Cloning and expression analysis of oleate dehydrogenase gene JrFAD2 in walnut

Abstract:【Objective】Walnut (Juglans regia L.) is an economically important nut tree in China with a long history of cultivation and a wide range of distribution. It is rich in fatty acids of which about 90% are unsaturated fatty acids which are essential nutrients for the people. The degree of unsaturated fatty acids is one of the important indexes to determine the quality of the oil in walnut for the characteristics of lowering cholesterol in the body of human The research on the mechanism of unsaturated fatty acid formation in walnut has also become a hotspot. The composition of the fatty acids in walnut and key genes related to high unsaturated fatty acid synthesis are of great significance for studying the synthesis of unsaturated fatty acids and optimizing the distribution ratio of fatty acids. As a critical enzyme in the lipid biosynthetic pathway, the fatty acid desaturase 2 (FAD2) takes part in the desaturated conversion of oleic acid (18∶1) to linoleic acid (18∶2). Currently, the FAD2 gene has been deeply studied in other plants, but it has not been reported in walnut. In this study, we cloned the walnut FAD2 gene to explore its role in the synthesis of unsaturated fatty acids in walnut in order to provide a basis for the analysis of the synthesis mechanism of high unsaturated fatty acid in walnut【. Methods】The components and rela- tive contents of fatty acids of the 20 walnut cultivars were determined by gas chromatography-mass spectrometry (GC-MS). High linoleic acid varieties and low linoleic acid varieties were used as materi- als, and the expression patterns of the fatty acid desaturase 2 (FAD2) in high and low linoleic acid variet- ies were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) based on the whole genome annotation information of walnut. The CDS sequence of the JrFAD2 gene was amplified by RT-PCR and its structure was analyzed by NCBI. MEGA7 was employed to build the phylogenetic tree of the FAD2. The expression vector 35S::JrFAD2 was constructed by pBI121 vector with GFP tag throughAgrobacterium-mediated transient expression system, and the expression of the walnut FAD2 in the epi- dermal cells of tobacco leaves was observed by using laser con-focal microscope【. Results】The fatty ac- ids of the 20 walnut varieties were mainly composed of linoleic acid, oleic acid, stearic acid and palmit- ic acid, of which unsaturated fatty acids were up to 90%. The content of linoleic acid was the highest, its average value was 58.63%, and its coefficient of variation was the smallest, 10.4%. The variation range of oleic acid was 24.17%-48.27%, and the coefficient of variation was the largest, 21.18%. There were some differences in the expression levels of the FAD2 in the high linoleic acid varieties and the low linoleic acid varieties, the expression level of the WALNUT_00002632-RA in high linoleic acid vari- eties‘Daihui’and‘Zhengduan 15’was significantly higher than those in the three low linoleic acid va- rieties (p ≤0.05), but there was no significant difference between‘Zha 343’and low linoleic acid vari- eties. The difference of the WALNUT_00027620-RA between‘Daihui’and the low linoleic acid variet- ies was significant. The expression level of the WALNUT_00011902-RA in the three high linoleic acid varieties was all significantly higher than those of the low linoleic acid varieties. The WALNUT_ 00011902-RA was selected as the gene to clone, and the CDS sequence of the JrFAD2 gene with a length of 1 152 bp was obtained, which codes for 383 amino acids, contains the functional domain of omega-6 fatty acid desaturase, and belongs to Membrane-FADS-like Super family. The JrFAD2 in wal- nut had higher similarity with the FAD2 in the other plants, and it was speculated that they had similar structure and function. Phylogenetic analysis showed that the walnut JrFAD2 was closely related to theVernicia fordii FAD2 and the soybean FAD2. The results of subcellular localization showed that the walnut FAD2 emitted fluorescence signal in the cytoplasm, which was presumed to be mainly located in the cytoplasm.【Conclusion】The fatty acids of walnut were mainly composed of linoleic acid, oleic acid, stearic acid and palmitic acid. The critical gene JrFAD2 in walnut may promote and regulate the syn- thesis of linoleic acid and the ratio of oleic acid and linoleic acid during the synthesis of the unsaturated fatty acids.