Cloning And Identification Of Aldehyde Dehydrogenase Genes From Brachypodium Distachyon For Regulating Ferulic Acid Biosynthesis In Cell Wall

The formation of lignin-ferulate-arabinoxylan complexes by ferulic acids(FA)covalently cross-linked with polymers of hemicellulose and lignin provides a molecular structure basis for rigid cell wall recalcitrance to biodegradation in grass plants.Therefore,insight into ferulic acid biosynthesis mechanism will make it possible to improve lignocellulose degradability by genetic transformation technique.In this study,two aldehyde dehydrogenase genes involving in ferulic acid biosynthesis in cell wall was cloned from a gramineous model plant Brachypodium distachyon,and their biological functions were tested preliminarily.The main results are as follows:1.The full-length c DNA sequences of Bd AD1 and Bd AD2 were isolated from Brachypodium distachyon using the RT-PCR method.The length of Bd AD1 is 1628 bp,which codes a protein of 500 amino acid residues,and Bd AD2 has 1767 bp coding 504 amino acid residues.Both of proteins belong to the aldehyde dehydrogenase family.The sequence identity between Bd AD1 and Bd AD2 is 69.44%.The identity between Bd AD1 and other homologous proteins from related species ranges from 84% to 91%,and that for Bd AD2 being from 68% to 70%,in which Bd AD1 and Bd AD2 have a higer similarity with the proteins from Triticum aestivum,Leymus chinensis and Hordeum vulgare.2.Subcellular localization analysis suggests that BdAD1 and BdAD2 proteins are expressed and targeted in both of plant cell nucleus and cytoplasm.Quantitative real-time PCR analysis reveals that Bd AD1 is expressed at higher level in Brachypodium distachyon mature leaf and at lower level in yong stem and root,while Bd AD2 is expressed at higher level in mature leaf and root and at lower level in yong stem and mature stem.3.The biological functions of Bd AD1 and Bd AD2 are prelimilarily identified using an in vitro test method.It is indicated that Bd AD1 has the catalytic activity of coniferylaldehyde dehydrogenase as well as sinapaldehyde dehydrogenase,which can oxidize coniferyl aldehyde and sinapic aldehyde into ferulic acid and sinapic acid,respectively,although its activity to coniferyl aldehyde is obviously higher than to sinapic aldehyde,and on the other hand,Bd AD2 only has the coniferylaldehyde dehydrogenase activity.