A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

利用细菌转座子构建适于家蚕的Bac-to-Bac快速基因表达系统

为了较好地解决家蚕杆状病毒基因表达系统(BmNPV expression system)在重组病毒构建和纯化过程中存在的重组率低、空斑分析技术繁琐、花费时间长等缺点,借鉴国外AcNPV Bac-to-Bac快速基因表达系统工作原理,对家蚕BmNPV基因组进行了改造。通过同源重组的方法,将含有单拷贝数细菌F复制子、插入有细菌转座子整合靶位点、编码LacZα肽的部分DNA片段和抗性选择标记基因的基因片段重组入家蚕BmNPV基因组,替换多角体蛋白基因,获得了家蚕BmNPV病毒穿梭载体BmBacm id;并利用供体质粒上的表达盒和细菌转座子以及细菌转座子的基因定位转移作用,在细菌体内实现外源目的基因向家蚕BmNPV基因组上的转移整合,快速完成重组BmN-PV病毒的构建。     

基于piggyBac转座子转hGM-CSF基因家蚕的研究

将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产生荧光蚕的比例分别为0.17%,0.15%。将次代荧光蚕与正常蚕交配后代(G1)的荧光蚕个体再相互杂交,连续进行多代选育,获得了稳定遗传的转hGM-CSF基因家蚕品系。     

利用转基因RNA干涉提高家蚕对BmNPV的抗性初探

基于探讨利用转基因RNA干涉技术使家蚕获得对核型多角体病毒抗性的目的,将家蚕核型多角体病毒(BmNPV)的DNA聚合酶基因、蛋白激酶(PK1)基因以及bro-d和orf1629基因的部分编码片段,分别以其反向重复的形式与家蚕Actin3启动子连接,构建了基于piggyBac转座子载体的转基因表达载体,通过显微注射于家蚕卵,获得了36~107个G1蛾区,得到了20~23头转基因阳性家蚕。经Southern杂交及RT-PCR分析表明,BmNPV的4个反向重复片段都已经导入家蚕基因组中,并且可以进行转录。攻毒实验结果表明,BmNPV的DNA聚合酶基因和PK1基因反向重复片段对病毒的增殖有抑制作用,从而使家蚕对BmNPV具有一定抗性;而BmNPV的bro-d和orf1629基因反向重复片段对病毒的增殖没有抑制作用。针对特定病毒的转基因RNAi技术有望使家蚕获得对该病毒的抗性。     

Tn5 transposon mutagenesis in Acidovorax citrulli for identification of genes required for pathogenicity on cucumber

An Acidovorax citrulli–cucumber pathosystem was established through which A. citrulli mutants with altered pathogenicity, generated by transposon mutagenesis, were identified on cucumber cotyledons. The A. citrulli group I strain FC440 was shown to grow faster in cucumber leaf tissues than a group II strain and was used for Tn5 transposon mutagenesis. A total of 2100 Tn5 insertional mutants were generated, and analysis of the mutant library showed that the transposon insertions were single, independent and stable. A conserved non‐flagellar type III secretion system (NF‐T3SS) ATPase gene hrcN was identified and confirmed to be essential for pathogenicity and functionality of NF‐T3SS in A. citrulli. Comparative sequence analysis of the HrcN protein and its homologues in other representative bacterial plant pathogens revealed that the NF‐T3SS of A. citrulli is close to that of Ralstonia solanacearum and Xanthomonas campestris, but distant from that of Pseudomonas syringae and Erwinia amylovora. The generated Tn5 insertional mutant collection is valuable for identification of genes required for A. citrulli pathogenesis, and the established A. citrulli–cucumber pathosystem will facilitate an improved understanding of A. citrulli biology and pathology.     

细菌转座子Tn5转座机理的研究进展

细菌转座子Tn5属于IS4家族，两端具有两段倒置的IS50序列。右末端(IS50R)编码转座酶(Tnp)和一个阻遏转座蛋白(Inh)；左末端(IS50L)存在一个赭石突变，不能编码有功能的转座酶。Tn5属于非复制型转座子，被广泛应用于发现新基因、分析基因功能和构建突变体库。但是在相当长的一个时期里，人们对其转座机理、转座热点等问题并不完全清楚。随着在体外具有活性的转座酶Tnp EK／LP(同时含有E54K和L372P两个突变点)的成功构建，以及对Tnp催化中心三维结构的深入研究，近几年对Tn5的转座机理才有了深刻了解。本文综述了Tn5的转座机制、Tnp结构、DDE模体和转座频率调控等研究的最新进展。     

鸭疫里默氏杆菌50S L27转座子插入突变株的生物学特性分析

为了鉴定鸭疫里默氏杆菌中与卡那霉素抗性相关的基因,本研究采用构建Yb2株转座子随机突变库结合卡那霉素抗性平板来筛选相关抗性基因。先以Yb2株为受体菌,携带质粒pEP4351的大肠杆菌BW19851(pEP4351)为供体菌,构建转座子Tn4351随机突变库。用含红霉素和卡那菌素的TSA进行筛选,得到卡那霉素耐药性降低的突变株DK-2;然后采用基因组步移的方法鉴定表明,转座子插入基因组上编码核糖50S L27亚基基因;再将携带野生株Yb2的50S L27基因的大肠杆菌-鸭疫里默氏杆菌穿梭表达载体pRES-50S L27导入突变株DK-2,构建得到了回复菌株cDK-2。结果表明:50S L27基因缺失对其生长无明显的影响,而DK-2突变株对卡那霉素、新霉素和利福平的敏感性均增强。本研究为进一步解析鸭疫里默氏杆菌50S L27亚基的功能奠定了基础。     

拟南芥的生物学特性及基因克隆策略

因拟南芥具有独特的生物学的特性，它被用作植物生物学研究的模式系统。国际间科学家们的使用在拟南芥研究中起着重要作用。虽然有许多方法可以得到基因，但转坐子标签技术使克隆基因更容易、简便。     

一个链霉菌中微型转座子系统的构建

针对链霉菌中现有转座子系统的一些缺陷,构建了一个链霉菌中的微型转座子质粒pHL265,它携带的转座酶基因tnpA在转座子mini-Tn4560A外部,降低了发生二次转座的可能性,并带有大肠杆菌与链霉菌进行属间接合转移的起始位点oriT,可通过接合转移的方式将其从大肠杆菌导入到链霉菌中.利用该转座子系统转座筛选得到天蓝色链霉菌M145的约1 000株转座突变株.选取其中的8株,经Southern杂交验证可知,该转座子的转座基本具有随机性,并在宿主DNA中稳定存在.此系统为链霉菌功能基因组的研究提供了新的技术手段.