Abstract
Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.
Highlights
Several strategies have been explored to clone DNA fragments from polymerase chain reaction (PCR) into desired vectors
A DNA fragment with the sequence GACAATAAGTC can be digested by the enzyme AhdI to generate a 39 T-overhang end. To introduce this sequence into the binary vector pCAMBIA 1300, two bridge fragments were amplified by PCR with the rice genomic DNA as a template using two primer sets as listed in Table S1 (See Methods in details)
The novel T-vector is suitable for cloning PCR products amplified by Taq DNA polymerase which can catalyze the addition of an adenine residue to the 39-end of its PCR fragment
Summary
Several strategies have been explored to clone DNA fragments from polymerase chain reaction (PCR) into desired vectors. One of the strategies is to incorporate restriction enzyme sites into oligonucleotide primers to create sticky ends by digesting the PCR products. The digestion efficiency is low for many restriction endonucleases when their recognition sequences are located within a few base pairs of the end of the PCR products [1]. The second strategy is to clone PCR products as blunt-ended fragments. The strategy requires an enzymatic processing to remove a 39 overhang adenosine which was generated by PCR Taq polymerase due to its template-independent terminal transferase activity [2]. The third strategy is to directly clone PCR products into a T-vector with 39-T overhang. The strategy is applicable to clone PCR products with 39-T overhang generated by some of thermostable DNA polymerases
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