Cloning And Functional Characterization Of Pollen-specific Gene ST901 From Potato Solanum Tuberosum L.

The gametophyte (pollen grain) contains all the genetic information required to unite with the female gamete at fertilization and to form a new sporephyte. 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.Screening of a potato genomic library with a full-length SB401 cDNA resulted in the isolation of six positive clones. One of clones, designated 901, was further characterized. Over 13 kb SacI restriction fragment was cloned into pGEM-7Zf (+), mapped for restriction endonuclease sites and an about 5.0kb fragment was further subcloned and sequenced. Through coding region specific primer, we amplied it's corresponding cDNA, named ST901.ST901 is 2889bp long, contains 1447bp putative promoter region within 5' upstream and three exons (475bp, 140bp, 39bp) and two introns (472bp, 2S3bp) in the coding region, encodes a hydrophilic protein of 217 amino acid residues with a molecular mass of 24kDa. There are four imperfect repeats V-V-E-K-K-N/E-E of which the core sequence is similar to MAP IB of mouse. RNA blot and RT-PCR analysis showed that this gene is expressed specifically in the mature pollen and can be classified as a late gene in pollen development. Genomic DNA blot analysis showed that ST901 is present in low copies in potato genome. Homologious analysis showed that it belongs to a small-member gene family.To study the expression pattern of ST901 gene in detail, the six promoter fragments were amplified by PCR and fused to the GUS report gene. The plant expression constructs were stably introduced into tobacco by Agrobacterium-media transformation. Histochemical GUS assay showed that the GUS staining was observed only in the mature pollen and germination pollen tube. There is no detectable GUS activity in other floral organs, leaves and stems. These results suggested that ST901 is a novel pollen-specific gene and the 288bp promoter fragment (-297?Xfrom the translation start codon ATG) is sufficient for pollen-specific expression and os-element regulatory of ST901 promoter was possibly concentrated in the region -297 to -9.To investigate the function of ST901 gene, we constructed four plant expression vectors with sense and antisense ST901 gene promoted by CaMV35S promoter or it'self promoter. Plant expression vectors were instructed into tobacco and potato by Agrobacteriwn-media transformation. To verify the integration of ST901 gene into transgenic plants genome, the stable transgenic plants were analyzed by PCR and Southern blotting. The aberrant phenotypes were observed in pollens and anthers of the transgenic plants. Most of pollen grains in the transgenic plant were distorted, shrunken, invagination and not stained with the solution of acetocarmine. In contrast, most of the pollen grains in the non-transformant have plump shape and were stained into red color uniformly. In transgenic potato plants with antisense ST901 promoted by it'self promoter, the normal pollen percent was reduced to 5.8% at the most, compare to the non-transformant plant. In the transgenic tobacco plants with sensegene promoted by CaMV35S, the anthers of four lines were changed into petals partly or totally or no anther at all. Another, the pollen sac and the microspore differentiation tissues are all aberrant, this make it's impossible to obtained normal pollen grains. All results showed that the ST901 gene plays an important role during pollen maturation and pollen tube germination.