Cloning of an APETALA3 homologous gene (PtAP3) from Populus tomentosa and genetic transformation of its sense and anti-sense constructs in tobacco

Abstract

A pair of primers were designed according to published literature on Populus trichocarpa gene (PTD), and PtAP3, an AP3 homologous gene from Populus tomentosa was isolated by PCR using genomic DNA of the male clone of P. tomentosa (L50) as a template. The result indicated that the sequence was 1,813 bp (BamH!`and Sac!` were introduced at the 5 2 and 3 2 end) including 7 extrons and 6 introns, coding 238 amino acids. It was found that there was 52%-82% homology to proteins from Lilium regale (AF503913), Petunia hybrida (AF230704), Gerbera hybrida (AJ009724), Rosa rugosa (AB055966), Malus domestica (AJ251116), and P. trichocarpa (AF057708) determined by blast analysis in the GenBank. There was a highly conserved MADS-box motif in the protein of PtAP3, so it was putatived to be a transcription factor. The result of Southern blot analysis indicated that there were double copies of PtAP3 or two members which had a high homology to each other in P. tomentosa (L50, male) genomic DNA, and there was single copy PtAP3 in P. tomentosa (5082, female) genomic DNA. Sense and antisense expression vectors of PtAP3 were constructed by PCR and restriction enzymes digestion identification, and transformed into tobacco (Nicotiana tabacum) by Agrobacterium GV3101 and LBA4404. Some transgenic tobacco plantlets were obtained by PCR identification. The results mentioned above have provided important data to understand the molecular mechanism of male flower development of P. tomentosa, and has contributed to the study on controlling flowering of P. tomentosa using genetic engineering.