, which acts as a biological slow-release system for TCBZ-SO and its precursor TCBZ to the posterior digestive tract where they are absorbed. The plasma concentration profiles of TCBZ-SO in both species were well described by a one-compartment open model with zero-order process of absorption and first-order process of elimination. The values of AUC_0-∞ and C _max of TCBZ-SO did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep. In the case of TCBZ-SO₂, t _max was the only parameter that did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep.     

DNA methylation status in tissues of sheep clones

Genomic imprinting and DNA methylation play an important role in mammalian development. Many cloned animals showed heterogeneous DNA methylation profiles. However, there are fewer reports in cloned lambs because of a lack of genomic imprinting information. In this study, we investigated DNA methylation patterns in CpG islands and differentially methylated regions of putative imprinted gene Peg10 and imprinted genes Dlk1, Igf2R and H19 in cloned lambs. Five organs from two cloned lambs died shortly after birth and two normal controls were investigated. We observed normal DNA methylation profiles in cloned lambs. The imprinted genes Dlk1, Igf2R and H19 in livers, kidneys, hearts, muscles and lungs of the two cloned lambs exhibited relatively normal DNA methylation, except for Peg10 showing some differences between controls and cloned lambs. Our results indicate that somatic cell nuclear transfer-produced sheep exhibited relatively normal DNA methylation pattern and experienced normal DNA methylation reprogramming at imprinted loci.     

废渣固态发酵生产酵母蛋白质饲料及其产业化发展措施

文章综述了近几年来采用废渣固态发酵技术生产酵母蛋白质饲料的研究进展,对其应用前景和产业化发展措施进行了探讨,并提出制订固态发酵产的酵母粉的质量评估指标与质量标准,以规范该产品的生产、品管和销售市场。     

70%噻虫嗪种子处理可分散粉剂和10%吡虫啉可湿性粉剂拌种对马铃薯甲虫的防效

利用药剂拌种处理马铃薯种薯后播种的方法,研究比较了2种新烟碱类杀虫剂对马铃薯甲虫的活性和防治效果。结果表明,按药剂有效成分18 g/100 kg种薯拌种,70%噻虫嗪种子处理可分散粉剂拌种出苗后60 d对马铃薯甲虫幼虫的生物活性和防效较高,卵块孵化率较低,死亡率在61.1%以上,防效达57.3%,孵化率为36%,这有利于降低马铃薯甲虫种群数量,减轻为害。而10%吡虫啉可湿性粉剂拌种出苗后60d对马铃薯甲虫幼虫生物活性和防效较低,卵块孵化率较高,死亡率在12.5%以下,防效为37.1%,孵化率为64.6%,对马铃薯甲虫的控制效果较差。     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

花烛体细胞胚胎发生及植株再生研究

 以花烛(Anthurium andraeanum Lind. ) 盆花品种无菌苗叶片、叶柄、根段为外植体, 对花烛体细胞胚胎发生及植株再生进行了研究, 建立了花烛体细胞胚胎发生体系。结果表明, 花烛叶片较其他外植体更适宜体细胞胚的诱导; 胚性愈伤组织及胚性结构诱导最适培养基为改良MS + 2, 4-D 2.0～3.0 mg·L ^{- 1} + KT 0.5 mg·L ^{- 1} +蔗糖2% +葡萄糖1% , pH 5.5, 暗培养40 d, 诱导率可达90.3%; 体胚发育适宜培养基为改良MS + 6-BA 0.5 mg·L ^{- 1} +蔗糖3% , 光强20 μmol·m ^-2 · s^-1 , 培养20 d后体胚萌发率达6219%; 萌发的体胚在发育培养基上继续培养30 d后均能发育成完整植株。对不同阶段培养材料的组织结构及超微结构的观察证明了花烛体胚发生过程。     

Doxycycline treatment for <Emphasis Type="Italic">Dirofilaria immitis</Emphasis> in dogs: impact on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Enterococcus</Emphasis> antimicrobial resistance

Doxycycline is an antibiotic that, in addition to the classic antibacterial use, is also prescribed to fight parasitic diseases, like heartworm disease in dogs. Despite the concern that the overuse of this antibiotic may decrease susceptibility of clinically important bacteria, the consequences of the prolonged doxycycline therapy in heartworm-infected dogs have never been studied before. We have analyzed the impact of this therapy on Staphylococcus aureus and Enterococcus antimicrobial resistance. In this study, 17 heartworm-infected dogs (10 that had completed the doxycycline treatment and 7 dogs that had not yet begun) were included. Twenty-four isolates of Staphylococcus aureus were obtained from two locations of each dog. After treatment, 73.3% of isolates were resistant to at least one antibiotic but only 22.2% of isolates before treatment. Most of doxycycline resistant isolates were obtained from dogs that have received treatment. Erythromycin resistance or intermediate susceptibility was detected in 45.6% of isolates, most of them from dogs after treatment. For Enterococci, 48 isolates were obtained from fecal samples (25 before treatment and 23 after treatment). Before treatment, 32% of isolates were resistant at least to one antibiotic while after, this data increase up to 65%. Comparing isolates before and after treatment, a clear increase in resistance to doxycycline (12% against 21.74%) and erythromycin (20% against 39.13%) was observed. Although the present work is a preliminary research, the results encourages the development of further studies to determinate the effect of prolonged doxycycline therapy on antimicrobial resistance.     

Newly developed SSR markers reveal genetic diversity and geographical clustering in Paeonia suffruticosa based on flower colour

Tree peony (Paeonia suffruticosa Andr.), considered by many to be the national flower of China, is famous for its ornamental, medicinal and culinary attributes. In this study, a total of 8,663 Simple Sequence Repeats (SSRs) were detected by means of a microsatellite search of unigene sequences identified from the de novo assembly of sequence data from different genotypes of tree peony from the Zhongyuan group. Among 100 randomly selected SSR markers, 25 were successfully amplified and showed polymorphism in 31 tree peony accessions. The number of polymorphic alleles ranged from 3 to 11 for each locus and the polymorphism information content value (PIC) ranged from 0.58 to 0.85, with a mean of 0.73, indicating a high level of discriminative capability. To analyse the genetic diversity, a phylogenetic tree was constructed, which demonstrated that tree peonies of similar flower colour were clustered together. The large number of tree peony SSR markers identified in this study will be valuable in studies in genetic diversity and linkage map construction, in gene localisation and cloning and in molecular-marker-assisted breeding in tree peony.