Cloning And Functional Athentication Of The Suberin Fatty Alcohol Synthase Gene In Brachypodium Distachyum

The suberin in plant root plays a crucial role in plant defense against a variety of biological and abiotic stresses.As a model one for studying wheat species,Brachypodium distachyum allows investigations on related genes and mechanisms about its drought resistance,which can further provide reference for genetic improvement of other important crops such as wheat.In this thesis,the composition and content of suberin in B.distachyum were analyzed by gas chromatography mass spectrometry(GC-MS)and gas chromatography(GC).The At FAR1 protein sequence was used as the query sequence to BLAST against B.distachyon proteome database,and the resulting candidate proteins related to the suberin fatty alcohol synthase were consequently identified by their sequence homolgoy.The expression characteristics of candidate genes in different tissues were also analyzed,among which,important ones were selected for cloning and functional verification.The major findings are listed as the follows:1.The suberin components in the root of B.distachyum are primary fatty alcohols,fatty acids,?-hydroxy fatty acids and ?,?-two carboxylic acid.The composition of suberin is similar to that of other species that were previously reported.However,there are yet some differences in amounts of different components.2.There are six candidate genes in the genome of B.distachyum encoding six proteins.Multiple comparison in amino acid sequences with AtFAR1 shows that only BdFAR4(Bradi3g20000)has more than 50% identitiy to AtFAR1,and the amino acid identities between BdFAR1(Bradi4g38460),BdFAR2(Bradi3g42780),BdFAR3(Bradi4g37860),BdFAR4(Bradi3g20000),BdFAR5(Bradi1g73877)and BdFAR6(Bradi4g37860)and AtFAR1 were 40.34%,42.30%,45.20%,50.80%,35.61%,and 44%,respectively.Through the bioinformatics analysis,it is speculated that these genes may have functions in synthesizing primary fatty alcohols.Three genes,including BdFAR1,BdFAR2 and BdFAR3 were previously cloned and their functions were found to be related with the synthesis of epidermal wax in the leaves of B.distachyum.Due to its expression is primarily in root,it is anticipated that the BdFAR4 gene may be involved in synthesizing suberin fatty in suberin of root.3.The heterologous expression of BdFAR4 in yeast indicates that BdFAR4 may function in encoding fatty acid acyl-CoA.After vector construct,transformation and product extract in yeast,it is found that the C20 and C22 fatty alcohols are synthesized in yeast strains with the transgene,rather than with the empty vector.4.The Heterologous expression of BdFAR4 gene in tomato implies that the the content of suberin fatty alcohol in roots of T1 transgenic offsprings are significantly increased.The chain length of C20 and C22 primary fatty alcohol are increased mostly.Compared to plants with the empty vector,fatty alcohols of suberin content in roots of the transgenic plants are significantly increased by 1.4-1.7 folds,the chain length of C20 primary fatty alcohol is increased by 1.3-1.8 folds,and the length of C22 primary fatty alcohol is increased by 1.4-1.8folds.The results show that BdFAR4 has the function in synthesizing suberin fatty alcohols,mainly primary fatty alcohols with chain lengthes of C20 and C22.