Cloning And Expression Analysis Of Bra1 Gene In Brachypodium Distachyon And Its Gene Editing System Establishment

The anti-degradation barrier of plant cell wall is a key limiting factor to the efficient conversion and utilization of crop straw biomass,and lignocelluloses feruloylationin the cell wall of Gramineaeis an important molecular mechanism for the formation of this anti-degradation barrier.Feruloyl transferase is one of the key enzymes responsible for transferring ferulic acid from the ferulic acid CoA to the arabinylxylan molecules which plays a key role in the connection between arabinoxylan and lignin and this enzyme is closely related to the barrier formation of plant cell wall against degradation.Therefore,fuction identification of feruloyl transferase genes in plant can provide important data for the development and utilization of grass energy crops as well as the recycle utilization of crop straw biomass.In this study,a putative ferulic acyltransferase gene Bra1 from Brachypodium distachyon was isolated and characterized.The main results are as follows:1.The full-length cDNA sequence of Bra1(5g14720)gene in Brachypodium distachyon was cloned from the mRNA of mature stems using RT-PCR technique,and the length of which was up to 1,369 bp.Bioinformatics analysis indicated that the open reading frame(ORF)of Bra1 gene encodes a deduced protein with 443 amino acid residues,with a theoretical molecular weight of 48.45 kDa.Prokaryotic expression and mass spectrometry analyses of recombinant protein also confirmed the correct translation of Bra1 gene.2.The amino acid sequence of Bra1 protein contains a HXXXD domain and a DFGWG conserved domain that are unique to the BAHD acyltransferase family,as indicates that Bra1 protein is a member of BAHD acyltransferase family.The phylogenetic analysis of 89 BAHD acyltransferases from Brachypodium distachyon suggested that these BAHD acyltransferases could be divided into four subfamilies,each of them containing several small groups.The Bra1 protein locates in subfamily I and does not belong to the same subfamily as the other previously characterized BAHD acyltransferases which are in the second group of subfamily III and whose functions are related to the metabolism of p-CA.So,Bra1 protein may be different in function from those previously characterized BAHD acyltransferases.3.The expression profiles of Bra1 gene and other 9 BAHD acyltransferase genes in the subfamily III or the subfamily I were analyzed using the real-time fluorescence quantitative PCR,and the result showed that Bra1 gene had a higher and more stable expression compared with other genes and its expression in the mature stem,leaf and spike was almost twice as high as that in younger tissues.The transient expression of Bra1-GFP fusion gene in the onion epidemal cells suggested that the subcellular localization of Bra1 protein located in the cytoplasm.4.Two target sites in Bra1 exon regions were selected to construct their gene editing vectors in plant based on CRISPR/Cas9 system.Both of gene editing expression vectors were introduced into Brachypodium distachyon mediated by Agrobacterium tumefaciens and several resistant plantlets were regenerated.Now,there are 3 positive transformed plants were identified by PCR amplification.The sequence analysis of the gene editing sites indicated that the Indel(insertion/delection)mutation was identified in the first gene editing site for these 3 plants with a single base deletion or insertion in two plants and only single base deletion in the third one,but that no mutation was found in the second gene editing site of Bra1 gene sequence for these three plants.