Identification And Expression Of Key MicroRNA And Their Target Genes Involved In Female Flower Differentiation In Early Fruiting Walnut (Juglans Regia L.)

Walnut?Juglans regia L.?is one of the four world nuts trees,which is an important economic forest tree in China,and is also the characteristic forest fruit of Xinjiang autonomous region.Walnut is divided into two categories of early fruiting and late fruiting.Generally,early fruiting walnut can blossom and bear fruit within three years,most blooming in two to three years,and a few species blooming in the first year.Early fruiting is the main characteristic of walnut,which has high inheritance ability.Study on flower bud differentiation of walnut will contribute great significance to the control and improvement of fruit varieties.In this study,transcriptome and small RNA sequencing libraries were constructed based on the flower buds in different differentiation stages?F₁,F₂ and F₃?and leaf buds during critical period?JRL?from early fruiting cultivar ‘Xinxin2'.Key miRNAs related to the differentiation of female flower buds were screened and their target genes were analyzed.The main results were summarized as follows.Transcriptome sequencing data was spliced into 132154 Unigenes.There were 2688 differentially expressed genes between female flower buds at different differentiation stages and 551 differentially expressed genes between female flower buds and leaf buds at critical stage.Total of 123 miRNAs were obtained through small RNA sequencing,including 51 known miRNAs and 72 novel miRNAs.Fifty-five differentially expressed miRNAs were obtained between female flower buds at different differentiation stages and female flower buds and leaf buds at critical stage,including 17 known miRNAs and 38 novel miRNAs.Jre-miR157a-5p—SPL,jre-miR160a-5p—ARF,jre-miRn69—AP2,jre-miR171b-3p/n49—SCL,jre-miR169b-5p—NF-YA and jre-miR396a-5p/b-5p—GRF were closely related to walnut flower bud differentiation,and mainly involved in physiological and biochemical processes,such as ubiquitin proteolysis,RNA degradation,polysaccharide degradation,plant circadian rhythm,photosynthesis and carbohydrate metabolism.Based on the small RNA sequencing data,RT-qPCR,geNorm,Normfinder and RefFinder were combined to screen the suitable reference genes for the quantitative analysis of miRNA and mRNA.Jre-miR394 a,jre-miR159 a and jre-miR159 c could be used as reference genes for the analysis of miRNA expression in flower buds at different stages of differentiation.5.8s rRNA and jre-miRn3 could be used as reference genes in leaf buds at different stages of differentiation.5.8s rRNA,jre-miRn3,jre-miR159b-3p and jre-miR159 c could be used as reference genes in different tissues.Jre-miR159b-3p,jre-miR159 c and jre-miR159 a could be used as reference genes in different cultivars.TUA,EF1 and TUB were suitable reference genes for flower buds at different differentiation stages.TUB and 18 S rRNA could be used as reference genes for leaf buds at different stages of differentiation.TUB and TUA could be used as the reference genes of different varieties.ACT2,18 S rRNA and GAPDH were suitable reference genes of different tissues.Association analysis was performed between the differentially expressed genes obtained by transcriptome sequencing and the differentially expressed miRNAs obtained by small RNA sequencing.Twenty-two differentially expressed miRNAs were negatively regulated by their target gene expression.RLM-5 'RACE was used to verify the target genes of jre-miR157a-5p and jre-miR160a-5p.RT-qPCR was used to verify the expressions of jre-miR157a-5p and jre-miR160a-5p and their target genes in female flower buds at different stages of differentiation,different tissues and different cultivars.Both jre-miR157a-5p and jre-miR160a-5p were expressed in all tissues,contrary to the corresponding target gene expression pattern.Sequence characteristics and evolutionary relationships of miR156/157 and miR160 families in nine plants including walnut,hickory,Arabidopsis,peach,apple sweet orange,papaya,grape and popular were analyzed.PlantCARE was used to predict the cis-elements of promoter sequences of jre-miR156/157 and jre-miR160 families.Common functional elements were identified,and promoter regions of different members of the miRNA family also had their own specific functional elements.The results showed that both jre-miR156/157 and jre-miR160 had synergistic and specific functions in walnut flower bud differentiation.Expressions of jre-miR156/157 and jre-miR160 families in female flower buds at different stages of differentiation and leaf buds at critical stage of differentiation were analyzed combining transcriptome sequencing data and RT-qPCR.There were different expression patterns among the same family members,which indicating that they played different roles in the process of flower bud differentiation.Forty-eight JrSBP genes were identified,among which 19 JrSBP genes were target genes of jre-miR156/157.JrSBPs and AtSPLs in the same group were functionally conservative.A large number of cis-elements,including light response elements,plant hormone response elements and stress response elements,were identified in the promoter region of JrSBP,indicating the expression of JrSBP gene in walnut was comprehensively regulated by light,plant hormone,stress and other factors.Expression profiles of 48 JrSBPs were divided into four categories,indicating that they had different functions during female flower buds differentiation.RT-qPCR showed the expression of JrSBP1/3/4/23/24/27/29/35 in female flower buds at critical differentiation stage was significantly higher than that in leaf buds at the same stage.JrSBP4,JrSBP23,JrSBP24 and JrSBP35 were significantly up-regulated in F₂ vs F₁.Expression of JrSBP23 increased during flower bud differentiation.JrSBP27 and JrSBP29 were down regulated in F₂ vs F₁.The results showed that JrSBP genes had diverse functions in walnut flower bud differentiation,and some of their functions had synergistic effects.