Identification And Preliminary Functional Analysis Of ZF-HD Gene Family In Woodland Strawberry

Woodland strawberry(Fragaria vesca)is the third kind of fruit tree crops which genome had been sequenced after grape and apple.Due to its small genome size(2 n=14,240 MB),short life cycle,small plant size and relatively easy cultivation and growth management,it has been used as a model system for studying strawberry instead of octoploid cultivated strawberry.The zinc finger Homeodomain(ZF-HD)proteins are plant specific transcription factors with homeodomain and zinc finger domain.The zinc finger is the sequence motif in which cysteine and/or histidine coordinate a zinc atom to form local peptide structures that are required for their specific functions and the HD is the DNA binding domain that consist of 60 amino acid.ZF-HD transcription factors play important roles during plant's growth and development.For example,they are involved in response to stress treatment,regulate flower development and regulate seeds longevity.Up to now,most of research about the ZF-HD family was done by using Arabidopsis,rice and tomato,and there are limited knowledge on ZF-HD family members in strawberry,and they may play important roles.Thus,identification of this gene family in strawberry,and analyse the genes function may provide important advancement to strawberry production and breeding.In this study,we use the woodland strawberry Yellow Wonder 5AF(YW)as the material.We isolated and identified ZF-HD gene family members,analyzed their expression patterns and tried to find out their functions.The major research results are as follows:(1)Identified 10 ZF-HD genes from woodland strawberry,most of them are intronless and there are two genes categorized as MIF subfamily.By using the website to predict the physiochemical property of them,we found that the PI of ZF-HDs range from 6.88 to 8.91 and molecular weight is range from 15687.62 Da to 42101.72 Da,the stability of the proteins is poor.(2)We used qPCR to detect the expression level of ZF-HD gene in different tissues.The result indicated that these genes are differentially expressed.Among them,the expression level of FvZF-HD7 is the highest,and the FvZF-HD10 is the lowest.The expression level of the two MIF genes FvZF-HD3 and FvZF-HD5 were undetectable,suggesting that they may be expressed in specific time or tissues.(3)We cloned 8 genes of this gene family from woodland strawberry and constructed the different expression vector.The result of transient expression of GFP-fusion vector in tobacco indicated that FvZF-HD1?FvZF-HD2? FvZF-HD4? FvZF-HD6? FvZF-HD7? FvZF-HD8 ?FvZF-HD10 proteins are all localized in nucleus,but for FvZF-HD9,GFP signal were not detected.(4)Transformed the overexpression vector into Arabidopsis and strawberry by stable transformation.In the overexpression lines of FvZF-HD10 in Arabidopsis,we find that the root is longer than the wild type.In the overexpression lines of FvZF-HD6 and FvZF-HD10,we find that the tolerance of drought is significantly improved during seeds germination stage.Thus,those genes may be involved in plant development or stress response.We used the leaf-disc method to do the stable transformation for strawberry plant and successfully induced the callus,bud and root.We are expecting to obtain stable transformation strawberry lines in the near future to further confirm roles of those genes in strawberry plant.