Cloning And Analysis Of A Novel Pollen-specific Gene From Potato And Transfer SBLR Genomic DNA Into Maize

The gametophyte (pollen grain) contains all the genetic information required to unite with the female gamete at fertilization and to form a new sporophyte. A relatively large number of genes are required to program pollen development. In order to establish the pollen-specific gene expression and regulation mechanism(s), it will be necessary to isolate and character more pollen-specific genes and promoters.Screening of a potato genomic library with a full-length SB401 cDNA resulted in the isolation of six positive clones. One of clones, designated 701, was further characterized. Over 9 Kb Sad restriction fragment was cloned into pGEM-7Zf (+), mapped for restriction endonuclease sites and an 4.5Kb fragment was further subcloned and sequenced.Sequence analysis showed 4459bp fragment contains 2320bp putative promoter region within 5' upstream of the genomic clone. A putative TATA box and CAAT box were located at -220-214 and -310-306 relatively to translation start site. SBLR gene contained three exons and two introns and encoded a protein of 211 amino acids, whose lysine composition is 18.93% (w/w). SBLR gene is a natural lysine-rich gene, which has been patented.To determine whether this 5' flanking region has promoter activity and to investigate its expression pattern, the series of fragments -2247bp, -1749bp and -1338bp was amplified by PCR and fused to the encoding region of gfp gene. The plant expression constructs were transformed into tobacco plants by Agrobacterium-media transformation. The transgenic plants were analyzed by PCR and Southern blotting to verify the presence of GFP coding sequence. In the GFP-positive plants, the expression of gfp gene under the control of the SBLR promoter was visualized in pollen tube, but no obvious expression in other tissues that have been detected, like leaves, roots and stems. The sequence was compared with nucleic acid databases, GenBank, EMBL and DDBJ by blastn servers, no significant matches to any other sequences was found, was regarded as a novel promoter. These results strongly suggested us that SBLR promoter is a novel pollen-specific promoter.To further localize c/s-elements regulating SBLR promoter activity, the progressive 5' deletions at position -1212, -1063, -842, -742, -456, -405, -345,-269 and -215 were fused to the GUS report gene and the plant expression constructs were stably introduced into tobacco by Agrobacterium-media transformation. SBLR promoter activity was determined by fluorimetric GUS assays and histochemical analyses, and the results demonstrated the cis-element regulatory of SBLR promoter was concentrated in the region -345 to -269.Plant expression vector 19ZKgH (seed-specific promoter 19Z::SBLR genomic DNA) and AkgH (Adhl promoter::SBLR genomic DNA) were transformed into maize embryonic callus by microprojectilebombardment. The stable transgenic maize plants were analyzed by PCR, Dot blotting and Southern blotting to verify the integration of SBLR gene into maize genome. We analyzed the content of lysine and crude protein in mature seeds of RI transgenic plants and found that content of protein and lysine had an increase of from 8.1% to 58.1% and from 3.2% to 54.8% respectively. In R2 transgenic plants, the lysine and the crude protein content were increased from 19.2% to 55.1% and from 3.2% to 51.6% respectively. Six transgenic lines with the increases of 30% in both protein and lysine content can be used further breeding. It is the first report transfering genomic DNA of lysine-rich protein gene into crop to improve the nutrition quality.RT-PCR and RT-PCR Southern blotting analyses of transgenic plants revealed the two introns derived from potato had been accurately and efficiently processed in transgenic maize plants and the average splicing efficiency was 98.60%. These results strongly determined an intron of dicot origin could be spliced accurately and efficiently in monocot plant.