Gene Cloning And Function Alanalysis Of BdHTA1in Brachypodium Distachyon

In this paper, Brachypodium distachyon diploid inbred line BD21was used as experimental material. We have completed the bioinformatics analysis of gene BdHTAl, comparison of the amino acid sequence and phylogenetic analysis of the13-member gene family. The experiment was aimed to explorer the potencials for BdHTAs to improve transformation efficiency and therefore, in the near future, to improve wheat genetic transformation.1. Cloning, amino acid sequence comparation and phylogenetic analysis in BdHTAsThe Brachypodium genome contained13histone H2A genes according to the bioinformatics analysis results. Comparation of the13Brachypodium histone H2A amino acid sequences indicated that they can be categorized into four majour groups. The members Bradi2g37330, Bradi2g23090, Bradilg66360.1, Bradilg66360.2, Bradi4g28560, Bradilg66370and Bradi4g06010had a SPKK motif in their C-terminal region. This motif was found in plant histone H2As that are cell cycle reglated. We set these into group I. The members Bradilgl0390and Bradi4g06010had a SQEF motif and were set into group II. The members Bradilg25390, Bradi25400and AtHTAl were set into group III. The rest including Bradilgl4960, Bradilg09060and Bradi3g26880were set into group IV. Among these members, Bradilg25390and. Bradilg25400were most likely the homologous gene of AtHTA1. The corresponding full-length cDNA of Bradilg25390was obtained in our experiment.2. Construction of overexpression vectors of Bradilg25390and AtHTA1, obtaining transgenic plants and plant transformation for complementation of the rat5mutantTo investigat the effect of HTA1on Brachypodium transformation, the Bradilg25390gene was ligated to a maize ubiquitin promoter. The Bradilg25390expression cassette was inserted into the T-DNA vector pGA3426. pGA3426was used as the vector control. Both AtHTA1and Bradilg25390were introduced into the Brachypodium accession BD21. The putative transgenic plants were confirmed by PCR analysis. For cDNA complementation analysis, histone H2A cDNA Bradilg25390and gene AtHTAl were ligated into the multicloning site of the T-DNA binary vector pBINPLUS behind the2×CaMV35S promotor. The various constructions were introduced into A. tumefaciens and used for floral dip transformation of rat5mutant plants. Transgenic rat5plants displayed restoration of transformation efficiency.3. Subcellular localization and different patterns of expression of Bradilg25390Compared to the empty vector which was expressed in nucleus, cytoplasm and cell membrane, the subcellular localization analysis showed that expression of Bradilg25390was only located at the nucleus in onion epidermal cells. The organ specific expression analysis by RT-PCR showed that the expression of Bradilg25390in roots, stems and pods was higher than in the leaves.4. The potentails for Bradilg25390to improve transformation efficiencyTo investigate the effect of Bradilg25390on transformation efficiency, the overexpression vectors of Bradilg25390. AtHTA1and GUS were conducted. The vectors were introduced into Brachypodium BD018, BD21, common wheat cv Longchun23using A. tumefkiens. The preliminary results showed that the overall GUS expressionwas stronger, and expression of Bradilg25390either transiently or in stably transformed cells improved both Brachypodium and wheat transformation efficiencies.