T-DNA insertional mutagenesis for functional genomics in rice.
We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.
Jeon
J S
JS
National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Republic of Korea.
Lee
S
S
Jung
K H
KH
Jun
S H
SH
Jeong
D H
DH
Lee
J
J
Kim
C
C
Jang
S
S
Yang
K
K
Nam
J
J
An
K
K
Han
M J
MJ
Sung
R J
RJ
Choi
H S
HS
Yu
J H
JH
Choi
J H
JH
Cho
S Y
SY
Cha
S S
SS
Kim
S I
SI
An
G
G
D001483:Base Sequence; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D018745:Genome, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic
D004269:DNA, Bacterial; C036483:T-DNA; D005966:Glucuronidase