Changes Of Cell Wall Of Grape With Soft Or Hard Texture During Fruit Development And Functional Analysis Of VvPME19 Gene In Fruit Softening

Grape is an important fruit crop,widely cultivated in China,and the planting area of table grape ranks first in the world.Grape is a kind of berry with thin skin,rich juice and high sugar content.It is easy to rot,browning and dropping during storage and transportation,which seriously reduces appearance quality and market value,and shortens storage and shelf life.The firmness of grape berry is one of the important indexes of table grape quality,which affects the storage,transportation and shelf life.Especially for the soft flesh grape,the firmness rapidly decreases after veraison,and the fruit texture becomes soft when ripening.The extremely short shelf life greatly affects the commodity and market value of grape.Therefore,it is of great significance for prolonging the shelf life and improving the quality of grapes to analyze the influencing factors in the softening process of soft and hard flesh grapes and to discover the key genes affecting the ripening and softening of grapes.In this study,hard flesh varieties ‘Dawn Seedless’ and ‘Shine Muscat’,and soft flesh varieties ‘Han Xiangmi’ and ‘Pinot Gris’ were used as materials to compare the correlation between the changes of cell wall component content,enzyme activity and fruit firmness during fruit ripening and softening.The gene structure and expression of VvPMEs gene family were analyzed,and the function of VvPME19,a key gene affecting fruit softening,was preliminarily analyzed.The main results are as follows:1.To investigate the relationship between components and microstructure of cell wall,and activities of their degradation enzymes,Vitis vinifera ‘Dawn Seedless’ with hard flesh and ‘Pinot Gris’ with soft flesh were used as material in this study.The cell wall components,microstructure as well as degradation enzyme activities were analyzed at 5 w,7 w,9 w,11 w,13 w after anthesis during grape fruit development.The results showed that the firmness of ‘Dawn Seedless’ and ‘Pinot Gris’ firstly increased and then decreased during fruit developing,and the firmness of ’Dawn Seedless’ was 3.22 times higher than that of ‘Pinot Gris’.The fruit firmness had a positive significant correlation with protopectin contents,and was negatively correlated with cellulase activity.During the ripening and softening of ‘Dawn Seedless’ fruit,the berry firmness was positively correlated to the content of propectin and hemicellulose,while the berry firmness of ‘Pinot Gris’ was positively correlated to the content of cellulose.The observation of cell wall microstructure showed that the cells were gradually uneven size and irregularly arranged with fruit development.At veraison,the cell wall of ‘Pinot Gris’ with soft flesh began to degrade,while the cell wall of ‘Dawn Seedless’ with hard flesh remained intact.2.Another 11 VvPME genes were identified by whole-genome analysis,numbered VvPME48-VvPME58.The 58 VvPME genes were divided into 5 groups.GroupⅠ-Ⅴcontained 7,15,1,18 and 17 VvPME genes,respectively.14 differentially expressed VvPME genes were isolated from the result of RNA-seq of ‘Shine Muscat’ and‘Han Xiangmi’ fruits at different development stages,and the expression of VvPME19 gene was the highest at four fruit development stages of the two varieties.At the rapid softening stage of 5 varieties with different firmness,the transcription level of VvPME19 was consistent with the change of firmness,which showed a trend of high before softening and low after ripening.3.Through agrobacterium-mediated genetic transformation of tomato,VvPME19 gene was transformed into tomato genome.PCR identification results showed that a total of 11T0 generation positive lines were obtained.q RT-PCR results showed that the expression of exogenous genes in VvPME19-1,VvPME19-2 and VvPME19-3 lines was high.q RT-PCR analysis of T1 plants showed that the transcription level of VvPME19 was higher in leaves and green fruits.Overexpression of VvPME19 significantly reduced fruit firmness and propectin content,and increased PME enzyme activity and WSP content.In green mature and veraison stage,the firmness of transgenic fruit was 11-18 N,while that of WT fruit was 20-21 N.Overexpression of VvPME19 significantly increased the PME enzyme activity of tomato fruits from green mature stage to 5 d after veraison,and the PME enzyme activity of transgenic lines was 2-4 μ mol/(min· g),while WT plants was 1-3 μ mol /(min· g).As for the content of propectin and WSP,in the green mature stage,the content of propectin in WT fruits was 0.9 mg/g,while that in transgenic plants was 0.3-0.6 mg/g.After veraison,the content of propectin in WT fruits was 0.2-0.4 mg/g,while that in transgenic plants was 0.1-0.3 mg/g.From green mature stage to 3 d after veraison,WSP content of WT fruit was 0.1-0.3 mg/g and that of transgenic fruit was 0.2-0.4 mg/g.