Functional Analysis Of The Fruit-specific Gene SlSAD8 From Tomato

China is a big country in tomato production and processing.Tomato seeds contain a large amount of unsaturated fatty acids,which is a source of high nutrient oils.It has been reported that stearoyl-ACP desaturase(Stearoyl-acyl carrier protein desaturase,SAD)involves in the regulation of fatty acid metabolism in soybeans(Glycine max),Arabidopsis thaliana,rice(Oryza sativa)and other species,but little is known in tomato(Solanum lycopersicum).In this study,a SlSAD8 gene was screened by bioinformatics and tissue expression pattern analysis,which was specifically expressed in fruits.The biological functions study of SlSAD8 in the regulation of tomato quality formation was performed by SlSAD8-knockout plants,which was obtained by CRISPR/Cas9 gene editing technology.The main findings are described as follows:(1)There are nine members of tomato SAD family,namely Sl SAD1-Sl SAD9,among which SlSAD8 is specifically expressed in fruits and seeds,and highest expressed in the fruit at Br+3 stage.The SlSAD8 is located on chromosome 6 and the full length of the gene is 1261 bp,which contains two exons and one intron.According to the phylogenetic tree and predicted protein structure,we found that SlSAD8 was most similar with Rc SAD1 in Ricinus communis and Gm SAD8 in soybeans.The result of subcellular localization showed that SlSAD8 was localized in chloroplasts and nucleus;(2)The knockout mutant of SlSAD8 gene was obtained by constructing SlSAD8CRSIPR-Cas9 vector and performing tomato genetic transformation.By measuring the SAD activity in WT and mutant fruits,it was verified that the SlSAD8 gene was undoubted knockout in tomato plants,and the SAD activity in the mutant plants was significantly lower than that in the wild type.According to the determination of fatty acids in fruits,the content of palmitic acid,stearic acid and linoleic acid in fruits and seeds of SlSAD8 knockout mutants were increased significantly by 2-3 times than that in WT plants.Compared with the wild type,the oleic acid content in the seeds of sad8-5line was decreased by 3-4 times,while the content in the fruits of sad8-3 and sad8-5lines were increased by 2-3 times;(3)By analyzing the shelf life and disease resistance of postharvest fruits,the fruits of sad8-3 and sad8-5 lines exhibited more slowly water loss and more resistant to storage than WT.Moreover,the mutant fruits also exhibited obvious resistance to Botrytis cinerea through regulating salicylic acid pathway;(4)According to transcriptome analysis,the knockout of S1SAD8 affected many physiological processes such as defensive reaction and fatty acid biosynthesis.And many genes involved in chloroplast function and photosynthesis function were also significantly down-regulated.The changes of these differentially expressed genes are consistent with the phenotypes,which allow us to verify the function of this gene from the transcriptome level,and also provides direction for the further study of this gene.In conclusion,this study was systematically analyzed the tomato SAD family and initially revealed the biological function of SlSAD8 gene.The study provides a new insight for enhancing tomato disease resistance and provides new track for breeding strategies aiming to improve high-nutrient seed oil of tomato.