The Preliminary Research Of The Function Of ZbFAD2 And ZbFAD3 In Alkylamide Compounds Biosynthetic Pathway In Zanthoxylum Bungeanum Maxim

Zanthoxylum bungeanum Maxim,a traditional cash crop belonging to the Zanthoxylum genus of the family Rutaceae,is a species of great interest because of its pungent taste and pharmaceutical properties value.Twenty-seven alkylamide compounds have been identified in Z.bungeanum as the primary pungent and medicinal active substances in Z.bungeanum.Alkylamides compounds were speculated as a conjugation of unsaturated fatty acids and valine or phenylalanine,but the biosynthesis pathway,pivotal precursors and key related genes of alkylamide compounds in Z.bungeanum have not been clarified.Previous studies analyzed the transcriptomes of these three varieties（Hancheng stingless,Fengxian Dahongpao and Fugu Huajiao）and 19 differentially expressed genes（DEGs）were speculated as the candidate genes involved in alkylamide compounds biosynthesis.Therefore,this study was aimed to prove the function of the candidate genes c99494 graph＿c0 and c102796 graph＿c0.The main results are as follows:1. The candidate genes c99494 graph＿c0 and c102796 graph＿c0 full-length c DNA sequences were analyzed by bioinformatics and they belong to unsaturated fatty acid dehydrogenase（named Zb FAD2 and Zb FAD3,respectively）by homologous sequence comparison.The ORF Finder analysis determined that the reading frames of the two genes were 1167bp and 1152bp,respectively.The phylogenetic trees were constructed from the Zb FAD2 and Zb FAD3 sequences with the amino acid sequences of 10 other plants.The results showed that Z.bungeanum and Citrus were categorized as one group cause of the closer relationship,but farther than Quercus and Jute.The analyses of amino acid sequence were speculated that the Zb FAD2 protein has 383 amino acids,a molecular weight of 44.16k Da,an isoelectric point of 8.04,a total hydrophilicity（GRAVY）of-0.013,and an instability index of 37.48.As the same,the Zb FAD3 protein has a total of 388 amino acids,a molecular weight of 44.79 k Da,an isoelectric point of 8.76,a GRAVY of-0.116,and an instability index of 33.76.2. The erokaryotic expression recombinant vectors T7-pET30a-Zb FAD2 and T7-pET30a-Zb FAD3 were constructed and transformed into E.coli BL21 expression strains.The recombinant proteins Zb FAD2 and Zb FAD3 were induced to express by IPTG.The molecular weigh of two purified proteins found to be about 44k Da through SDS-PAGE,nearly equal to the predicted.The substrate reaction and product identification of protease by gas chromatography showed that recombinant protein Zb FAD2 can react with substrate oleic acid（C18:1△⁹）to produce linoleic acid(C18:2△^9,12)and Zb FAD3 can react with substrate linoleic acid to produceα-linolenic acid(C18:3△^9,12,15),and the results were same as their homologous sequence which were FAD2 can catalyse oleic acid to LA and FAD3 can catalyse linoleic acid toα-linolenic acid.3. The plant overexpression recombinant vectors 35S-pCAMBIA2300-Zb FAD2 and35S-pCAMBIA2300-Zb FAD3 were constructed and transformed into Nicotiana benthamiana and Arabidopsis thaliana by Agrobacterium-mediated method,respectively.The contents of fatty acids in transgenic A.thaliana were determined by gas chromatography,and it was found that the seeds of Zb FAD2 transgenic A.thaliana had decreased oleic acid content and increased linoleic acid content compared to wild type,while the linoleic acid content of Zb FAD3 transgenic A.thaliana seeds was reduced and theα-linolenic acid content was increased.It showed that Zb FAD2 catalyzed the reaction of oleic acid to produce linoleic acid and Zb FAD3 catalyzed the reaction ofα-linoleic acid to produceα-linolenic acid in accordance with prokaryotic expression.The HPLC results showed that the content of hydroxy-α-sanshool in Zb FAD2 transgenic N.benthamiana（0.2167±0.0026 mg/g）was significantly higher than wild-type（0.0875±0.0049mg/g）but the Zb FAD3 transgenic N.benthamiana（0.0535±0.0037 mg/g）was lower than wild-type.The two candidate genes and the synthesis of unsaturated fatty acids were related to the synthesis of alkylamide compounds.