Screening Of Differentially Expressed Genes And Proteins For Photosynthetic Characteristics Of Malus Sieversii Seedlings In Response To Freezing Stress

In this experiment,tissue culture seedlings of Malus sieversii were used as materials to study the effects of-3℃ simulated freezing stress for 0 h,3 h,6 h,12 h and 24 h on the morphology,electrical conductivity(REC),malondialdehyde(MDA)content,chlorophyll(Chl)content,and the effect of the maximum quantum yield(Fv/Fm)parameters of photosystem II and the performance index(PIabs)of photosystem II based on absorbed light energy of Malus sieversii seedlings.The changes of photosynthetic pigments and photosynthesis-related genes and proteins were analyzed by transcriptome and proteome,which provided a reference for further research on the changes of photosynthesis pathway-related genes and proteins in Malus sieversii under freezing stress.The main findings are as follows:1.Malus sieversii seedlings began to shrink when the leaves were treated for 12 h at-3 ℃ to simulate freezing injury.Compared with CK,the chlorophyll content of treatment 24 h decreased significantly,and chlorophyll a/b increased significantly.Under different treatments,such as 0,3,6,12 and 24 hours,MDA content and REC value increased significantly,while Fv/Fm and PIabs values decreased significantly.2.A total of 13320 differentially expressed genes were detected by transcriptome sequencing.Compared with control,the number of differentially expressed genes after 0 h,3 h,6 h,and 12 h treatments were 4280,6213,7003,and 11103,respectively,there were 2256 differentially expressed genes in the four comparison groups.During the 0 h-12 h treatment,the number of differential genes increased gradually.GO enrichment analysis found that 11 of the top 20 GO Terms were related to photosynthesis and photosynthetic pigments.There were 103 differentially expressed genes related to photosynthesis,and 28 differential genes in the four comparison groups.Among them,3 genes related to chloroplast oxyalkylene reductases were down-regulated,and the rest 25 genes related to zeaxanthin epoxidase,dephytyl chlorophyll such as oxygenase,15-cis phytoene synthase,light-harvesting pigment protein,and chlorophyll(ide)b reductase vinyl reductase were down-regulated.58 differentially expressed genes involved in photosynthesis regulation were screened,including 18 genes responding to photosystem II,12 genes responding to photosystem I,13 genes related to the photosynthetic electron transport chain,3 genes related to ATP synthase and 12 differentially expressed genes related to cytochrome b6 f.At the same time,Psb Q(Msi＿13A005910)and Psb Y(BGI＿novel＿G011171)genes were significantly up-regulated under freezing stress.By q RT-PCR verification of the seven photosynthesis-related differential genes with high expression,it was found that the expression levels of these genes had the same trend as the results of transcriptome sequencing,which indicated that these seven genes related to photosynthesis responded to the freezing stress.3.Through proteome sequencing,it was found that the number of quantitative proteins was 8502,and there were 251 differential proteins in the four comparison groups.Compared with CK,there are 75,109,108 and 68 differentially expressed protein in 0 h,3 h,6 h and 12 h treatment,respectively,and there are 12 differentially expressed in protein group.Enrichment analysis showed that differentially expressed protein was mainly involved in flavonoid metabolism,sugar metabolism,photosynthesis and other metabolic pathways.Among them,there are 4 differentially expressed proteins related to photosynthesis,Msi＿15B021960(Pet H),Msi＿15B000520(Pet E),Msi＿15B039440(Psa N),Msi＿10A022940(Psa K),and 1 differential expression protein(Msi＿03A014670(CHl D,BCHD))related to chlorophyll synthesis.It shows that these proteins play an important role in freezing stress.Correlation analysis of transcriptome and proteome data under simulated freezing damage at-3°C found that 7935 genes were quantified in both transcriptome and proteome,and there was a certain correlation between them,with the average pearson correlation coefficient of 0.031.The related genes are mainly involved in tyrosine metabolism,α-linolenic acid metabolism,tropane,piperidine and pyridine alkaloid biosynthesis,phenylalanine metabolism,isoquinoline alkaloid biosynthesis and other pathways.According to cluster analysis,these were divided into six categories,indicating that there is a certain correlation between the transcriptome and the proteome.Among them,there are 14 related genes related to photosynthesis,but only one related differential gene(Msi＿15B039440,Psa N),and 21 genes related to chlorophyll synthesis expressed at both the transcription and protein levels,but no differentially expressed genes.