A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation.
In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes-one for the selection gene Hyg and the other for the target gene Gus-to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1and T2generations: Gus only, Hyg only, Gus+Hyg, and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Gus in the Hyg and Gus lines was not amplified. Genome walking confirmed that the Hyg and Gus T-DNA cassettes were independently inserted in different regions of the tobacco genome. Thus, the two T-DNA cassettes were integrated randomly as independent loci into the tobacco genome. The results of reverse transcription-PCR indicated that Hyg could normally be expressed in the roots, stems, and leaves of transgenic lines, and the resistance test showed that all Hyg transgenic lines could grow in the presence of 50mg/L hygromycin. All Gus transgenic lines showed obvious blue coloration in enzyme activity tests, indicating that the Gus gene could be normally expressed in all the lines. Therefore, the super twin T-DNA vector (pTRIDT313-g) exhibits independent integration, heredity, and normal gene function from two T-DNA cassettes. This vector could be a useful and valuable tool in the production of marker-free transgenic lines.
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Qiang
Q
Yang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Mei
M
Deng
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Ling-Ling
LL
Zhang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Xiao-Wei
XW
Zhang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Le-Ning
LN
Wang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Hu
H
Chen
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Jian
J
Ma
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Peng-Fei
PF
Qi
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Electronic address: qiantaojiang@sicau.edu.cn.
Qian-Tao
QT
Jiang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Xiu-Jin
XJ
Lan
Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Yu-Ming
YM
Wei
Key Laboratory of Southwestern Crop Germplasm Utilization, Ministry of Agriculture, Sichuan Agricultural University, Ya'an, Sichuan 625014, China.
You-Liang
YL
Zheng
D060054:Agrobacterium / Q000502:physiology*; D016386:Chromosome Walking; D004269:DNA, Bacterial; D015870:Gene Expression; D023061:Gene Order; D008040:Genetic Linkage; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics*; D016254:Mutagenesis, Insertional; D010641:Phenotype; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics / Q000382:microbiology; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA
Border sequence; Cotransformation; Southern blot; Tobacco; Twin T-DNA vector