具有III型分泌系统的Pseudomonas moraviensis FP1761植物益生功能及其全基因组分析

 本研究从节瓜根际分离到一株具有防病促生功能的荧光类假单胞菌FP1761。该菌株对部分植物病原真菌和细菌具有拮抗能力,能够解钾、解有机磷和无机磷,可产生氨、蛋白酶、嗜铁素、吲哚乙酸。生物测定表明菌株FP1761可显著促进小麦生长。生理生化、平均核苷酸相似度、16S rDNA和多基因分析将FP1761鉴定为摩拉维亚假单胞菌(Pseudomonas moraviensis)。菌株FP1761基因组草图全长6.12 Mb,(G+C)含量为59.9%,共编码5467个基因序列。将该菌株与种内3个代表性菌株进行泛基因组和核心基因组分析,共产生 4 357个共有基因,菌株FP1761特有基因327个。利用antiSMASH对菌株次生代谢基因簇进行预测,发现其含有8个潜在的次生代谢产物基因簇。其中两个基因簇与嗜铁素pyoverdine合成相关,未见聚酮类合成基因。基因组分析发现,该菌株具有与病原性假单胞菌相似的III型分泌系统,但丢失了效应蛋白调控因子hrpS和转运相关的hrpH和hrpK1基因。对全基因组扫描,菌株FP1761仅保留了病原性假单胞菌的保守效应蛋白AvrE和HopAA1-1。FP1761是目前已发现的唯一具有III型分泌系统的摩拉维亚假单胞菌。本研究表明摩拉维亚假单胞菌FP1761具有潜在的植物防病促生功能,但其III型分泌系统与植物益生互作机制有待进一步解析。     

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R

1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks.

2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum.

3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks.

4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.     

1株库蠓源版纳病毒的分离与全基因组序列分析

为分析云南省虫媒病毒的种类与遗传特征,在云南省师宗县采集库蠓进行病毒的分离与鉴定;通过全长cDNA扩增与高通量测序技术获取病毒全基因组序列,进行序列比对与系统发生树构建。结果显示,从采集的库蠓样本中分离出1株可在C6/36细胞上引起细胞病变的毒株（YNSZ043）,病毒基因组为分节段双链RNA,琼脂糖凝胶电泳呈"2-4-3"的带型特征;电镜观察可见直径为70~80 nm,呈"指环状",表面具有纤维突起的病毒粒子。全基因组测序结果显示,YNSZ043毒株为版纳病毒（Banna virus,BAV）,基因组大小为20 683 bp,由Seg-1（3 762 bp）至Seg-12（861 bp）12个基因节段组成,与中国BAV毒株各基因节段的核苷酸序列相似性在64.8%~99.6%之间,氨基酸序列相似性在58.8%~100%之间,在系统发生树上YNSZ043毒株与中国分离的BAV聚为一簇,形成独立的中国进化支系。对决定BAV基因型的Seg-12分析结果显示,YNSZ043毒株属于A2基因型,该毒株的Seg-5/VP5与越南分离BAV毒株的核苷酸和氨基酸序列相似性高达97.1%和97.6%,表明该毒株的Seg-5基因节段很可能与越南毒株之间发生了基因重配。研究结果丰富了中国BAV的基因组序列,为开展云南省BAV的流行病学研究提供了参考。     

Ace and ace-like genes of invasive redlegged earth mite: copy number variation,target-site mutations,and their associations with organophosphate insensitivity

Background

Invasive Australian populations of redlegged earth mite, Halotydeus destructor (Tucker), are evolving increasing organophosphate resistance. In addition to the canonical ace gene, the target gene of organophosphates, the H. destructor genome contains many radiated ace-like genes that vary in copy number and amino acid sequence. In this work, we characterise copy number and target-site mutation variation at the canonical ace and ace-like genes and test for potential associations with organophosphate insensitivity. This was achieved through comparisons of whole-genome pool-seq data from alive and dead mites following organophosphate exposure.

Results

A combination of increased copy number and target-site mutations at the canonical ace was associated with organophosphate insensitivity in H. destructor. Resistant populations were segregating for G119S, A201S, F331Y at the canonical ace. A subset of populations also had copy numbers of canonical ace > 2, which potentially helps overexpress proteins carrying these target-site mutations. Haplotypes possessing different copy numbers and target-site mutations of the canonical ace gene may be under selection across H. destructor populations. We also detected some evidence that increases in copy number of radiated ace-like genes are associated with organophosphate insensitivity, which might suggest potential roles in sequestration or breakdown of organophosphates.

Conclusion

Different combinations of target-site mutations and (or) copy number variation in the canonical ace and ace-like genes may provide non-convergent ways for H. destructor to respond to organophosphate selection. However, these changes may only play a partial role in organophosphate insensitivity, which appears to have a polygenic architecture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Complete Genome Sequencing and Analysis of Rehmannia Mosaic Virus Isolate from Shanxi Province

Using double-stranded RNA(dsRNA) technology and sequence-independent amplification(SIA), the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV) Shanxi isolate(ReMV-SX) was sequenced. Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV). The full-length of the obtained ReMV-SX sequence(GenBank accession no. JX575184) was 6 395 nt, containing four open reading frames(ORFs). The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0% homologous to ReMV in Tobamovirus subgroup I, while only 49.8%-58.9% homologous to the isolates in subgroups II and III of the same genus. Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship. The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa.     

一株牦牛源产细菌素植物乳杆菌SWUN5815全基因组测序及序列分析

旨在分析一株牦牛源植物乳杆菌SWUN5815的全基因组序列测序,并对该菌株的功能特性基因进行挖掘。基于PacBio RSII测序平台对乳杆菌SWUN5815进行全基因组测序分析和GO、KEGG、COG和NR数据库进行基因组基本功能注释。结果表明:1)植物乳杆菌SWUN5815的基因组大小为3.27 Mb,GC含量44.59%;2)预测到3 258个编码基因,编码基因总长度为2 814 147bp,平均长度为864bp;3)编码基因通过功能数据库对该菌株基因组基本功能注释和代谢通路基因信息注释得知,SWUN5815基因组中包括5个基因参与抗氧化活性过程,2个基因参与免疫过程;基因组中包含了抗生素、生物降解等代谢过程;可调控免疫和炎症的相关通路;基因组中含有18种已知细菌素基因;有4个合成ABC型细菌素转运系统的功能序列。综上,SWUN5815全基因组测序及分析挖掘其特异性功能基因有利于研究其益生特性提供基础,为菌株作为抗生素替代品的益生菌和饲料添加剂的应用研究提供了重要的参考数据。     

Genomic characterization of multidrug-resistant extraintestinal pathogenic Escherichia coli isolated from grain culture soils

Multidrug-resistant (MDR) Escherichia coli, mainly extraintestinal pathogenic E. coli (ExPEC), has been widely reported in infections worldwide. In agricultural soils, manure is a hotspot for the dissemination of antimicrobial resistance genes (ARGs) and pathogenic bacteria; however, MDR bacteria have also been reported in soils with no history of manure use. In addition, cross-resistance and co-resistance have been described as responsible for the metal-driven selection of bacteria resistant to antimicrobials. Therefore, the aim of this study was to analyze three MDR E. coli isolates obtained from Brazilian grain culture soil samples with no history of manure use by whole-genome sequencing. The MDR E. coli isolates were recovered from soils from corn and coffee fields, and presented resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulphonamides, and dihydrofolate reductase inhibitor. Resistome analysis showed ARGs to several antimicrobials (i.e., β-lactams, tetracyclines, aminoglycosides, sulphonamides, trimethoprim, phenicols, fosfomycin, and macrolides) as well as several metal resistance genes and antibacterial biocide resistance genes. In addition, known mutations in quinolone-resistance-determining regions of GyrA (Ser83Leu and Asp87Asn), ParE (Ser458Thr), and ParC (Ser80Ile) were also detected. Virulome analysis showed the presence of virulence genes (lpfA, mcmA, gad, mchF, iroN, cma, and iss) associated with ExPEC. Multidrug-resistant ExPEC isolates were assigned to phylogenetic group B1. The presence of MDR B1-ExPEC in soil samples shows the ability of these isolates to survive in soils. This study reports for the first time some sequence types (i.e., ST345, ST448, and ST1146) of MDR E. coli in Brazilian soils. Therefore, these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on whole-genome sequencing worldwide.     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。     

中国樱桃InDel标记开发及其在蔷薇科果树中通用性评价

对栽培和野生中国樱桃进行De novo基因组测序和比对，在全基因组范围内检测筛选出50 617个InDel位点，并挑选均匀分布的200个标记进行验证，其中，27个标记在中国樱桃种内表现出多态性。利用多态性标记，对不同来源的192份中国樱桃（168份栽培和24份野生）种质进行基因型分型，共检测到60个等位基因（N_a），平均每个标记2.2个；基因多样性指数（H_s）为0.010 ~ 0.500，平均0.239；多态性信息含量（PIC）为0.010 ~ 0.375，平均0.198，表明中国樱桃遗传基础相对狭窄。根据聚类结果和地理分布将192份中国樱桃种质大致划分为5个群体。筛选出在蔷薇科李亚科樱属、李属、桃属、杏属、苹果亚科梨属、苹果属和蔷薇亚科草莓属、悬钩子属等重要果树共8个属156个个体中表现出较好通用性标记34个。物种与中国樱桃亲缘关系越近，InDel标记的多态性越高。     

三倍化复制对白菜花粉特异表达候选基因的影响

【目的】研究三倍化复制事件后白菜花粉特异表达候选基因的进化情况,为研究白菜的花粉特异表达基因提供理论依据。【方法】利用SynOrths软件及拟南芥花粉特异表达基因集,通过共线性分析获取白菜中的花粉特异表达候选基因。通过InterproScan获取这些候选基因的GO注释,并将这些GO注释划分到与花粉相关的13个大类。然后统计每个GO的基因数占不同拷贝类型基因总数的比例,对不同拷贝类型间进行比较。进一步根据白菜3个亚基因组集,将这些候选基因划分到不同的亚基因组上。通过分析白菜花粉特异表达候选基因中串联重复基因与拟南芥基因的共线性关系,推测这些串联重复基因是在芸薹属特有的三倍化事件之前形成,还是在其之后形成。【结果】通过对拟南芥中的1 651个花粉特异表达基因进行共线性分析,在白菜中总共找到了1 962个花粉特异表达候选基因。拟南芥特异基因集里有182个串联重复基因,而白菜包含了137个串联重复基因。白菜与拟南芥在花粉特异表达基因数目上没有明显差异,因此推测这些基因的大部分拷贝可能在三倍化复制事件后发生了丢失。549个拟南芥花粉特异表达基因在白菜上找不到相应的共线性基因,白菜在进化过程中可能丢失掉了这部分基因。拟南芥花粉特异表达基因中有898个串联重复基因在白菜中对应单或多拷贝的非串联重复基因。在白菜中对应单拷贝基因的拟南芥基因数目为480个,而且所占比例高达53.5%。另外,322个基因在白菜中对应两拷贝,比例约为35.8%。而在白菜中对应三拷贝的拟南芥基因只有96个,大约占总数目的10.7%。在白菜中,单拷贝和两拷贝的基因数目显著高于三拷贝,这说明三倍化后白菜中大部分花粉特异候选基因发生了丢失,部分两拷贝和三拷贝的基因也可能处于进化选择的过程中。白菜三拷贝基因功能在7个GO分类上的比例高于单拷贝和两拷贝,而白菜两拷贝在所有GO分类上都有分布。白菜花粉特异候选基因中的大部分串联重复基因可能在三倍化事件以后发生了基因丢失,变成了非串联重复基因。也有部分非串联重复基因在三倍化事件之后形成了串联重复基因。【结论】白菜花粉特异候选基因在芸薹属特有的三倍化事件之后处于进化之中,并且三倍化事件可能促进了3种拷贝类型基因的功能差异。