Sequencing Analysis Of MicroRNA And Transcriptome And Genetic Transformation Of BpmiR408 In Betula Platyphylla Under Drought And Salt Stress

Drought and salinity are important environmental factors affecting normal physiological and biochemical metabolism of plants.It is important to study the complex and efficient stress response mechanism of Betula platyphylla for breeding new varieties with drought and salinity tolerance.In this study,the transcriptome and microRNA of B.platyphylla under drought and salt stress were sequenced.The BpmiR408 gene and its target gene BpCBPl,which can respond to drought and salt stress,were screened out,and the function of BpmiR408 and BpCBP1 in drought and salt tolerance was further studied.The main findings are as follows:1.The results of RNA-seq analysis of B.platyphylla under drought stress and salt stress showed that 3224 differentially expressed genes(DEGs)were screened under drought stress,3799 DEGs under salt stress and 1668 DEGs under both conditions.There were 308 differentially expressed transcription factors(TFs)under drought stress,253 under salt stress and 157 under both conditions.Go analysis showed that DEGs were mainly enriched in hormone-related responses and signaling pathways under drought stress.Under salt stress,DEGs were mainly concentrated in the items of metabolism,enzyme activity and ABA response.The results of KEGG enrichment analysis showed that under drought stress,DEGs was mainly enriched in signal transduction and substance synthesis-related metabolic pathways,while under salt stress,DEGs was mainly involved in signal transduction and photosynthesis-related metabolic pathways.2.miRNA-seq analysis of B.platyphylla under drought stress and salt stress showed that a total of 190 miRNAs were identified in B.platyphylla under drought and salt stress,and 221 target genes were predicted,of which 49 were TFs.There were 30 and 25 differentially expressed miRNAs under drought and salt stress,respectively.Three target genes were up-regulated in RNA-seq analysis under drought and salt stress.The target gene GO analysis of differentially expressed miRNAs showed that DEGs were mainly related to growth and development,oxidation-reduction process and ion binding activities under drought and salt stress.KEGG analyzed the target genes differentially expressed in mirnas involved in the metabolism of oxidative phosphorylation,Cysteine and methionine metabolism under drought and salt stress.There were 18 miRNA differentially expressed under both drought and salt stress.Especailly,BpmiR408 was down-regulated under both stresses,and its predicted target gene BpCBPl/2 was reported to be involved in stress response.3.BpmiR408 and its target gene BpCBP1 functions in response to both drought and salt stress were analyzed.BpmiR408 sequence analysis showed that its mature sequence was highly conserved.The pre-BpmiR408 sequence is the closest relative to the Mtr-miR408 of Medicago truncatula.Under drought and salt stress,the POD activity in transient overexpression plants of BpmiR408 was 77.36%and 66.22%,the PRO content was 60.10%and 37.34%,and the H2O2 content was 1.02 and 1.3 times of the control,respectively.The expression of BpCBPl in transient overexpression plants was only 65%of that in control plants.5’RLM-RACE experiment showed that BpCBP1 was the target gene of BpmiR408.The transient overexpression of BpCBP1 changed the POD activity of the plant.The contents of PRO under drought stress and salt stress were 1.17 and 1.52 times of the control,and the contents of H2O2 were 67.98%and 49.21%of the control,respectively.In addition,the BpmiR408 overexpression vector was stably transformed into B.platyphylla.At present,resistant lines have been obtained,and the PCR test was positive,which laid a foundation for further exploring the response mechanism of BpmiR408 to drought and salt stress.