Expression Analysis Of Auyophagy-related Gene Family And Sreening Of Interacted Proteins Of VvATG7 In Grape

The ovules of stenospermocarpy grapes fail to develop completely after fertilized,which is accompanied by programmed cell death(PCD).Autophagy is a important protein degradation pathway in eukaryotic organisms,and involved in many processes such as plant development,senescence,stress response and cell death.So far,many autophagy-related genes had been studied in various plants,but most of them focus on senescence and stress response.Research on the role of autophagy in the seed formation process have rarely reported.We identified ATG genes in grape,and analyzed their expression profiles via RT-PCR technologies in tissues and ovule development to study the role of autophagy in grape.The main results were as follows:1.We identified 33 grape ATGgenes in grape genome via bioinformatics approaches,which protein sequence length vary from 94 to 2003 amino acids.The phylogenetic analysis indicatedthat grape ATG genes are closer to Arabidopsis and tobacco than to monocotyledonous rice.Phylogenetic analysisand exon-intron structure analysis in grape demonstrated that grape ATG proteins with similar strutures tended to cluster together,indicating the Vv ATGgeneswere more conservative during grape evolution.Chromosomal localization and synteny analysis showed that the Vv ATG genes was distributed in the 18 chromosomes of grape,and only segmental duplications played a crucial role in the expansion of Vv ATG gene family.17 pairs of Vv ATG genes were located in the linear region of grape and Arabidopsis,so we can predict Vv ATG functionsbased on the known At ATG function.2.Results of sq RT-PCR show that the transcripts of each ATGin Vitis vinifera cv.Pinot Noir could be detected in all tissues(root,stem,leaf,flower,alabastrum,pericarp,tendril,ovule),indicating a universalrole of autophagy in the process of grape development.The expression patterns of Vv ATG genes between seeded(V.viniferacv.Pinot Noir)and seedless(V.vinifera cv.Thompson Seedless)varieties during the ovule development were totally different.Results of q RT-PCR show that most of Vv ATGs present higher expression level in Thompson Seedless,which may imply the autophagic activityin Thompson Seedless is relatively active,so we speculate that Vv ATGs might be associated with seedless grapes ovule abortive process.Among them,ATG7 is a like ubiquitin-activating enzyme,which is indispensable in the formation of autophagosomes.The expression level of Vv ATG7 gene in Thompson Seedlesswas significantly higher than that in Pinot Noirfrom 20 to 30 days after full-bloom,which was down-regulated in PN instead of a peak in TS.3.We predicted10 functional partners of Vv ATG7 in grape genome.Vv ATG7,Vv ATG10 and Vv ATG12 genes were cloned from Vitis vinifera cv.Pinot Noir,encoding 711,230,and 94 amino acids,respectively.Yeasttwo-hybridtestsshowedthat both Vv ATG10 and Vv ATG12 couldinteractwith Vv ATG7.