Identification And Characterization Of Genes Involved In Gibberellins Pathway And DaDELLA In Dioscorea Alata

Water yam(Dioscorea alata),belonging to the family dioscoreaceae,is an important food crop in tropical and subtropical areas,which has high nutritional value.A single fully-grown tuber of water yam could have more than 50 kilogram,and yield per mu could reach thousands kilogram.Research on tuber development could lay a foundation for mining and utilizatation genetic resources of this species.In our previous research,we found that gibberellin promotes tuberization and tuber enlargement of water yam.In this study,different concentrations of gibberellin(1 mg/ L,10 mg / L,20 mg / L,100 mg / L)were used to treat(tuberiation late)and Da40(tuberization early)which had disctinct difference on tuberization day.After 45 days treatment,all treatments of different gibberellin concentrations promoted the tuber formation,stem elongation and leaf morphological changes of water yam,but there were phenotypic differences for the plants treated with the same concentration of gibberellin.The plants with elongated stem had relatively later tuberization day or smaller tuber than those without stem elongation.Higher gibberellin concentration leads to more obvious stem elongation.RT-q PCR assays for genes involved in gibberellin biosynthesis and metabolism indicated that the expression levels of gibberellin biosynthesis genes were declined after tuberization,while gibberellin treatment made the expression decreasing of GA2 ox and GA3 ox earlier,which implied the biosynthesis of active gibberellin reduced after tuberization.Morever,the expressions of gibberellin receptors(GIDs)were significantly decreased after gibberellin treatment,which were negatively regulately by gibberellin.Because DELLA is a key negative regulator in the gibberellin pathway,we systematically screened the GRAS gene family in water yam,and focused on the sequence characteristics of the DELLA subfamily members.Based on BLASTP and HMMER analysis,75 Da GRAS genes were identified.Through multiple sequence alignment and phylogenetic tree construction,the GRAS family proteins were divided into 7 subfamilies,among which LISCL subfamily had the most GRAS members.Three DaDELLAs were identified according to the conserved DELLA domain,among which DaDELLA1 has incomplete DELLA domain,DaDELLA3 has one amino acid variation for DELLA-motif,only DaDELLA has conseved DELLA-motif and VHYNP-motif and is homologous to Os SLR1.Analysis of transcriptome datasets from gibberellin-treated water yam,we found that DaDELLA2 had relatively high expression levels in all tissues of water yam,and showed a reduced expression after gibberellin treatment.We chose DaDELLA2 and DaDELLA3 for transcriptional function analysis and found both of which had a strong auto-activation ability.Moreover,different concentrations of auto-activation inhibitors(5mg / ml,10 mg / ml,30 mg / ml,50 mg /ml,60 mg / ml)could not inhibit their transcriptional activity.Deletion of DaDELLA2N-terminal sequences leads to the variation of its transcriptional activity.Yeast two hybrid assay demonstrated that DaDELLA2 without partial N-terminal sequences did not interate with EIN,GID1C-1,ac GID1C-4,LSH11,and LSH2.For interaction with Da GID genes(GID1C-1 and GIDC-4),N-terminal deletion may influence their interaction domain.This study provides the basis for the analysis of DELLA mediated gibberellin regulation of water yam development.