Expression Patterns Of VvACO Genes In Vitis Vinifera And Over-expression Of VvACO2 Gene In Arabidopsis

Berries detachment,decay and rachis browning were common problems during grape storage and transportation,which seriously affects post-harvest storage and economic benefits.The length of storage time depends on the speed of berries ripening and senescence,in which ethylene plays an important role during this process.Therefore,the decreasing of ethylene biosynthesis can delay the ripening and senescence of fruits and improve fruit quality,and then increase the value of the grape fruits.ACO is a key enzyme in the ethylene biosynthesis pathway and plays an important role in the reguLation of biosynthesis of ethylene.In order to study the possible roles of ACO genes in grape fruit growth and development,Vitis vinifera L.'Black Beet' and Vitis vinifera L.'Xiang Yue' were used as materials to determine the ethylene release of fruit and rachis during fruit growth and postharvest storage,and to analyze the spatiotemporal expression of three members of the VvACO gene family.Plant expression vector of VvAC02 was constructed,and transformed into Arabidopsis for gene function verification.The main findings are as follows:1.The ethylene release content in berries and rachis of 'black beet' and 'Xiang Yue'during postharvest storage was detected.The resuLts showed that ethylene release level was low in berries of two grape varieties.The rate of ethylene release was higher in the earlier stage of berries development,and then decreased gradually.The ethylene release rate was lower than 1.0 ?L kg-1 h-1,and showed a gradual decline trend.The amount of ethylene release of berries approached to 0 at mature.The peak of ethylene release in rachis of two cuLtivars appeared at 56 days after blooming.But there is an ethylene release peak in rachis of 'Black Beet' at the 2nd day of storage,and the content of ethylene release increased by 3 times in 'Black Beet' which was significantly higher than 'Xiang Yue'.2.Respiration rate in rachis was higher than in berries during the whole physiological development period and post-harvest storage in 'Black Beet' and 'Xiang Yue',and there was no significant difference in respiration rate of rachis and fruit in two cuLtivars.3.During the physiological development of 'Black Beet' and post-harvest storage of fruits,the quantitative real-time PCR resuLts of VvACO1 gene in berries showed that the expression level of VvACO1 gene had a significant positive correlation with the ethylene release content of berries,the correlation coefficient was 0.919.However,there was a significant negative correlation of expression level of VvACO1 gene in rachis and the content of ethylene released from the berries,and the correlation coefficient was-0.691.Correlation was not detected between other genes and ethylene release from berries and rachis.The expression level of VvACO1 gene in 'Xiang Yue'berries was significantly positively correlated with the amount of ethylene released from the fruit,and the correlation coefficient was 0.923.The expression of other genes was not associated with the release of ethylene from the berries and rachis.4.VvACO2 was overexpressed in Arabidopsis,the transgenic Arabidopsis thaliana showed significant dwarfing,slow growth throughout the growth stage with delayed flowering.In the whole development stage of transgenic Arabidopsis,the VvAC02 gene was highly expressed,but the amount of ethylene released has no significantly difference with wild type,which indicating the post-transcriptional reguLation in transgenic Arabidopsis.5.A total of 25,799 genes were obtained from transcriptome sequencing of transgenic VvACO2 Arabidopsis thaliana and wild-type Arabidopsis thaliana samples with 4 weeks of seedling age,and including 6506 differential expressed genes,of which 2344 were up-reguLated and 4162 were down-reguLated.Analysis of differential expressed genes revealed that overexpression of VvACO2 gene in Arabidopsis caused downreguLation of AtACSl,AtACS4,AtACS5,AtACS11 and AtACO1,up reguLation of AtACS2 and AtACS7.Expression of genes in signal transduction of ethylene,AtCTRl,AtSIMKK and three AtEIN3 were down-reguLated;the expression levels of AtERF1,AtERF2 and two AtEIN3 genes were up-reguLated.This indicates that the overexpression of VvAC02 gene causes a change in the transcriptional level of related genes in ethylene synthesis and signal transduction pathway in Arabidopsis,which is one of the reasons for the no significant change in ethylene release.In conclusion,this study revealed that grape VvACO1 gene is a key functional gene in fruit growth and development.Overexpression of VvACO2 gene can resuLt in dwarfing of Arabidopsis,but it has little effect on ethylene release rate.