Tapiscia Sinensis Sucrose Non-fermenting 1-related Protein Kinases 2.3(TsSnRK2.3) Participates In The Initial Exploration Of Fruit Wintering

Tapiscia sinensis is an ancient tree species endemic to China.It takes about 15 months for its fruit to develop,including an entire winter.The long development period of fruits is one of the reasons for the weak natural regeneration ability of Tapiscia sinensis.In recent years,some studies on the wintering development of Tapiscia sinensis fruits are still in their infancy,and studies based on the stress of adversity have not been reported before.Therefore,we will focus on the stage of fruit development stagnation to recovery development to explore the molecular mechanism of overwintering.In this study,we recorded and recognized the development cycle of the Tapiscia sinensis fruit by morphological observation and paraffin section,so as to understand the structural change rules in the development process.Then,we selected fruits from four periods for transcriptome sequencing.Based on the whole genome data of the gall pepper tree Tapiscia sinensis and transcriptome sequencing results,and combined with bioinformatics analysis methods,we obtained a macroscopic understanding of the changes inside the cells in this process from a molecular perspective.Then,based on previous studies on abiotic stress of ABA signal transduction pathways,Sn RK2 genes in the pathway were identified by the significance parameters of several different expression genes(DEGs)in the transcriptome.Through the comparison and identification of severalSnRK2 gene sequences,the prediction of the physicochemical properties and the high-level structure of proteins,the functional and structural characteristics of severalSnRK2 genes were understood.Next,we further analyzed the expression pattern of the most significant TsSnRK2.3 gene using real-time fluorescence quantitative PCR technology,and prepared arabidopsis thaliana protoplasts for subcellular localization of TsSnRK2.3 protein.Finally,we overexpressed the TsSnRK2.3 gene in arabidopsis thaliana,and preliminarily verified the function of this gene to explore the anti-stress function of this protein under low temperature and salt stress.The results are as follows:(1)Morphological observation and paraffin section showed that the fruit of Tapiscia sinensis had entered the development stagnation stage around the middle of July.With the decrease of the temperature,the external suppository of the approximately "gourd-like" wintering structure became thicker and thicker,and the color gradually deepened.In addition,the number of mucous cavities inside the fruit also increases,and this structural change prepares the fruit for the winter and the development of the next year.In march of the following year,the fruit began to develop and the mucus cells in the enlarged receptorum were rapidly reduced.(2)A total of 23,988 genes were annotated after transcriptome data was assembled.Among them,the KEGG pathway enriched in groups B and C was the most abundant,and the number of DEGs was the highest,containing 9,497 annotated genes in total.In addition,they were enriched in 126 pathways,including 2307 DEGs,and the largest number of DEGs were enriched in Plant hormone signal transduction,Carbon metabolism,Starch and sucrose metabolism,Biosynthesis of secondary metabolites and Biosynthesis pathways of amino acids.Therefore,we obtained a batch of genes related to fruit development and overwintering,which laid a foundation for the follow-up study on the regulation of extralong fruit development cycle.(3)In combination with ABA studies in abiotic stress,four differentially expressedSnRK2 genes were selected from the ABA signal transduction pathway,and their differentially expressed levels were evaluated.Meanwhile,we identified four TsSnRK2 genes,which were named as TsSnRK2.1,TsSnRK2.3,TsSnRK2.4 and TsSnRK2.6.And their ORF was around 1050 bp,encoding about 350 amino acid residues.Next,the relative molecular weight of the protein is around 40 KDa,and the isoelectric point is between 5 and 6.In addition,functional domain prediction of the proteins showed that all four proteins were more than 70% similar to threonine/serine protein kinase.In addition,the most significant difference of evm.tu.scaf?470.27 was quantitatively analyzed,and the results showed that the expression of this gene was inversely proportional to the temperature in combination with the annual temperature of that year.Finally,by preparing the protoplast of arabidopsis thaliana,we found that the TsSnRK2.3 protein was localized in the nucleus.(4)We overexpressed TsSnRK2.3 gene in arabidopsis thaliana and then treated transgenic seedlings under low temperature and high salt stress.The results showed that the roots of transgenic seedlings under both stresses were longer than those in the control group.Therefore,we speculate thatSnRK2.3 gene of Tapiscia sinensis may be one of the important members involved in fruit wintering,and it may also improve the salinity tolerance of plants.