Primary Study Of Long-Chain Fattyalcohol Oxidases In Higher Plants

When some industrial yeast species are utilizing alkanes and/or long chain fatty acids as carbon source for growth, they first metabolize alkanes and/or long chain fatty acids to carboxylic acids or dicarboxylic acids throughω-oxidation system located at endoplasmic reticulum membrane, and then provide carbon source or energy to organism byβ-oxidation.Duringω-oxidation, the methyl end of the molecule is oxidized successively by a cytochrome P450 alkane/fatty acid hydroxylase, a hydrogen peroxide-generating alcohol oxidase, and an aldehyde dehydrogenase, producingω-alcohol,ω-aldehyde, andω-fatty acid, respectively.The P450 gene which encodes the enzyme that catalyzes the first step of the reactions using the alkanes or long-chain fatty acids as carbon source belongs to CYP52 gene family. There exist many homologous genes of CYP52 gene family in plants. Most of this kind of genes are related to resistance against disease and stress. Since the enzymes similar to those catalyzing the first step of theω-oxidation in yeast exist in plants, whether the enzymes similar to FAOs in yeast also exist in plants and what are their functions?This research mainly focuses on gene sequences analysis, biochemical and physiological functions of FAOs in three higher plants including Arabidopsis thaliana, Lotus japonicus and Oryza sativa.There are four long-chain fatty alcohol oxidase genes found in Arabidopsis thaliana genome using the amino acid sequence of the FAO in yeast to search the data-base of the Arabidopsis thaliana. These four genes are designated as AtFAO1 (At1g03990), AtFAO3 (At3g23410), AtFAO4a (At4g19380) and AtFAO4b (At4g28570). We have obtained the full-length ORF of three AtFAOs except AtFAO3 by RT-PCR.The Lotus japonicus EST database was searched against AtFAOs full-length amino acid sequences. One EST fragment was detected. A 1.4 kb fragment was cloned by 3'-RACE using the primers which were designed according to this EST and the cDNA that is from the apexes of Lotus japonicus as template. The corresponding full-length cDNA was obtained by screening a cDNA library of L. japonicus using the 1.4 kb fragment as probe. The LjFAO1 genomic DNA was amplified by PCR, to give a product of 3.6 kb in length. Comparison between the LjFAO1 cDNA and genomic DNA reveals that the LjFAO1 contains 3 exons and 2 introns. In addition, the LjFAO2 was obtained by screening the same cDNA library.The genome of Oryza sativa was also searched against the amino acid sequences of FAO genes in Arabidopsis, by tblastn and blastp. It shows that there are four FAO genes in genome of Oryza sativa. One is located at the chromosome 2 and it is designated as OsFAO1. The other three named as OsFAO2, OsFAO3 and OsFAO4 respectively are all at the chromosome 10.Analysis of the ten FAO gene sequeces mentioned above indicates that most of the FAO genes which exist in higher plants are composed of three exons and two introns, and they all contain five conserved domains in their amino acid sequences. Evolutionary study shows that the differentiation of FAOs in higher plants follows the differentiation of the monocotyledon and dicotyledon.Enzymes assay were carried out after over-expression of the above mentioned genes by BL21(DE3) . The results show that only the AtFAO4b and LjFAO1 have the ability to catalyze the oxidation of the long-chain fatty alcohol. Enzyme kinetics analysis shows that AtFAO4b and LjFAO1 both have substrate specificity. After the proteins purification using Ni-NTA columns and electrophoresis analysis, single protein band was obtained respectively.RT-PCR was performed to check the expression patterns of the AtFAO3 and AtFAO4b. The results show that the AtFAO3 and AtFAO4b are both expressed in the whole plant but respective transcription level is variant in different tissues. Semi-quantitative RT-PCR results also show that the AtFAO3 and AtFAO4b have different responses to cold stress. T-DNA insertion mutants of AtFAO3 and AtFAO4b were inoculated with pathogen, P. syringe pv. tomato DC3000. AtFAO3 T-DNA insertion mutant shows no obvious change whereas AtFAO4b T-DNA insertion mutant shows higher susceptibility to infection. These results indicate that AtFAO3 and AtFAO4b both can catalyze the long-chain fatty alcohol oxidation but they may carry out different functions in vivo, and AtFAO4b may have important impact on lipid metabolism and cell wall development in A. thaliana.RT-PCR analysis shows that the LjFAO1 is expressed in the whole plant, with the highest expression level in apex and the lowest expression level in the siliques. The LjFAO1 gene is down-regulated by cold stress in both the apexes and the cotelydons of the 8-day old seedlings.Analysis of the OsFAO genes expression data downloaded from the public data-base shows that the OsFAO1 and OsFAO4 have higher expression level while OsFAO2 and OsFAO3 have lower expression level. Further more the four FAO genes have different responses to stresses.All the results mentioned above indicate that the FAO genes exist ubiquitously in higher plants. Research focusing on the Arabidopsis thaliana,Lotus japonicus and Oryza sativa show that this kind of genes are related with the resistance against disease and stresses.