Cuticular Wax Analysis Of Leaf And Cloning Of Alkane Biosynthesis Enzyme Genes CER1 And CER3 In Brachypodium Distachyum

Cuticular wax plays an important role in protecting plants against all kinds of biotic and abiotic stresses.B.distachyum is an important model plant for wheat crops,it can provide reference for related study of wheat by studying the drought resistance mechanism of cuticular wax and related genes of alkane biosynthesis in B.Distachyum.In this paper,cuticular wax of B.distachyum leaf after drought stress were analyzed by gas chromatography mass spectrometry?GC-MS?and gas chromatography with flame ionization detection?GC-FID?.The structure of leaf wax crystals were also observed by scanning electron microscope?SEM?.At the same time,the water loss rate and chlorophyll permeability of leaf were analyzed.The AtCER1 and AtCER3 protein sequence were subjected to BLASTP search using the B.distachyon genome database,candidate genes related to alkane biosynthesis were identified in B.distachyon genome by bioinformatic analysis of homologous sequences.Some homologous genes were cloned according to their expression levels in different tissues and under abiotic stresses.Finally we transformed these homologous genes into plant overexpression vector for researching alkane biosynthesis of B.distachyum in the future.The major findings were listed as follows:1.Crystal structure of both adaxial and abaxial surface of B.distachyum cultivar Bd21leaf was flake,and the total content of its leaf cuticular wax was 11.39±1.40?g/cm².Twenty-two chemical compounds were identified and divided into primary alcohol,aldehyde,alkane and fatty acid.The main wax compound was primary alcohol,while C₂₆6 alcohol content was the most dominant,with small amount of aldehyde,alkane and fatty acid,in which the fatty acid content was very rare.2.Drought stress had no influence on the type of wax compounds,but it could increase the content of cuticular wax compound in B.distachyum leaf,especially the alkane content with 20.49%increment compared with control,which mainly reflected in the increase of C₂₅,C₂₇7 and C₃₃3 alkane content.The content change of primary alcohol was followed by an increase of 16.08%compared with the control,which mainly reflected in the increase of C₂₂,C₂₄,C₃₀0 and C₃₂2 alcohol content.While the content changes of aldehyde and fatty acid were less affected and increased by 13.15%and 7.14%compared with the control,respectively.Meanwhile,the structure of wax crystal on both adaxial and abaxial surface of B.distachyum leaf was also unchanged,but the amount of wax crystal was increased after drought stress,which might be related to the increase of primary alcohol content in leaf wax.The decrease of leaf permeability resulted in both water loss rate and chlorophyll permeability of leaf occured more slowly after drought stress,which might be related to the increment of alkane content in leaf wax.3.There were five homologous genes of CER1 and three homologous genes of CER3 in B.distachyon genome,which coded seventeen homologous proteins,but only thirteen homologous proteins harbored the FAH domain with three His-rich motifs and WAX2C-terminal domain.The multiple alignments of protein sequences showed that five BdCER1?BdCER1-1 to BdCER1-5?proteins had more than 50%amino acid identity,compared to AtCER1 protein,while three BdCER3?BdCER3-1 to BdCER3-3?proteins had more than60%amino acid identity,compared to AtCER3 protein.They were divided into CER1 protein group and CER3 protein group,and closely clustered together with homologous genes from rice and maize in the phylogenetic tree.All above results preliminarily suggested that these eight candidates had function of alkane synthetase.4.Tissue expression analysis suggested that both BdCER1-1 and BdCER1-3 had relatively high expression levels in spike and glume.Moreover,BdCER1-3 was also highly expressed in section.Interestingly,BdCER1-4 was predominantly expressed in leaf and had low or no expression level in other tissues.Other genes had low or no expression levels in all tissues.These suggested that BdCER1-4 was mainly involved in leaf alkane biosynthesis,while BdCER1-3 was involved in section alkane biosynthesis.Meanwhile,BdCER1-3 was also involved in alkane biosynthesis of spike and glume with BdCER1-1 and played more important role.5.Stress expression analysis indicated that both BdCER1-4 and BdCER1-5 were up-regulated under drought,PEG or NaCl stress,while both BdCER1-3 and BdCER3-3 were up-regulated under drought,PEG,ABA or NaCl stress.These suggested that BdCER1-4 and BdCER1-5 differed from BdCER1-3 and BdCER3-3 in response to drought stress and salt stress.Other genes had no obvious expression change under different stresses.6.BdCER1-1,BdCER1-3,BdCER1-4,BdCER3-1 and BdCER3-3 were successfully cloned from B.distachyon leaf or peduncle,and BdCER1-1,BdCER1-4,BdCER3-1 and BdCER3-3 were successfully transformed into plant overexpression vectors pCXSN.