Study Of EjFAD8 On The Regulation Of Lipid Metabolism And Cold Resistance In Loquat

Loquat(Eriobotrya japonica Lindl.)is an evergreen fruit tree native to southwestern China and has been cultivated in China for more than 2200 years.The loquat fruit is sweet,sour,juicy and nutritious,and has high commercial value.Loquat is native to hot and humid subtropical regions,and its phylogeny is influenced by the climatic environment and is very sensitive to low temperatures.However,loquat differs from other fruit trees in that its phenological period is generally autumn and winter flowering and fruit setting,so its flower and young fruit development is highly susceptible to low temperature stress,which leads to reduced yield.Therefore,the selection of cold-tolerant loquat varieties is one of the important objectives of breeders.In a previous study,the F1 generation of triploid loquat obtained by crossing tetraploid loquat(B431)with diploid wild loquat(GZ23)was more cold-tolerant and had higher photosynthetic efficiency than its parents.In the present study,analysis of transcriptomic and lipidomic data revealed that the fatty acid desaturase gene Ej FAD8 was closely associated with low temperature.the FAD8 gene has been reported in many species,and its expression level is regulated by temperature and is responsive to low temperature in most species.In this study,we analyzed the expression levels of Ej FAD8 in different ploidy loquats by q PCR and demonstrated that Ej FAD8 was significantly up-regulated at low temperatures,especially in triploid loquats.Subsequently,the gene function of Ej FAD8 was further analyzed by cloning Ej FAD8 and constructing a plant expression vector to transfer it into Arabidopsis thaliana;and constructing a TRV vector to transfer it into loquat seedlings.The main findings were as follows:1.Transcriptome and lipidome analysis of different ploidy loquat under low temperature stress:To clarify the reasons for the higher cold resistance of triploid loquat than its parents,we analyzed transcriptomic and lipidomic data of different ploidy loquats before and after treatment.The results showed that the transcriptome data illustrated that,among a series of genes involved in lipid metabolism,Ej FAD8 was most significantly up-regulated in triploid loquat,significantly higher than in diploid and tetraploid loquat.Subsequent analysis of the lipid data also showed that lipids associated with Ej FAD8 metabolism were significantly up-regulated in all triploid loquats.Thus,the key gene for cold tolerance in triploid loquat,Ej FAD8,was obtained.2.Cloning,bioinformatics analysis and subcellular localization of Ej FAD8.:Ej FAD8 was annotated in the loquat genome as a fatty acid desaturase gene,and a 1155 bp fragment was amplified from c DNA of triploid loquat leaves and named Ej FAD8.sequence comparison and phylogenetic tree analysis showed that Ej FAD8 was highly homologous to Pb FAD8 in white pear(Pyrus bretschneideri)of Rosaceae and Md FAD8 in apple(Malus domestica).Subcellular localization showed that the fluorescent signal of Ej FAD8 was present with chloroplasts,which could indicate that Ej FAD8 is a chloroplast-localized protein.。3.Effect of heterologous overexpression of Ej FAD8 on cold resistance in Arabidopsis thaliana:Arabidopsis heterologously overexpressing the Ej FAD8 gene showed a dwarfed plant phenotype.Under low temperature treatment,the transgenic Arabidopsis plants did not show significant wilting and leaf dehydration compared with wild-type Arabidopsis,and resumed normal growth three days after the return to normal temperature.Moreover,the root system of the Ej FAD8 transgenic plants was longer than that of the wild-type at 0℃.The ion leakage of the transgenic lines was lower than that of the wild type at 0℃.And the chlorophyll content of the leaves of the transgenic lines was higher than that of the wild type.Staining of Arabidopsis leaves under low temperature treatment with NBT and DAB showed that transgenic Arabidopsis had lower reactive oxygen content under low temperature.Quantitative analysis of Arabidopsis before and after low temperature treatment by q PCR also showed that the expression of genes related to cold resistance and those promoting lipid unsaturation metabolism were significantly up-regulated in the transgenic strain under low temperature relative to the wild type.The lipid data also indicated that Ej FAD8 increased unsaturated fatty acid content in Arabidopsis leaves thereby improving cold tolerance in Arabidopsis.The analysis of the Ej FAD8 gene promoter showed that the Ej FAD8 promoter has many hormone-related binding elements,and the treatment of Arabidopsis with abscisic acid also showed that the Ej FAD8 transgenic Arabidopsis is better adapted to the environment with abscisic acid.4.Effect of VIGS inhibition of Ej FAD8 expression on loquat:The TRV vector was constructed and injected into the cotyledons of loquat seedlings,which were subjected to q PCR analysis to demonstrate that Ej FAD8 gene expression was repressed in VIGS gene silenced loquat strains.The loquat leaves before and after treatment were subjected to transcriptomic sequencing.The transcriptomic sequencing results showed that the VIGS gene silenced loquat strain showed more genes down-regulated at low temperature and more genes enriched in environmental stress response compared to the control.