Identification Of MiR164 And Its Target NAC Family Genes And Expression Analysis Of Related Genes During Flower Formation In Walnut

Object: Walnut is one of the main horticultural industries in Xinjiang,and its economic value is mainly realized by dried fruit.Therefore,the normal flowering and fruiting of walnuts is particularly important.The study of walnut flower formation is of great significance to its variety improvement and stable yield and efficiency.Previous studies have shown that miR164 regulates the formation and development of floral organ primordia and meristem by regulating NAC gene.At the same time,NAC transcription factors play a regulatory role in regulating the number of stamens and floral organ development.In order to have a clearer understanding of the molecular mechanism of walnut flowering process,and to explore the regulation of miR164 and NAC family on walnut flowering process,it is necessary to identify and study the miR164 and NAC family in walnut.Methods: Taking the morphological undifferentiated stage of female flower bud,the initial stage of morphological differentiation and the differentiation stage of female flower primordium as the sampling time points,combined with the observation of free-hand sectioning,the walnut ‘Wen 185’ was collected on June 2,June 6 and June 10,respectively.Female flower buds,male flower buds and leaf buds.miR164 family in walnut was identified by smallRNA sequencing and analyzed by sequence alignment,evolutionary analysis,target gene prediction,and real-time quantitative PCR detection.At the same time,using bioinformatics methods,through sequence alignment,evolution analysis,motif prediction,collinearity analysis,protein interaction and real-time quantitative PCR analysis,the walnut NAC transcription factor family was identified and analyzed.The NAC gene was cloned and analyzed.In addition,the regulation mode of miR164 on target NAC genes in walnut was explored,and the network regulation prediction based on the jre-miR164-NAC regulation mode in walnut was performed.Results:(1)Two miR164 members in walnut were obtained by smallRNA sequencing.Sequence alignment showed that the mature sequences of miR164 were highly conserved with a length of 21 nt,and there were only individual base differences at the 3’ end among the sequences,with the largest difference mainly at the 21 st base.However,the precursor sequences of miR164 were quite different.Evolutionary analysis found that jre-miR164 had the closest relationship with juglandaceae species.Gene expression analysis of jre-miR164 indicated that it may affect the formation and development of young organs in flower buds and leaf buds by regulating target NAC genes.(2)Eight target genes of jre-miR164 were predicted by the online website psRNATarget,of which six are members of the NAC family,which belong to the NAC1,CUC1 and CUC2 genes in the NAC1 subfamily and NAM subfamily of walnut,respectively.Moreover,they are highly consistent with NAC1,CUC1 and CUC2 genes in Arabidopsis thaliana.Through RT-q PCR analysis,it was found that the regulation mode of jre-miR164 in walnut on JrNAC1,Jr CUC1 and Jr CUC2 genes was inhibition.(3)A total of 121 NAC family members were identified in the whole genome of walnut,which were divided into 15 subfamilies.Sequence analysis showed that there were differences in motifs in each subfamily.Synteny analysis showed that JrNAC genes had multiple homologous genes in Arabidopsis,grape and poplar,and 26 JrNAC genes had gene duplications in the walnut genome.Expression analysis showed that JrNAC115 and JrNAC8 genes were significantly expressed in female flower buds,male flower buds and leaf buds,while other genes were also up-regulated to varying degrees,suggesting that these JrNAC genes might play a key role in flower and leaf bud development.In addition,the possible effects of other genes involved in the regulation of miR164/NAC on this regulation were discussed,and based on this,the possible network regulation patterns between genes involved in the regulation of jre-miR164/JrNAC in walnut were predicted.(4)The highly expressed JrNAC gene in flower buds was cloned,and a full-length 805 bp JrNAC38 gene was successfully obtained.The analysis found that it was a non-transmembrane protein and had no signal peptide.It is composed of four structures of corners and random coils.RT-q PCR analysis revealed that JrNAC38 gene may play a regulatory role in the development of walnut flower and leaf buds.Conclusion: jre-miR164 in walnut may affect the formation and development of walnut pistil primordia,stamens and leaf bud primordia by regulating NAC1,CUC1 and CUC2,and may also affect the establishment of juvenile organ boundaries.In addition,members of other subfamilies of NAC may also be involved in the development of flower and leaf buds,and there may be other genes regulating jre-miR164/JrNAC.