Functional Study Of Suberin Fatty Alcohol Synthase Gene BdFAR4 In Brachypodium Distachyon

Suberin is a secondary metabolite of plants composed of glycerol,aliphatic,phenolic compounds,etc.,which is widely found in the roots of plants,but also in the epidermis and periderms.It,together with the cutin existing above ground,constitutes a hydrophobic barrier formed by plants to adapt to complex environmental changes.Suberin is related to various resistances of plants.Research on suberin can help to explain the mechanism of plant resistance and improve plant resistance.However,most of the related research currently has focused on Arabidopsis and there are few reports in other species.Previous studies have shown that the Bd FAR4 gene in Brachypodium distachyon has been confirmed to be involved in the synthesis of primary fatty alcohol of suberin in yeast and tomato,but the detailed functional study of this gene still needs to be revealed.Brachypodium distachyon is an important model plant for wheat crops.The study of Bd FAR4 can provide a reference for the related research of wheat crops.Based on the previous research,this paper carried out further research on the function of Bd FAR4.We used CRISPR/Cas9 gene editing technology and overexpression technology in combination with genetic transformation of Brachypodium distachyon to verify gene function;performed prokaryotic expression of the Bd FAR4 protein;identified subcellular location of Bd FAR4 protein in rice protoplasts and tobacco epidermal cells through subcellular localization technology;performed various abiotic stress treatments on Brachypodium distachyon and explored the expression changes of Bd FAR4 under various stress conditions.The main conclusions of this article are as follows:1.The CRISPR/Cas9 system successfully edited the Bd FAR4 gene sequence of Brachypodium distachyon Bd21 plants,resulting in a variety of mutation types,including single-base insertion,deletion,substitution,and deletion of large fragments.The positions of the mutations were all related to the target sequence and concentrated at 3～5 bp upstream of the PAM sequence.2.The main components of suberin in Brachypodium distachyon roots are fatty acids,primary fatty alcohols,ω-hydroxy acids and α,ω-dicarboxylic acids.The CRISPR/Cas9 mutant lines and the Bd FAR4 overexpression lines were not significantly different in the contents of these components compared to the wild-type plants.The contents of C20 and C22 primary fatty alcohols in the CRISPR/Cas9 mutant lines were significantly lower than those in the wild-type plants,and the content of C20 primary fatty alcohol decreased the most and could hardly be detected;the contents of C20 and C22 primary fatty alcohols in the overexpression lines were significantly higher than those in the wild-type plants.These results indicated that Bd FAR4 is specifically involved in the synthesis of C20 and C22 primary fatty alcohols in Brachypodium distachyon root suberin.3.The complete prokaryotic expression Bd FAR4 protein was constructed,and the size of the expressed product was about 55 KDa.The Bd FAR4 protein was localized in the endoplasmic reticulum by subcellular localization in Arabidopsis protoplasts and tobacco epidermal cells.4.The expression of Bd FAR4 in Brachypodium distachyon was tissue-specific,with low expression levels in leaves,internodes,spikelets,nodes,sheaths,and glumes,and high expression in roots.The expression level increased first and then decreased with the root development.5.Drought,PEG,cold,Na Cl,ABA and wounding could all induce Bd FAR4 up-regulation.Among them,Bd FAR4 was most up-regulated by wounding,and the least was up-regulated by cold stress.It had the fastest response to drought stress,and the slowest response to wounding.