Establishing a gene trap system mediated by T-DNA(GUS) in rice.
Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless beta-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 T(0) plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hyg(r)) 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 T(1) 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.
Chen
Shi-Yan
SY
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.
Wang
Ai-Min
AM
Li
Wei
W
Wang
Zong-Yang
ZY
Cai
Xiu-Ling
XL
D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics*; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D005821:Genetic Techniques; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006720:Homozygote; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA; D005966:Glucuronidase