Molecular Mechanism Of Ps-miR156i And Its Target Gene PsSPL4 Regulating Pistil Abortion In Prunus Sibirica

Prunus sibirica is an important economic forest species that has wide applications in food,medicine,and industry.During the cultivation and production process,the phenomenon of pistil abortion often occurs,which greatly reduces the yield and seriously restricts the development of the industry.Based on the previous work of our research group,this study used the flower buds of Prunus sibirica during the crucial period of pistil abortion,constructed Small RNA and transcriptome sequencing libraries,and identified differentially expressed miRNAs and genes related to pistil abortion.The SPL gene family was subjected to bioinformatics analysis.Ps-miR156 i and its target gene PsSPL4 were cloned,expression vectors were constructed,and they were heterologously transformed into Arabidopsis to verify their biological functions.The aim was to reveal the molecular mechanism of pistil abortion in Prunus sibirica.The main research results were as follows:(1)The soluble protein and soluble sugar contents of flower buds in Prunus sibirica with abortive pistils during the exposing white stage,initial blooming stage,and full blooming stage were significantly or extremely significantly lower than those with normal pistils.This suggested that pistil abortion was related to insufficient nutrient supply.A total of 41 hormones were detected in flower buds of Prunus sibirica with abortive and normal pistils.Cytokinins were the most commonly detected hormones(25 types),followed by auxins(13 types)and gibberellins(three types).The total amount of auxins and gibberellins during the exposing white stage,initial blooming stage,and full blooming stage of flower buds with abortive pistil were extremely significantly lower than those with normal pistils.The total amount of cytokinins during the exposing white stage and initial blooming stage was also extremely significantly higher than those of normal pistils.This suggested that pistil abortion was related to insufficient supply of auxins and gibberellins that promote pistil development,such as auxins and gibberellins,while the accumulation of cytokinins that inhibit pistil development was abundant.(2)Small RNA sequencing was performed on the flower buds of Prunus sibirica with abortive and normal pistil.455 miRNAs were identified between abortive and normal flower buds of Prunus sibirica,including 124 differentially expressed miRNA.A total of 48 miRNA families have been identified in known miRNAs,including miR171＿1,miR395,miR159,miR160,miR482,and miR156 contained a large number of members.99.47% of known miRNAs predicted target genes,and a total of 3196 target genes were predicted for 189 miRNAs.Real-time fluorescence quantitative PCR detection confirmed that the expression trend of miRNAs was consistent with Small RNA sequencing.(3)Transcriptome sequencing was performed on the flower buds of Prunus sibirica with abortive and normal pistil.1950 differentially expressed genes were identified among the flower buds of Prunus sibirica with abortive and normal pistil.GO enrichment analysis showed that differentially expressed genes were mainly involved in metabolic process,cellular process,biological regulation,stress responses,and signal transduction.KEGG pathway enrichment analysis showed that differentially expressed genes were mainly involved in plant-pathogen interaction,starch and sucrose metabolism,and plant hormone signal transduction.A total of 901 transcription factor genes were identified among the differentially expressed genes,and they belong to 51 families.Real-time fluorescence quantitative PCR detection confirmed that the expression trend of differentially expressed genes was consistent with transcriptome sequencing.Combined analysis of Small RNA and transcriptome sequencing mined combinations of 7 miRNAs and 20 target genes with opposite expression patterns.(4)Eleven members of the SPL gene family containing SBP-conserved domains were identified from the Prunus sibirica genome.Ps SPLs were distributed on six chromosomes,ranging in molecular weight from 18438.55 to 102386.08 Da,with the amino acid count of 162 to 923 aa and the isoelectric point ranging from 5.99 to 9.62.The number of exons in Ps SPLs was 2 to 10.They were non-transmembrane hydrophilic proteins.Ps SPLs displayed collinearity with other Rosaceae species and clustered with orthologous genes of Prunus persica.The promoter regions of Ps SPLs contained regulatory elements related to meristem expression,abscisic acid response,gibberellin response,and MYB binding sites involved in drought induction and light response.The expression of Ps SPLs had certain tissue specificity.(5)Ps-miR156 i and its target gene PsSPL4 were successfully cloned,and plant overexpression vectors were successfully constructed.The interaction between Ps-miR156 i and its target gene PsSPL4 was verified using RLM-5′RACE and tobacco transient transformation technology.Ps-miR156 i negatively regulated the expression of PsSPL4.Based on agrobacterium mediated genetic transformation of Arabidopsis,the pistil length,silique length,and the number of seeds per silique of the Ps-miR156 i overexpression lines were significantly lower than those of the wild-type.The pistil length,silique length,and the number of seeds per silique of PsSPL4 functional recovered lines were significantly higher than those of the spl4 mutant.It was confirmed that Ps-miR156 i regulates pistil abortion in Prunus sibirica by inhibiting the expression of the target gene PsSPL4.