| Zanthoxylum armatum DC.is a kind of inportant economic tree in China.Due to the medicinal and edible value,it is also one of the pillar industries for rural revitalization in southwestern China.And it has great efficiency in economy,ecology and society.However,in recent years,a large number of male flowers have been differentiated from Z.armatum DC,which is mainly parthenogenetic,and the proportion of male flowers has increased year by year,seriously restricting the industrial development of Z.armatum DC.Analyzing the internal mechanism of male and female flower differentiation in Z.armatum DC can provide a basis for formulating prevention and control techniques for male flowers in Z.armatum.Class C genes of floral organ differentiation have a decisive regulatory role in the differentiation and development of stamens and stamens.Therefore,this study combined transcriptome and genomic data from Z.armatum DC,cloned and analyzed the coding sequence and related bioinformatics characteristics of the C class gene ZaAGAMOUS(ZaAG)in Z.armatum DC,identified the functions associated with the nutrition and reproductive growth using the ectopic overexpression of ZaAG in Arabidopsis thaliana,analyzed the interacted proteins of ZaAG,and explore the key endogenous factors and enrichment pathways significantly regulated by the ZaAG gene based on RNA-seq and endogenous hormone detection analysis among the flower buds of wild type and ZaAG overexpression lines.Additionally,the genetic regulatory network related to the differentiation of male and female flowers via ZaAG pathways in Z.armatum was comprehensively revealed,which will provid a rich theoretical support and data reference for breeding a high-quality and high-yield cultivation of Z.armatum.The main conclusions of this study are as follows:(1)The C-terminal transcriptional activation region of the ZaAG coding sequence(ZaAGd)from the transcriptome dateset of Z.armatum is incomplete,and the N-terminal MADS domain of the AG homologous gene in the genome is incomplete.Combined the transcriptome data and genomic data,it was successfully obtained a complete ZaAG encoding sequence of Z.armatum,encoding 244 amino acids,containing a MADS domain composed of 77 amino acids and a K-box domain composed of 89 amino acids.The results of bioinformatics analysis indicate that ZaAG is an unstable hydrophilic protein with the highest genetic relationship with CsAG in the Rutaceae plant Citrus sinensis.A plenty of transcription factor binding sites related to plant reproductive growth had been identified in ZaAG sequence,such as MYB,LFY,and AP2,and some other members were also obtained and involved in plant stress resistance responses and endogenous hormone signal transduction pathways.In addition,the results of qRT-PCR showed that ZaAG was mainly expressed in the reproductive organs of Z.armatum DC,and was significantly higher in male flowers than in female flowers during the floral differentiation process.(2)The overexpression of ZaAG significantly inhibits the vegetative and reproductive growth of Arabidopsis thaliana,resulting in a significantly reduced plant size,curly moss leaves,shortened childhood,significantly advanced flowering,reduced floral organs,abnormal differentiation of stamens and pistils,extremely significantly shortened fruit pods,and significantly reduced seed setting.However,there is no significant phenotypic difference between the overexpressed strains of ZaAGd and wild type Arabidopsis thaliana,suggesting that a complete C-terminal domain is crucial for ZaAG to play its biological functions.(3)Using ZaAG protein as a bait,a total of 25 candidate interaction proteins were obtained from the yeast two-hybrid screening library of Z.armatum DC.Further yeast two-hybrid and bimolecular fluorescence complementary analysis showed that the regulatory factor ZaICE1,annotated with low-temperature response and stamen differentiation,strongly interacts with ZaAG protein.The expression of AtICE1(a homologous gene of ZaICE1 in Arabidopsis)was significantly increased in ZaAG transgenic lines,and the expression patterns of ZaICE1 and ZaAG were basically consistent for exogenous stress.The above results indicate that ZaICE1 interacts directly with ZaAG to play a role in the process of plant reproductive growth induced by exogenous stress.(4)Based on the plant hormone content and RNA-seq analysis using the floral samples of wild type and ZaAG overexpression lines of Arabidopsis thaliana,the results showed that the the contents of cytokinin,gibberellin,jasmonic acid,and abscisic acid was significantly reducted in the ZaAG overexpression lines,while the contents of auxin and salicylic acid were significantly increased.A total of 2456 differentially expressed genes were obtained from RNA-seq,1734 of which were down-regulated and 724 of which were up-regulated.All differentially expressed genes were significantly enriched in plant endogenous hormone and carbohydrate metabolism pathways,especially cytokinin and auxin biosynthesis and signal transduction pathways,as well as polygalacturonase metabolism pathways.These results indicate that overexpression of ZaAG significantly affects the biosynthesis and signal transduction processes of endogenous hormones in plants,such as cytokinins and auxin pathways.(5)To the investigate the patterns of gene related to cytokinin and polygalacturonase metabolic pathway,the floral samples at key time points during the development of male and female flowers of Z.armatum DC were detected by qRT-PCR analysis.The results showed that the expression abundance of genes related to cytokinin biosynthesis during male flower differentiation was higher than that of female flowers,and genes related to polygalacturonase metabolic pathway were also significantly upregulated during male flower differentiation.The results suggest that the male flower differentiation of Z.armatum DC may be related to cytokinin biosynthesis and polygalacturonase metabolic pathways. |
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