Promoter trapping in tobacco and Arabidopsis.

A promoter trapping system based on the promoter trap vector pDeltaGUSBin19 was studied for the identification and isolation of developmentally regulated plant genes. pDeltaGUSBin19 contained a promoterless gusA gene and a nos promoter-driven nptII gene. The previously unknown regions of pDeltaGUSBin19 were sequenced. A method of high frequency transformation of Arabidopsis thaliana by Agrobacterium-tumefaciens was developed. The T-DNA of pDeltaGUSBin19 was introduced into tobacco and Arabidopsis to generate a large number of transformed lines. The levels and patterns of gusA gene activation in diverse organs and cell types of transgenic plants were analysed. Line atvt1 exhibited GUS fusion activity in the tapetum and vascular tissues. It contained a single copy of the T-DNA and the gusA gene fusion was transcribed as a fusion transcript. Wild-type genomic and cDNA clones corresponding to the tagged gene were isolated using a molecular probe generated by IPCR of genomic sequence flanking the T-DNA. The native mRNA was approximately 4.4 kb. The foil length cDNA of this gene was cloned, sequenced and analysed. It encoded a putative nucleic acid helicase, designated HVT1 (Helicase in Vascular tissue and Tapetum). Of a predicted 1291 amino acid residues, HVT1 is homologous to the Drosophila MALELESS, human RNA helicase A and bovine nuclear DNA helicase proteins, and represents the first identified member of a new subgroup within the mle group of the DEAH protein family. Low stringency genomic Southern blot analysis indicates there exists another structurally related gene in Arabidopsis. The value of promoter trapping as a complement to other approaches of gene isolation and possible function of the HVT1 protein is discussed.