Isolation And Functional Analysis Of A Wheat Pollen-specific Promoter

Wheat is the foremost staple food crop in the world which provides both calories and proteins to over 35%of the human population.Wheat is the second most important staple crops in China.Isolation and characterization of wheat anther/pollen-specific genes and their promoters are immensely important,and has potential applications in crops biotechnology and their improvement.It would not only help us to understand gene regulation in the sexual reproduction process,but also can be used to induce sterility in the experimental plants in breeding programs,to avoid any horizontal gene transfer.But the study of wheat anther/pollen development is lagging far behind other agricultural crops, such as rice and maize.At present,only several genes involved in wheat anther/pollon development were reported.But no information about anther/pollen-specific promoter from wheat is available in the scientific literature.With the aims to get a wheat pollen-specific promoter,the upstream regulatory region of TaPSG719 gene,a wheat pollen-specific gene,was isolated and analyzed in transgenic wheat and tobacco.The main results were as follows:(1) Based on the mRNA sequence of TaPSG719 gene reported,a 1,024 bp DNA sequence was isolated from wheat Bobwhite.Alignment of the two sequences showed that they were identical and no intron was found in the coding region of TaPSG719 gene. Based on the DNA sequence of TaPSG719 gene,a 1,776 bp promoter sequence upstream the translation start site was isolated by inverse-PCR technique.Putative functional promoter elements were analyzed by the PLACE database and the PlantCARE database. The sequences contained the basic elements,such as TATA-box and CAAT-box.Two motifs AGAAA and GTGA were detected which are known to be involved in pollen/anther-specific expression.There were ten copies of AGAAA motifs and eight copies of GTGA motifs.Many copies of AGAAA and GTGA motifs suggested that this promoter may be a pollen/anther-specific promoter.(2) To study the regulatory characteristics of TaPSG719 promoter,two vectors containing 1.7 kb promoter sequence and one vector containing 1.0 kb promoter sequence were constructed and named p1.7GFP,p1.7GUS and p1.0GUS respectively.Transgenic wheat plants harboring p1.7GFP were obtained by particle bombardment technique.GFP fluorescence was detected in pollens of transgenic wheat.It meant that TaPSG719 promoter could start GFP gene expression in wheat pollen.Vectors p1.7GUS and p1.0GUS were transformed to tobacco respectively by Agrobacterium-mediated method and 45 transgenic plants were obtained.Histochemical GUS staining and fluorescence quantitative analysis showed that TaPSG719 promoter was a pollen-specific promoter which can drive gene expression in the middle stage of pollen development till anther mature.Although TaPSG719 gene is species-specific as it has homology with only wheat and closely related species,its promoter still has the tissue-specific activity in tobacco. And the 1.0 kb fragment upstream the coding region of TaPSG719 gene was enough to drive GUS expression exclusively and specially in pollen.TaPSG719 promoter was anther/pollen-specific promoter first isolated from wheat. So far further detailed study of this promoter would shed light on understanding the function of TaPSG719 gene and sexual reproduction in wheat.TaPSG719 promoter drives the gene expression only in pollen grains would be useful for producing male-sterile plants in which the pollen is normal during microspore development but aborts at the late stage of pollen development.Therefore,it has potential applications in agriculture,especially in male-sterility.