Abstract
We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU14 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline-based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.
Highlights
Bacteriophage λ relies on the site-specific recombination reaction to integrate the phage DNA by the BP clonase into the bacterial chromosome and excise it out by the LR clonase [1]
The minimal requirement for the component of tetracycline resistance gene was tested by the tetracycline-resistance comparison and the plasmid stability experiment in E. coli and A. tumefaciens
We previously constructed a series of kanamycin-based binary Ti vectors pLSU1 to 5 to improve the transformation frequency and plasmid yield in E. coli and A. tumefaciens for A. tumefaciens-mediated transformation of higher plants [22,25]
Summary
Bacteriophage λ relies on the site-specific recombination reaction to integrate the phage DNA by the BP clonase into the bacterial chromosome and excise it out by the LR clonase [1]. The BP clonase reaction for DNA integration is catalyzed by the phage integrase and integration host factor. Two attB sites (21 to 25 bp) at the ends of a target DNA fragment (or a PCR product) recombine with two attP sites of the Gateway donor vector (pDONR), resulting in generation of two attL sites (96 bp) in an entry vector (pENTR) concomitant with transfer and integration of the target DNA [2]. The LR clonase reaction for DNA excision is catalyzed by the phage excisionase, integrase, and integration host factor. Two attL sites flanking the target DNA in the entry vector recombine with two attR sites of a destination vector (pDEST), resulting in creation of two attB sites in an expression vector (pEXPR) and excision/transfer of the target DNA fragment. Succession of four Gateway vectors, donor, entry, destination, and expression vectors are bacterial kanamycin-based plasmids
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