Wide mapping of a T-DNA insertion site in oilseed rape using bulk segregant analysis and comparative mapping

Doubled haploid oilseed rape lines segregating for a transgene inducing herbicide resistance (bar gene) were investigated for the wide mapping of the T-DNA insertion site. Bulk segregant analysis using presence/absence and intensity polymorphisms between the bulks, as well as comparative mapping with a linkage group deriving from another cross, led to the identification of 11 random amplified polymorphic DNA (RAPD) markers tightly or loosely linked to the bar gene. Ten RAPD loci out of 11 were located on the same side of the bar locus, strongly suggesting that the T-DNA integrated in a telomeric or subtelomeric position. The eleventh RAPD marker exhibited a strong segregation distortion, which could be the result of a heteroduplex formation. Comparison of the linkage groups obtained from the two crosses showed different recombination rates between markers, possibly reflecting differences in parental genetic backgrounds. Consequences and potential applications in transgene dispersal safety assessment studies are discussed.     

发根农杆菌Ri质粒的研究和利用

本文综述了作为遗传转化高等植物载体的发根农杆菌Ri质粒,在其结构、功能及用作基因导入方面的研究进展,并讨论了被转化植物的利用策略。     

Identification of Pathogenicity Defective Mutants from a T-DNA Insertion Mutant Library of Verticillium dahliae

[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes. [Method] In total, 294 T-DNA insertion mutants of V. dahliae were tested for their virulence using a cotton infection assay. Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant. DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR. [Result] Based on the virulence assay, the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991. The Southern blot assay revealed that only one mutant contained two T-DNA insertions, while the remaining eight mutants harbored a single T-DNA insert. An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991. The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17. Furthermore, the pathogenicity-related genes were cloned from strain Vd991. [Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V. dahliae on a genome-wide scale. This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V. dahliae.     

木薯地毯草黄单胞菌转化子T-DNA插入模式分析

随机挑取30个已构建的木薯地毯草黄单胞菌(Xanthomonas axonopodis pv.manihotis)Tn5转化子,利用hi Tail-PCR技术获得Tn5转座子插入位点的侧翼序列64条。其中,有19个转化子获得了插入位点左右两端的侧翼序列,11个转化子获得了单侧侧翼序列。序列分析结果发现,插入位点集中在低GC含量区,特别是49%~50%的区域;Tn5转座子倾向于插入基因编码区内;Tn5转座子的插入会形成9 bp正向重复序列,且插入方向没有偏好性,9 bp正向重复序列的上下游碱基位点中,-5,-2,1,4,5,6,9,11位可能对转座酶的识别起关键作用。     

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system ...     

拟南芥长链脂肪醇氧化酶(AtFAO3)在抗病防卫反应中的作用分析

长链脂肪醇氧化物酶(FAO)基因编码一个与膜结合,包含黄素,产生H_2O_2的长链脂肪醇氧化酶.在拟南芥基因组中包含4个FAO同源基因.然而,拟南芥FAO在响应非生物和生物环境胁迫中起何种作用还不得而知.本研究中,我们分析了拟南芥AtFAO3在植物防卫病原细菌Pseudomonas syringae pathovar(pv.)tomato strain DC3000(Pst DC3000)中的作用.拟南芥原生质体细胞瞬时表达AtFAO3偶联GFP融合蛋白表明,AtFAO3定位于细胞膜.与野生型植株相比,拟南芥AtFAO3基因T-DNA插入突变体Atfao3在正常条件叶片中H_2O_2含量下降,在氧胁迫或生物胁迫下积累活性氧含量减少.接种病原细菌Pst DC3000后,与野生型植株相比,Atfao3突变体体内细菌繁殖数量增加,叶片病害症状加重,防卫相关基因PR1、PR2和PAL表达减弱.我们基于以上T-DNA突变体分析结果表明,AtFAO3在植物对病原细菌防卫中起重要作用.     

一种农杆菌介导稻瘟病菌的遗传转化

以农杆菌C58C1及携带潮霉素抗性的质粒pBIG2RHPH2为介导,以广泛不致病的野生型稻瘟病菌CY2为出发菌株,开展稻瘟病菌T-DNA插入转化条件的研究。农杆菌OD600为0.1条件下,乙酰丁香酮(AS)200μmol/L,用IM培养基(pH5.2)共培养。结果表明,在潮霉素、头孢噻肟钠和壮观霉素为200μg/mL,2mm滤纸条筛选培养,转化效率最高,平均可获得329.0个转化子/1×104个孢子。通过对转化子的继代稳定性和PCR检测,证明转化子均获得了T-DNA插入片段,且能稳定遗传。采用接种法,在不同遗传背景的44个抗稻瘟病的水稻近等基因系接种8000个转化子,获得20个致病突变体。     

Screening of conidium development mutant of Botrytis cinerea and functional analysis of the related gene

A novel conidium development mutant was obtained by screening the transformants of Botrytis cinerea produced by Agrobacterium tumefaciens mediated method, which lost the ability of producing conidia. The flanking sequence of T-DNA insertion site was acquired by TAIL-PCR technology, and then, the T-DNA insertion in the second exon of BC1G_02800.1 confirmed by BLAST between the flanking sequence and the known sequence in the B. cinerea gene database. The mutant gene was identified as BC1G_02799.1 located in the upstream of BC1G_02800.1 gene by RTPCR. The DNA full-length sequence of BC1G_02799.1 was 1951 bp and contained 1848 bp coding region, which encoded a 615 amino acids putative protein similar to ABC-transporter, and the function of BC1G_02799.1 gene was unknown to date. Phenotype analysis of the mutant found that the mutant strain colony was white, grew slowly, and did not produce conidium and sclerotia on PDA medium but showed a stronger pathogenicity to tomato leaves and successfully increased the enzyme activity related to pathogenicity compared to the wild type strain. The results suggested that the BC1G_02799.1 gene was involved in the conidium development, the sclerotia formation, and pathogenicity in B. cinerea. Our research will facilitate in understanding the molecular mechanism of conidium development, sclerotia formation, and pathogenic in B. cinerea.     

拟南芥atcwinv1基因T-DNA插入纯合突变体PCR鉴定及表型观察

以拟南芥atcwinvl 基因T-DNA插入纯合突变体和野生型植株为材料,比较研究了2种基因型植株在营养期和生殖期的形态差异.结果表明:拟南芥atcwinvl 基因T-DNA插入纯合突变体(简称突变体)较野生型萌发率平均下降5.88个百分点;突变体在44 d抽薹,较野生型延后4d;分支数平均4支,较野生型下降20.84...     

Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

To find new genes involved in fungal pathogenicity,a mutant(B11)exhibiting enhanced pathogenicity was isolated from an Agrobacterium-mediated transformed Magnaporthe oryzae mutant library.Southern blotting analysis showed that T-DNA insertion in the B11 genome was a single copy.TAIL-PCR and sequence alignment analyses revealed that a putative gene locus MG01679 was interrupted by the T-DNA fragment.By using the PCR-based method,the DNA and cDNA of the mutant gene MG01679 was cloned and sequenced.The open re...