Gibberellin Influences Tuberization Time In Dioscorea Alata L. And Characterization Of Differentially Expressed Genes

Dioscorea alata L（greater yam）is an important tuber crop with high yield and high nutritional value in the tropical and subtropical regions.In addition to intrinsic factors influencing tuber development in D.alata,plant hormones will also have a certain effect on its production.As an important plant hormone,gibberellins can regulate tuber development and affect the yield of tubers and oligosaccharides.Exploring the molecular mechanism of gibberellin regulating the development of D.alata tubers can provide a theoretical basis for controlling the tuber formation time and increasing tuber yield.In this study,we analyzed the effect of gibberellin treatment on D.alata transcription levels after applying gibberellin and paclobutrazol treatments to tissue-cultured seedlings via RNA-seq.The genes related to the gibberellin pathway were mined and characterizaed their expression profiles under GA₃ treatment.The expression patterns of key genes differentially expressed under GA₃ treatment were verified by RT-q PCR assays.The main results are as follows:1.Greater yam seedlings showed promoted tuberization process after using different concentrations of GA₃（1 mg/L～20 mg/L）.The concentration of GA₃is 10 mg/L showed the most significant influence on tuber formation.After 60 days of GA₃ treatment,the tuberization ratio and the tuber diameter reached 93.17%and 10.29 mm,which had a significant increase by 23.44%and 5.31 mm comparing with the control treatment.Relatively higher GA₃ concenitration（20 mg/L）also promoted tuber formation and inhibited the vegetative growth of the seedlings significantly.Moreover,gibberellin inhibitor-polytrisazole PP₃₃₃（10 mg/L）treatment significantly inhibited the tuber formation,which suggested that gibberellins could have positive influence on tuber formation.2.RNA-seq was performed on 34 D.alata samples derived from 15 days and 60 days after H2O,GA₃ and PP₃₃₃ treatment,and 2339 differentially expressed genes（more than two-folded change,p value<0.05）were identified,which were distributed in 37 KEGG functional pathways,and significantly enriched in hormone signaling Pathway,starch and sucrose metabolism pathway,photosynthesis and other pathways;cis-acting factors prediction in promoter regions of these differentially expressed genes demonstrated that836 contained gibberellin response elements.After analyzing the gene expression pattern of starch synthesis pathway,we found that the expression levels of genes in the starch synthesis pathway were significantly increased after gibberellin treatmentment tuber tissues.The results suggested that gibberellin treatments induced the expression of genes in this pathway,which might affect the development of tubers.3.q RT-PCR assays were used to detect the effect of GA₃ treatment on gene expression in the gibberellin pathway and we found that the expression levels of most genes were decreased significantly after 6 h and 24 h of GA₃treatment;the transcriptome data showed that the expression level of GA20ox2 was decreased after 60 days of gibberellin treatment while the expression of GA2ox6 was induced and the expression level of most genes had no significant change.Moreover,we identifed homologous genes in D.alata,which were stably influenced by gibberellins in Arabidopsis thaliana,and found that DaLSH gene was specifically expressed in tubers.q RT-PCR results showed that short-term treatment of gibberellin would inhibit the expression of this gene.However,the expression change of DaLSH in the transcriptome of 60 days of gibberellin treatment was not significant.Co-expression analysis of 34 transcriptome datasets showed that DaLSH and genes in the starch and carbohydrate metabolism pathway were in the same co-expression module,which suggested that DaLSH might involved in the expression regulation of starch synthesis genes in tuber tissues.4.We analyzed the transcriptome data of D.alata to obtain the gene sequences of tuber-specific expressed genes DaLSH1 and DaLSH2,and successfully cloned and constructed the over-expression vector and subcellular localization vector.The constructed DaLSH1 overexpression vector was transformed into D.alata.Among the candidate transgenic plants obtained at present,there are obvious tissue swelling in the axillary buds,while the control samples is the growth of young leaves directly.The similarity of organ production indicates that DaLSH1 and DaLSH2 of D.alata may participate in the differentiation and growth of lateral buds（side branches）of D.alata.Since the tubers of D.alata are metamorphosis stems from the growth and differentiation of lateral buds,and DaLSH1 and DaLSH2 are specifically expressed in tubers,we speculate that they have an important role in tuber development.