Highly efficient generation of T-DNA insertion lines and isolation of flanking sequence tags (FSTs) of Brachypodium distachyon

Hongjiang Han; Guoan Shen; Tianyue An; Bo Song; Suzhen Zhao; Xiaoquan Qi

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자료유형: 학술저널

저자정보: Hongjiang Han (Institute of Botany Chinese Academy of Sciences) Guoan Shen (Institute of Botany Chinese Academy of Sciences) Tianyue An (Institute of Botany Chinese Academy of Sciences) Bo Song (Institute of Botany Chinese Academy of Sciences) Suzhen Zhao (Institute of Botany Chinese Academy of Sciences) Xiaoquan Qi (Institute of Botany Chinese Academy of Sciences)

저널정보: 한국식물생명공학회 Plant Biotechnology Reports Plant Biotechnology Reports 제12권 제4호

발행연도: 2018.8

수록면: 237 - 248 (12page)

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Brachypodium distachyon (Brachypodium) has been developed as a model system for the temperate grasses. Hence, establishing a large insertion mutant population and identifying T-DNA insertion sites are imperative for functional genomics studies. Currently, Brachypodium has an established T-DNA collection resource, but it is far less than needed. In addition, the conventional methods for the isolation of flanking sequence tags (FSTs) characterizing the T-DNA inserts, such as TAIL-PCR, adapter ligation-mediated PCR, and inverse PCR, are time-consuming, laborious, and expensive. In this study, Agrobacterium-mediated transformation system was optimized to generate T-DNA mutants. Approximately 7000 T-DNA insertion lines were obtained. Furthermore, a simple and highly efficient method was developed to isolate T-DNA flanking sequence tags (FSTs). The procedures simplified the multi-step reaction that is required for the conventional inverse PCR and several reactions were conducted within a microscale reaction system in one tube. It is flexible to isolate FSTs for either individual or large numbers of T-DNA lines. To rapidly process the large-scale sequence data, a serial of Perl scripts were developed using the Perl Programming Language (http://www.perl.org). Using these methods, a total of 794 flanking sequences were isolated and analyzed in detail. Although the methods were developed in the process of isolating and analyzing T-DNA flanking sequences of Brachypodium, it can be applied to other plants.

#Brachypodium distachyon #Agrobacterium-mediated transformation #T-DNA insertion #Flanking sequence tag (FST) #Modified iPCR

참고문헌 (42)

참고문헌 신청

An S / 2003 / Generation and analysis of end sequence database for T-DNA tagging lines in rice / Plant Physiol 133:2040~2047

An T / 2016 / Brachypodium distachyon T-DNA insertion lines: a model pathosystem to study nonhost resistance to wheat stripe rust / Sci Rep 6:25510

Bragg JN / 2012 / Generation and characterization of the Western Regional Research Center Brachypodium T-DNA insertional mutant collection / PLoS One 7:e41916

Brutnell TP / 2015 / Brachypodium distachyon and Setaria viridis: model genetic systems for the grasses / Annu Rev Plant Biol 66:465~485

Camacho C / 2009 / BLAST+: architecture and applications / BMC Bioinformatics 10:421

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