Screening Of Genes Responsed To NaCl Stress In Malus Sieversii Seedlings By MicroRNA And Transcriptome Sequencing

In this experiment,Malus sieversii seedlings of tissue culture were used as the test materials.The transcriptome and Small RNA sequencing of the leaves and roots in Malus sieversii seedlings treated with150 mmol·L^-1NaCl for 6 h and 48 h were performed,All Unigenes obtained were functionally annotated in GO and KEGG databases,and finally verified by qRT-PCR to screen out the related genes of Malus sieversii in response to NaCl stress,To provide a reference for further exploring the action mode of Malus sieversii seedlings in response to NaCl stress.The results are as follows:1.Analysis of transcriptome sequencing data of leaves in Malus sieversii seedlings treated with 150mmol·L^-1NaCl for 6 h and 48 h revealed that:Compared with the control,there were 713 differentially expressed genes（DEG）in the leaves of Malus sieversii seedlings treated with NaCl for 6 h,among which295 DEGs were up-regulated and 418 DEGs were down-regulated in response to NaCl stress.At 48 h,the differentially expressed genes were 3364,with 1745 DEGs up-regulated and 1619 DEGs down-regulated.GO was mainly enriched in oxidoreductase activity,catalytic activity,PS II,photosynthetic membrane,thylakoid,oxidation-reduction process,photosynthesis,lignin metabolic process,etc.KEGG functional annotation showed that photosynthetic activity and plant hormone signal transduction pathways were significantly enriched in leaves of Malus sieversii seedlings with NaCl stress.qRT-PCR verified 6photosynthetic genes PsaE,PsaN,PsbP,PsbW,PsbO,psb27-1,and 4 genes related to plant hormone signal transduction PP2C37,SAPK2,PIF4,and SRK2A,which played a certain role in Malus sieversii responded to NaCl stress.2.Analysis of transcriptome sequencing data of roots in Malus sieversii seedlings treated with 150mmol?L^-1NaCl for 48 h revealed that:Compared with the control,there were 3808 differentially expressed genes in the root system,among which 1057 DEGs were up-regulated and 2751 DEGs were down-regulated in response to NaCl stress.According to KEGG analysis,2095 differential genes in leaves and roots of Malus sieversii have been annotated,involved 44 pathways.The most significant pathways are glycolysis/gluconeogenesis,pentose phosphate pathway,fructose and mannose metabolism,pyruvate metabolism,etc.Through transcriptome sequencing and qRT-PCR verification,it was found that after Malus sieversii was subjected to NaCl stress,the expression levels of TPI,FBPase,pckA,and ppdK genes were down-regulated in leaves and up-regulated in roots during glycolysis participate in NaCl stress response.3.Sequencing of small RNA in leaves of Malus sieversii seedlings treated with 150 mmol?L^-1NaCl for 6 h and 48 h showed that:Compared with the control,3 miRNA differentially expressed,2 up-regulated and 1 down-regulated during the treatment with NaCl for 6h,and the corresponding number of target genes was 64.When NaCl was treated for 48 h,differential expression of 13 miRNA was found,4 were up-regulated,9 were down-regulated,and the number of corresponding target genes was 108.The differential target genes were classified and annotated by GO and KEGG.When NaCl treatment for 6 h,GO enrichment mainly included:inositol pentakisphosphate 2-kinase activity,oligosaccharyltransferase activity,tRNA wobble adenosine to inosine editing,xylan biosynthetic process,etc.When NaCl treatment for 48 h,GO enrichment mainly included:oxidoreductase activity,DNA binding,Apoplast,secondary metabolic process,etc.KEGG accumulate in the N-glycan biosynthesis pathway.qRT-PCR verified that when NaCl treatment for 6 h and 48 h,the expression levels of mdm-mir390f and its target genes HF10976,mdm-mir171i and its target genes HF33844,mdm-mir399j and its target genes HF11095 were negatively correlated.These three miRNAs were involved in the response process of Malus sieversii to NaCl stress.4.Sequencing of small RNA in roots of Malus sieversii seedlings treatment with 150 mmol?L^-1NaCl for 48 h showed that:Compared with the control when NaCl was treated for 48 h,The number of differentially expressed miRNAs in the root system was 4,among which 1 was up-regulated,3 were down-regulated,and the number of target genes was 19.Malus sieversii roots GO mainly enriched in sulfate adenylyltransferase activity,adenylyltransferase activity,Membrane,inorganic anion transport,sulfur compound metabolic process.KEGG Pathway mainly enriched in Monobactam biosynthesis,Selenocompound metabolism,MAPK signaling pathway,Plant-pathogen interaction pathways.qRT-PCR verified that in the root system and leaves,there was a negative correlation between mdm-miR156o and its target gene HF27440,mdm-miR396b,and its target gene HF18932.These two miRNAs were involved in the response of Malus sieversii to NaCl stress.