An Insertional Mutagenesis System for Analyzing the Chinese Cabbage Genome Using Agrobacterium T-DNA

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.

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“…For example, Yu et al . 49 recently reported the development of 3400 T-DNA insertion mutagenesis lines in Chinese cabbage. Stephenson et al .…”

Section: Resultsmentioning

confidence: 99%

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Abé

Narusaka²,

Sasaki

et al. 2011

DNA Research

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“…Our analysis using in-house generated 1,114 and 4,030 FSTs from T-DNA or Tos17 inserted lines of rice, respectively, highlights the applicability of FSTVAL. Further analysis using preexisting rice T-DNA insertion sequences (27,621) showed FSTVAL can be applicable even in large-scale insertional mutagenesis screens such as those on-going in Chinese cabbage [13], cotton [14], poplar [15], and maize [16,17]. …”

Section: Discussionmentioning

confidence: 99%

“…Rice and Arabidopsis FST-based insertion mutant databases are freely available for searching a mutant of interest (GABI-Kat [12], Salk Institute [6]). For other plant species, such as Chinese cabbage [13], cotton [14], poplar [15], and maize [16,17], similar projects have been developed by large-scale insertional mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

FSTVAL: a new web tool to validate bulk flanking sequence tags

Kim

Lee

et al. 2012

Plant Methods

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BackgroundInformation about a transgene locus is one of the major concerns in transgenic research because expression of the transgene or a gene interrupted by the integration event could be affected. Thus, the flanking sequences obtained from transgenic plants need to be analyzed in terms of genomic context, such as genic and intergenic regions. This process may consist of several steps: 1) elimination of a vector sequence from the flanking sequence, 2) finding the locations in the target genome, and 3) statistics of the integration sites. These steps could be automated for flanking sequences from several dozens of transgenic plants generated in an ordinary targeted gene expression strategy. It would be indispensable in a genome-wide mutagenesis screen using T-DNA or transposons because these projects often generate several thousands of transgenic lines and just as many loci of the transgene among the transgenic plants.ResultsWe present an open access web tool, flanking sequence tags validator (FSTVAL), to manage bulk flanking sequence tags (FSTs). FSTVAL automatically evaluates the FSTs and finds the best mapping positions of the FST against a known genome sequence. The statistics, in terms of genic and intergenic regions, are presented as a table, a distribution map, and a frequency graph along the chromosomes. Currently, 17 plant genome sequences, including Arabidopsis thaliana, Oryza sativa, and Glycine max, are available as reference genomes. We evaluated the utility and accuracy of the tool with 5,144 rice FSTs. The whole process, from uploading the sequences to generating tables of insertions, required a few minutes, with less than 4 clicks in the web environment.ConclusionsRun for 1 year and tested over 1,000 times, we have confirmed FSTVAL efficiently handles bulk FSTs. FSTVAL is freely available without login at http://bioinfo.mju.ac.kr/fstval/.

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“…The ability to regenerate transgenic plants from these transformed cells is also vital for successful transformation. In B. rapa, Agrobacterium-mediated transformation of cotyledonary explants has led to the generation of stable transgenic plants with over-expression of N-acyl-homoserine lactonase (AHL-lactonase) 14 , rice leucine-rich repeat protein 15 , pineapple fruit bromelain gene (BAA) 16 , and GUS reporter gene 17 ; hypocotyl segments were inoculated with Agrobacterium suspension in MS liquid medium for T-DNA insertional mutagenesis 18 and metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants 19 , and a mannose selection system 20 ; and peduncular segments from flower stalk were used to transform polygalacturonase-inhibiting protein 2 (PGIP2) 21 . In B. napus, an efficient protocol for Agrobacteriummediated transformation has been described using seedling explants that is applicable to various Brassica varieties 22 .…”

Section: Agrobacterium-mediated Transformation Of Brassica Cropsmentioning

confidence: 99%

In planta transformation of Brassica rapa and B. napus via vernalization-infiltration methods

Bai¹,

Wu²,

Mao³

et al. 2013

Protocol Exchange

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In planta transformation has been widely used for gene transfer in the model plant Arabidopsis. Unlike Arabidopsis, the genus Brassica that comprises many important vegetable, oil seed and weed crops are vernalization-required, cross-pollinated, and self-incompatible. The protocols for in planta transformation established for Arabidopsis are not directly applicable to these crops. We take advantage of Arabidopsis vacuum infiltration to establish vernalization-infiltration method for transformation of Brassica crops. The germinated seeds of Chinese cabbage and oilseed rape are exposed to cold treatment for a period of vernalization; the floral buds of the early inflorescences are opened by hand before Agrobacterium infection; Agrobacterium-infected inflorescences are infiltrated with vacuum; the transformed stigmas are pollinated with the non-infected pollens of siblings; and hygromycin resistance is used to select the transformants. Using the vernalization-infiltration method, we have identified 52 homozygous transgenic lines from the cultivars of Chinese cabbage and oilseed rape.

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An Insertional Mutagenesis System for Analyzing the Chinese Cabbage Genome Using Agrobacterium T-DNA

Cited by 16 publications

References 34 publications

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

FSTVAL: a new web tool to validate bulk flanking sequence tags

In planta transformation of Brassica rapa and B. napus via vernalization-infiltration methods

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