Screening And Analysis Of Genes Related To The Synthesis Of Unsaturated Fatty Acids In Walnut(Juglans Regia L.)

Walnut?Juglans regia L.?,one of the most important woody oil-bearing tree species,has been widely cultivated in the world.Walnut has high oil content with rich unsaturated fatty acids which are beneficial to human health and are favored by consumers.The composition and proportion of fatty acids are closely related to walnut oil quality.Therefore,the optimization of walnut oil quality is of great significance for walnut breeding.In this study,gas chromatography mass spectrometer?GC-MS?was used to detect the fatty acids components to explore the differences of fatty acids among 20 different walnut varieties.Transcriptome sequencing technology was used to analyze differentially expressed genes of ‘Zhonghefeng' at different development stages,in order to find key genes that are related to the synthesis of unsaturated fatty acids in walnut.The main contents and results of this study are as follows:1.The fatty acids composition and contents of 20 different walnut varieties were studied,the results indicated that four major fatty acids were detected in walnut kernel,respectively named linoleic acid,oleic acid,stearic acid and palmitic acid,of which oleic acid content was the highest,followed by linoleic acid content and unsaturated fatty acids content was up to 90%.There was highly significant negative correlation between linoleic acid and oleic?P<0.01?.The variation coefficient of oleic acid was the largest with 19.98% and that of linoleic acid was the smallest with 9.89%.2.Transcriptome sequencing and analysis were based on kernel of 'Zhonghefeng' which has high oleic acid content.The results showed that,the transcriptional level of genes related to the synthesis of unsaturated fatty acids in ‘Zhonghefeng' showed differences among different development stages.After the analysis of functional category of differentially expressed genes?DEGs?,a total of 16 genes related to the synthesis of unsaturated fatty acids in walnut were selected.It included 4 ACCase homologous genes?WALNUT₀0001126-RA,WALNUT₀0006339-RA,WALNUT₀0001038-RA,WALNUT₀0018192-RA?,1 MCAT homologous gene?WALNUT₀0005699-RA?,3 KAS homologous genes?WALNUT₀0008175-RA,WALNUT₀0025602-RA,WALNUT₀0000456-RA?,1 LACS9 homologous gene?WALNUT₀0022757-RA?,1 FATA homologous gene?WALNUT₀0009941-RA?,1FATB homologous gene?WALNUT₀0020080-RA?,3 SAD homologous genes?WALNUT₀0005946-RA,WALNUT₀0019717-RA,WALNUT₀0023519-RA?and 2 FAD2 homologous genes?WALNUT₀0002632-RA,WALNUT₀0011902-RA?.SAD homologous gene WALNUT₀0019717-RA and FAD2 homologous gene WALNUT₀0011902-RA were typical genes of fatty acids synthesis,which have been widely reported.The qRT-PCR supported the reliability of transcriptome results.3.We selected two genes for cloning and analysis,including 1 SAD homologous gene?WALNUT₀0019717-RA?and 1 FAD2 homologous gene?WALNUT₀0011902-RA?.The results showed that the amino acid sequence encoded by walnut SAD?WALNUT₀0019717-RA?was the most similar to that of pecan and sesame.The amino acid sequence encoded by walnut FAD2?WALNUT₀0011902-RA?has the highest similarity with soybean.The results of subcellular localization showed that walnut SAD was mainly located in cytoplasm and nucleus,while FAD2 waslocated in cytoplasm.