TY - JOUR
T1 - Activation tagging approach in a model legume, Lotus japonicus
AU - Imaizumi, Ryujiro
AU - Sato, Shusei
AU - Kameya, Nanako
AU - Nakamura, Ikuo
AU - Nakamura, Yasukazu
AU - Tabata, Satoshi
AU - Ayabe, Shin Ichi
AU - Aoki, Toshio
N1 - Funding Information:
Acknowledgements The authors thank Dr. Hitoshi Kobayashi, Niigata Agricultural Research Institute, for help in constructing the tagging vector pEKB35SEXTra, and Dr. Naoto Yabe, the University of Tokyo, for useful advice on TAIL-PCR. This research was partially supported by a Grant-in-Aid for Scientific Research) (C) 14540604 from Japan Society for the Promotion of Science and a Grant-in-Aid for the 21st century COE Program “Bioresource utilization based on microbial symbiotic systems” from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
PY - 2005/12
Y1 - 2005/12
N2 - We constructed T-DNA insertional lines of a model legume, Lotus japonicus, using a multifunctional vector for gene and exon activation tagging. The vector had the CaMV 35S promoter together with two additional enhancer elements, the start codon, and splice donor and acceptor sites facing the left border, in anticipation of the activation of T-DNA flanking genes and forced expression of flanking exons. The improved transformation technique yielded more than 3,500 lines, including 45 dominant mutant candidates with abnormal phenotypes with respect to aerial parts, nodules, and roots. Among the 44 selected lines, one copy of T-DNA was inserted into the genome of 37 lines (84%). The T-DNA flanking regions of seven lines were isolated by thermal asymmetric interlaced (TAIL)-PCR or reverse transcription (RT)-PCR, and the corresponding genomic clones were analyzed. The transcripts of four genes adjacent to T-DNA out of 11 genes tested were increased in the T1 generation, demonstrating that gene and exon activation effects by the newly developed tagging vector are heritable. The T-DNA insertional population of L. japonicus will provide legume-specific dominant mutants.
AB - We constructed T-DNA insertional lines of a model legume, Lotus japonicus, using a multifunctional vector for gene and exon activation tagging. The vector had the CaMV 35S promoter together with two additional enhancer elements, the start codon, and splice donor and acceptor sites facing the left border, in anticipation of the activation of T-DNA flanking genes and forced expression of flanking exons. The improved transformation technique yielded more than 3,500 lines, including 45 dominant mutant candidates with abnormal phenotypes with respect to aerial parts, nodules, and roots. Among the 44 selected lines, one copy of T-DNA was inserted into the genome of 37 lines (84%). The T-DNA flanking regions of seven lines were isolated by thermal asymmetric interlaced (TAIL)-PCR or reverse transcription (RT)-PCR, and the corresponding genomic clones were analyzed. The transcripts of four genes adjacent to T-DNA out of 11 genes tested were increased in the T1 generation, demonstrating that gene and exon activation effects by the newly developed tagging vector are heritable. The T-DNA insertional population of L. japonicus will provide legume-specific dominant mutants.
KW - Activation tagging
KW - Legume
KW - Lotus japonicus
KW - Thermal asymmetric interlaced (TAIL)-PCR
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U2 - 10.1007/s10265-005-0231-5
DO - 10.1007/s10265-005-0231-5
M3 - Article
C2 - 16273423
AN - SCOPUS:29244459161
VL - 118
SP - 391
EP - 399
JO - Journal of Plant Research
JF - Journal of Plant Research
SN - 0918-9440
IS - 6
ER -