Effect Of Ethylene On The Biosynthesis Of Squalene And β-sitosterol In Torreya Grandis Nut During Post-harvest Ripening Storage

T.grandis ‘Merrilli’ belongs to the gymnosperm Taxaceae family Torreya.It is the general name of the cultivar of excellent varieties of Torreya grandis through artificial grafting.Its seed kernels are rich in various nutrients and biologically active compounds.Among them,squalene and β-sitosterol are rich in content,and have high medical value such as antioxidant,anti-inflammatory,cholesterol lowering,antitumor and so on.However,the biosynthesis of squalene and β-sitosterol in Torreya grandis,as well as the changes in their contents and the regulatory mechanism during the post-ripening process of Torreya grandis are still unclear.Therefore,this study intends to identify and explore the biosynthetic pathways of squalene and β-sitosterol in Torreya grandis by studying the changes in the content of squalene and β-sitosterol in the seeds of different Torreya grandis provenances and combining with transcriptomics analysis.Potential mechanism of differential accumulation of squalene and β-sitosterol in provenances;At the same time,by studying the changes in the content of squalene and β-sitosterol during the postripening process of Torreya grandis,the effect of ethylene on the biosynthesis of squalene and β-sitosterol and its mechanism of action were explored.The research through this paper will lay a foundation for improving the theory and technology of the Torreya grandis nut industry.The main findings are as follows:(1)The accumulation of squalene and β-sitosterol in Torreya grandis has obvious provenance specificity.Transcriptome analysis obtained a total of 60,372 unigenes,of which more than 60% of unigenes were successfully annotated.The identification and expression analysis of differentially expressed genes showed that 39 candidate genes were involved in the biosynthesis of squalene and β-sitosterol in the seeds of Torreya grandis.Among them,the expression patterns of the genes related to mevalonate(MVA)and methylerythritol phosphate(MEP)pathways showed that both pathways promoted isopentenyl diphosphate(IPP)and dimethyl dimethyl in different varieties of Torreya grandis Upstream allyl diphosphate(DMAPP)biosynthesis.In addition,Tg SQS and Tg SMT1 may be the key targets for the regulation of squalene and sitosterol biosynthetic pathways in Torreya grandis.There is a significant positive correlation between fluorescence quantitative PCR analysis and RNA-Seq results;subcellular localization reveals that Tg FPS :: GFP fluorescence signals are observed in both the plasma membrane and the nucleus.(2)Ethylene is beneficial to the cracking process of Torreya grandis.The exogenous ethephon treatment induced the biosynthesis of endogenous ethylene in Torreya grandis.The aril cracking rate increased significantly,the seed starch and soluble sugar content decreased significantly,and the soluble protein content and fatty acid content increased significantly.After 9 days of ethephon treatment,the content of saturated fatty acids decreased,and the content of unsaturated fatty acids(linoleic acid)increased significantly.It showed that ethylene promoted the nutrient conversion and unsaturated fatty acid accumulation of Torreya grandis seeds,which was conducive to the ripening and cracking of Torreya grandis and the nutrient quality of nuts.(3)Ethylene promotes the synthesis and accumulation of squalene during the the post-harvest ripening process of Torreya grandis.Under the conditions of ethephon treatment,the content of squalene was significantly higher than that of the control group and the 1-MCP treatment group,while the content of β-sitosterol decreased slightly,but the overall level was not much different from that of the control group.Transcriptome differential gene expression analysis found that after Ethephon treatment,the expression of endogenous ethylene synthesis and signal transduction related genes in Torugula tomentosa was upregulated,and the squalene biosynthesis gene 3-hydroxy-3-methylglutaryl-COA reductase HMGR,Isopentenyl diphosphate isomerase IPI,geranyl diphosphate synthase GGPS,etc.are up-regulated,while the β-sitosterol synthesis gene squalene epoxidase SQE,cycloatenol synthase CAS,sterol methyl Transferase SMTs showed down-regulated expression.Correlation analysis found that the expression levels of HMGR,IPI,and GGPS were positively correlated with squalene content under ethylene treatment,and the correlation coefficients were 0.90,0.71,and 0.75,respectively.In summary,ethylene promotes the biosynthesis of endogenous ethylene during the cracking process of Torreya grandis,and also promoting the expression of key genes in squalene biosynthesis,inhibiting the expression of genes related to β-sitosterol synthesis,and ultimately promoting the synthesis and accumulation of squalene.