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t-dna Integration Category and Mechanism in Rice Genome

Journal of Integrative Plant Biology 2005年第3期47卷 350-361页

作者： JiangWANG LinLI Zhen-YingSHI Xin-ShanWAN Lin-ShengAN Jing-LiuZHANG StateKeyLaboratoryofPlantMolecularGenetics InstituteofPlantPhysiologyandEcologyShanghaiInstitutesforBiologicalSciencestheChineseAcademyofSciencesShanghai200032China

Abstract: t-dna integration is a key step in the process of plant transformation, which is proven to be important for analyzing t-dna integration mechanism. the structures of t-dna right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the t-dna right border occurred mainly on five nucleotides “tGACA” in inverse repeat (IR) sequence of 25 bp, especially on the third base “A”. However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the t-dna right border, which made the t-dna, carrying vector sequences, integrated into the rice genome. these results seemingly suggest that the IR sequence of the right border plays an important role in the process of t-dna integration into the rice genome, but is not an essential element. the appearance of vector sequences neighboring the t-dna right border suggested that before being transferred into the plant cell from Agrobacterium, the entire t-dna possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the t-dna right border homologous with plant dna and filler dnas were frequently discovered in the integrated position of t-dna. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler dna, through mutual twisting, and then the t-dna was integrated into plant chromosome through a partially homologous recombination mechanism. the appearance of filler dna would facilitate t-dna integration. the fragments flanking the t-dna right border in transformed rice plants could derive from different parts of the inner t-dna region; that is, disruption and recombination could occur at arbitrary positions in the entire t-dna, in which the homologous area was compar

关键词： integration mechanism rice (Oryza sativa L.) right border t-dna

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Distribution of t-dna carrying a Ds element on rice chromosomes

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Science China(Life Sciences) 2004年第4期47卷 322-331页

作者： WANG Jiang LI Lin WAN Xinshan AN Linsheng ZHANG Jingliu National Key Laboratory of Plant Molecular Genetics Institute of Plant Physiology & EcologyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200032China

Over 3000 rice plants with t-dna carrying a Ds element were constructed by Agro-bacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of t-dna (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler dna between the right border of t-dna and flanking sequence of rice genome. type I sequences were the most common and showed canonical in-tegration that t-dna right border was followed by rice genome sequence with or without filler dna of no more than 50 bp, while type II sequences displayed a vector-genome combination that t-dna right border was followed by a vector fragment and then connected with rice genome sequence. the location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLASt analysis. the 340 Ds insertions at an average in-terval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. the frequency of t-dna (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of t-dna (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.

关键词： Ds element t-dna mutagenesis rice(Oryza sativa L.).

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Establishing a Gene trap System Mediated by t-dna(GUS) in Rice

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Journal of Integrative Plant Biology 2008年第6期50卷 742-751页

作者： Shi-Yan Chen Ai-Min Wang Wei Li Zong-Yang Wang Xiu-Ling Cai State Key Laboratory of Plant Chinese Molecular Genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200032 China Graduate School of the Chinese Academy of Sciences Beijing 100039 China School of Life Sciences Xuzhou Normal University Xuzhou 221116 China

two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promotsriess β-glucuronidase （GUS） reporter gene in the t-dna region. transformation of these two plasmids into the rice variety Zhonghua 11 （Oryza sativa ssp. japonica cv.）, mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 to plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistsnt （hygr） homozygotes were screened and the copy number of the t-dna inserts was determined in the GUS-positive transgenic plants. the flanking sequences of the t-dna were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of t-dna were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the t1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. the results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

关键词： β-glucuronidase gene trap rice gene t-dna

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Illumina Sequencing technology as a Method of Identifying t-dna Insertion Loci in Activation- tagged Arabidopsis thaliana Plants

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Molecular Plant 2012年第4期5卷 948-950页

作者： Joanna K. Polko Mohamed-Ramzi temanni Martijn van Zanten Wilbert van Workum Sven Iburg Ronald Pierik Laurentius A.C.J. Voesenek Anton J.M. Peeters Plant Ecophysiology Institute of Environmental Biology Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands ServiceXS BV Plesmanlaan ld 2333 BZ Leiden The Netherlands Department of Cell Biology and Molecular Genetics Maryland Pathogen Research Institute (MPRI) 3128 Biosciences Research Bldg University of Maryland College Park MD 20742 USA Molecular Plant Physiology Institute of Environmental Biology Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands

Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially t-dna activation tagging pop- ulations are frequently employed. these populations are gener- ated using vectors containing multiple copies of the constitutive 35S promoters derived from cau and often result in isolation i flower mosaic virus （35S CaMV） of dominant gain-of-function alleles （Weigel et al., 2000; Nakazawa et al., 2003）. this allows the study of members of large gene families that are often func- tionally redundant and, therefore, hard to identify in loss-of- function screens. Moreover, due to the dominant nature,

关键词： t-dna 激活标签拟南芥测序技术插入位点 35S启动子识别中国农业大学

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A Gγ subunit promoter t-dna insertion mutant——A1-412 of Magnaporthe grisea is defective in appressorium formation,penetration and pathogenicity

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Chinese Science Bulletin 2006年第18期51卷 2214-2218页

作者： LIANG Shen WANG Zhengyi LIU Pengjuan LI Debao Institute of Biotechnology Zhejiang University Hangzhou 310029 China

In our previous studies,a nonpathogenic mutant of Magnaporthe grisea,A1-412,which was defective in appressorium formation,penetration and infectious growth,was obtained by t-dna insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Se- quence analysis of the genomic dna franking t-dna isolated by tAIL-PCR technique showed that t-dna was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. the amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that ex- ogenous cAMP could induce appressorium formation,although the formed appressoria were abnormal in shape and unable to penetrate onion epidermis or rice leaves. Moreover,few perithecia were observed when A1-412 was crossed with the appropriate mat- ing-type strain. the above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis,mating and pathogenicity in M. grisea.

关键词：形态发生插入突变致病性病毒 t-dna

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农杆菌介导转化黑曲霉分生孢子及产孢缺陷突变子t-dna插入位点分析

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微生物学报 2011年第2期51卷 270-275页

作者：黄文杨洪江秦慧彬工业微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院天津300457

【目的】利用农杆菌(Agrobacterium tumefaciens)t-dna系统,建立转化黑曲霉(Aspergillus niger)分生孢子的方法,构建t-dna插入突变子文库,为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105... 详细信息

【目的】利用农杆菌(Agrobacterium tumefaciens)t-dna系统,建立转化黑曲霉(Aspergillus niger)分生孢子的方法,构建t-dna插入突变子文库,为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105,诱导转化黑曲霉分生孢子,筛选具有潮霉素抗性的突变子。分析抗性稳定突变子菌株的表型,采用反向PCR方法分析t-dna插入位点相邻位置的序列,并推测突变基因可能具有的功能。【结果】实验获得具有稳定潮霉素抗性转化子193株,转化率为5.6×102转化子/108分生孢子。部分转化子表型出现较为明显改变,其中一株不能产孢,对其t-dna插入位点序列分析比对结果显示,突变基因属于超级转运家族(major facilitator superfamily,MFS)。【结论】本研究建立的农杆菌转化黑曲霉分生孢子平台,结合t-dna插入突变位点分析,可以为黑曲霉基因组功能注释研究提供一种简便有效的途径。

关键词：黑曲霉分生孢子农杆菌介导转化 t-dna 反向PCR

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农杆菌介导转基因植物t-dna的整合方式

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遗传 2011年第12期33卷 1327-1334页

作者：杨琳付凤玲李晚忱四川农业大学玉米所成都611130

农杆菌介导的遗传转化已被广泛应用于植物转基因研究。作为外源基因的载体,农杆菌t-dna片段在植物基因组中的整合方式,不仅影响外源基因的整合效率及稳定性,还会影响外源基因的表达特性。文章就农杆菌介导的t-dna整合的两种主要模式、... 详细信息

农杆菌介导的遗传转化已被广泛应用于植物转基因研究。作为外源基因的载体,农杆菌t-dna片段在植物基因组中的整合方式,不仅影响外源基因的整合效率及稳定性,还会影响外源基因的表达特性。文章就农杆菌介导的t-dna整合的两种主要模式、规律及相关研究手段进行综述,为农杆菌介导的转基因及t-dna插入突变等相关研究提供借鉴。

关键词：农杆菌 t-dna 侧翼序列整合转基因

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Induction of chromosomal inversion by integration of t-dna in the rice genome

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Journal of Genetics and Genomics 2010年第3期37卷 189-196页

作者： Chuanfeng Zhu,Jiahe Wu,Chaozu He State Key Laboratory of Plant Genomics,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China [a] State Key Laboratory of Plant Genomics Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China

transfer dna （t-dna） of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments, including inversions. However, there is very little information concerning the inversion. the present study re- ports a transgenic rice line selected from a t-dna tagged population, which displays a semi-dwarf phenotype. Molecular analysis of this mutant indicated an insertion of two tandem copies of t-dna into a locus on the rice genome in a head to tail mode. this insertion of t-dna resulted in the inversion of a 4.9 Mb chromosomal segment. Results of sequence analysis suggest that the chromosomal inversion resulted from the insertion of t-dna with the help of sequence microhomology between insertion region of t-dna and target sequence of the host plant.

关键词： Agrobacterium tumefaciens-mediated transformation (AtMt) t-dna chromosomal inversion

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t-dna transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

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High technology Letters 2008年第1期14卷 92-97页

作者：迟彦 Ping Wenxiang Li Shanshan Zhu Jing Ma Xi Gao Fengshan Zhou Dongpo Key Laboratory of Microbe Heilongjiang University Harbin 150080 P.R. China Key Laboratory of Bio-organic Chemistry Dalian University Dalian 116622 P. R. China

Agrobacterium tumefaciens-mediated dna transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-resistant gene cassette between the right and left borders of t-dna, Optimal co-cultivation of *** with A. tumefaciens containing pBI121-43 led to 110- 130 hygromycin-resistant transformants per＂ million eonidia. Putative transformants were found to be mitotically stable. the molecular analysis of transformants demonstrated the random integration of single copy of the t-dna into the host genome. this transformation system serves as a basic tool for insertional mutagenesis in N. sylviforme fusant HDF-68, and the development of such svstem lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.

关键词： taxol t-dna insertional mutagenesis

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稻瘟病菌t-dna插入方法优化及其突变体分析

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生物工程学报 2003年第4期19卷 419-423页

作者：李宏宇潘初沂陈涵赵长江鲁国东王宗华福建农林大学生命科学学院福建农林大学植物保护学院福州350002

优化了农杆菌介导转化稻瘟病菌获得t dna插入突变的条件 ,包括选择转化子的潮霉素B用量 ,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比 ,不同转化阶段培养基的选择等。转化 1× 10 6 个孢子平均可获得约5 0 0个左右的转化子 ,PCR和t... 详细信息

优化了农杆菌介导转化稻瘟病菌获得t dna插入突变的条件 ,包括选择转化子的潮霉素B用量 ,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比 ,不同转化阶段培养基的选择等。转化 1× 10 6 个孢子平均可获得约5 0 0个左右的转化子 ,PCR和tAIL PCR检测表明约 85 %转化子中含t dna插入。对 15 2 0个突变体进行形态变异观察 ,发现菌落颜色突变的有 15个 ;随机取 5 8个突变体进行比较 ,发现产孢量减少的 4个 ,孢子萌发率降低的 8个 ,附着胞形成率降低的 9个 ;还获得对水稻品种C10 1LAC(Pi 1)和 75 1 12 7(Pi 9)致病的突变体。

关键词：稻瘟病菌 t-dna 插入方法突变体功能基因组学

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