Homologous Cloning And Functional Analysis Of GASA1 Gene In Faba Bean

Faba bean(Vicia faba L.)is the fourth largest winter legume crop in the world.In temperate climate region,faba bean often need to grow over winter,so leading to a longer breeding period,and the delayed flowering time will reduce land use and economic benefits,so it is important to promote the flowering time for faba bean production.Therefore,exploration of flowering molecular mechanism in faba bean,excavation of related genes,breeding new varieties and enriching germplasm resources are the key objectives of the breeder and researchers.Flowering time is controlled by both endogenous and exogenous factors,among which gibberellin pathway is one of the endogenous regulation pathways,a high gibberellin level can promote flowering.GASA(gibberellic acid stimulated in Arabidopsis)protein is a kind of low molecular weight protein regulated by gibberellic acid,which is involved in many growth and development pathways including flowering time in planta.In this research,we use faba bean variety ’Tongcanxian No.6’ as our experimental material.Homologous cloning was carried out,and primers were designed based on the sequence of MtGASA13(LOC11422708)from Medicago truncatula,which is closely related to faba bean,to amplify a GASA gene in faba bean cDNA sample.Consequently,a VfGASA1 gene containing a complete ORF was obtained,which is the first GASA family gene ever cloned in faba bean.Via sequence alignment,subcellular localization,spatiotemporal expression analysis and functional verification,we studied the regulatory mechanism on flowering through gibberellin signaling of VfGASA1 and preliminarily explored the theoretical basis of faba bean flowering.The results were listed as follows:1.VfGASA1 is a member of the GASA family of higher plants,which has a GASA domain at the N-terminal.VfGASA1 shows the highest homology with MtGASA13.It’s a secretory protein,and the subcellular localization result indicated its location in the apoplast.2.Tissue expression analysis showed that VfGASA1 was highly expressed in leaves,flowers,young pods and seeds,with the highest expression in leaves and young pods,while the expression was very low in stems,and almost not detected in roots,the result reflected a specific expression pattern of VfGASA1.In addition,the expression of VfGASA1 was remarkably sensitive to environmental factors.Under different abiotic stress treatments such as low temperature(4℃),high temperature(40℃),750 mM PEG and 150 μM NaCl,the expression level of VfGASA1 was rapidly induced,and the expression changed over treatment time.3.Heterologous overexpression of VfGASAl in Arabidopsis delayed flowering time,but there was no difference between the homologous mutant gasa15 and the wild type(Clo-0).After treated with exogenous GA3,the flowering time of transgenic plants showed not difference from that of wild type.The qPCR analysis showed that the expression level of FT,the "florigen" gene was significantly reduced in transgenic plants,and the FLC,a flowering inhibition gene,was notedly increased,while the expression level of CO,regulator of FT was not changed.GAI,member of DELLA genes in Arabidopsis,was significantly up-regulated in the overexpression lines,indicating that VfGASA1 indirectly regulated the expression of GAI,thereby inhibiting the expression of FT and delaying the flowering time.4.Some seeds of overexpression plants developed abnormally,and the size of seed became smaller and the shape of seed deformed.Besides,the skotomorphogenesis of overexpression plants was enhanced according to the increasing length of hypocotyls under darkness.Taken together,VfGASA1 is a pleiotropic gene.In summary,this study cloned the VfGASA1 gene of faba bean for the first time and completed the bioinformatics analysis.Through the heterologous expression of VfGASA1 in Arabidopsis,it is found that VfGASA1 may indirectly promote the expression of GAI to inhibit FT expression and delay flowering time.The observation of other phenotypes shows that VfGASA1 is a multifunctional gene,may affect seed development and enhance skotomorphogenesis.