Cloning And Functional Analysis Of Transcription Factor VvHDZ27 In Vitis Vinifera L.cv Thompson Seedless

The seedless grape is deeply loved by consumers and has an economic significance.In seedless grape,studying formative causes of seedlessness plays an important role in developing high-quality seedless grape,but the forming mechanism of seedlessness has not been revealed.Based on results of previous studies from our research group,VvHDZ27,which was differentially expressed in ovules at different developmental periods in seedless grape Thompson Seedless and seeded grape Pinot Noir,was selected as a studying object to explore biological functions on ovular development,further providing a theoretical foundation for elucidating the regulatory mechanism of ovular abortion in seedless grape.The main results were as follows:1.The relative expression of VvHDZ27 was analyzed in ovules at different developmental stages after flowering,in somatic embryos,and in different tissue of Thompson Seedless and Pinot Noir through q RT-PCR,which pointed out that VvHDZ27 had much higher expression in ovules at 20,25,30,35,40,45,50 days after flowering in Thompson Seedless than in Pinot Noir.In Thompson Seedless,transcript levels of VvHDZ27 were relatively higher in globular embryos and cotyledon embryos than in other embryos including proembryogenic masses,heart embryos and torpedo embryos.Besides,in Thompson Seedless,VvHDZ27 had much higher expressing levels in root,leaf and flower rather than stem and tendril.2.The coding sequence of VvHDZ27 had 747 base pairs and encoded 248 amino acids,which contained a homeobox domain and a leucine-zipper domain.The phylogenetic analysis manifested that VvHDZ27 belonged to HD-Zip I subfamily and was closely related to At HB7 and At HB12,as well as being similar with conserved sequence of 17 members from HD-Zip I subfamily.Transforming protoplasts of Nicotiana benthamiana certified that VvHDZ27 was a nuclear protein,and the yeast GAL4 system suggested that VvHDZ27 had transcription activity in yeast.3.VvHDZ27 was transformed into model plant “Micro-Tom” through plant genetic transformation technique and steady overexpressing lines were obtained by PCR,q RT-PCR and Western Blot.Observering flower structure in overexpressing lines of VvHDZ27(OE-VvHDZ27)and wild type(WT)pointed out that both of flower structure were consistent and complete.Also,results of paraffin section indicated that both of ovary structure were intact and normal in OE-VvHDZ27 and WT.The pollen viability was significantly lower in OE-VvHDZ27 than in WT through making use of Alexander Stain solution to detect pollen viability.Surveying inner structure of ripen fruits suggested that seeds of WT were densely and linearly arranged on placenta,but seeds in OE-VvHDZ27 were few and scattered on the placenta.A large number of abnormal seeds appeared in ripen fruits of OE-VvHDZ27 and had not complete yellow seed coat as well as being very small.Through fixed and ptransparent treatment,these abnormal seeds was divided into two categories: one type had a complete embryonic structure,and another kind didn?t have an embryonic structure but no embryo marks were found.The relative content of salicylic acid(SA)was apparently higher in fruits at 10,15,20 and 25 days after flowering in OE-VvHDZ27 than in WT through using Liquid Chromatograph Mass Spectrometer to detect contents of endogenous hormone.4.Promoters of VvEDS1,VvSAED1,Sl EDS1 and Sl SARD1 involved in biosynthesis of SA in Thompson Seedless and Micro-Tom were p VvEDS1,p VvSARD1,p Sl EDS1 and p Sl SARD1,as well as being cloned.Members of HD-Zip I subfamily recognize and bind TAATTA cis-element,TAATTA cis-element is similar to ATTAATT.Analyzing the sequence of p VvEDS1,p VvSARD1,p Sl EDS1 and p Sl SARD1 indicated that p VvEDS1,p VvSARD1 and p Sl SARD1 had three,six and six TAATTA/ ATTAATT cis-elements respectively,but Slp EDS1 didn?t have these cis-elements.The yeast one-hybrid assay,LUC assay and GUS assay demonstrated that VvHDZ27 bound p VvEDS1 and p VvSARD1 as well as promoting activity of p VvEDS1 and p VvSARD1.Nevertheless,VvHDZ27 didn?t bind p Sl EDS1 and p Sl SARD1,and could not affect activity of p Sl EDS1 and p Sl SARD1.