Cloning Of CDNA Fragment Of Cytosolic Ascorbate Peroxidase And Its Expression During The Accumulation Of Vitamin C In Rosa Roxburghii Tratt

Rosa roxburghii Tratt,a Chinese lately-domesticated fruit tree,has brought about great interest worldwide due to its extremely high content of L- ascorbic acid(AsA) in fruit.In order to clarify the physiological and molecular mechanism of AsA accumulation in R.roxburghii,R.roxburghii Tratt cv. 'Guinong 5' was used to investigate the relationships between AsA accumulation and related enzyme activities,the gene expression of cytosolic ascorbate peroxidase(cAPX),during the fruit development, the storing after collecting and the process of mechanical wounding treatment.The main results were shown as follows.During fruit development of the R.roxburghii,AsA accumulated continually flowing a trend of slow-fast-slow accumulating rate.In Guiyang,AsA accumulated with a high rate during the period from the end of June to the early of August.For the other organs,the stem has the highest content of AsA,then the flower and leaf,and the root has the lowest content of AsA.In the early stage of fruit growth,low activities of ascorbate oxidase(AAO) and ascorbate peroxidase(APX) were found. However,the enzymatic activities of AAO and APX were not detected in other organs.418 bp cDNA fragment encoding cAPX was isolated from fruit of R.roxburghii by the method of RT-PCR.Sequensing analysis shows that 87%-97%identity in amino acid sequence to that of strawberry,rosa,cucumber,peanut and pea,etc.RT-PCR showed that the expression of cAPX was significantly different in different organs.The transcription level of cAPX in fruit,leaf and flower were significantly higher than that in root,stem, respectively.During the development of fruit;the cAPX gene expressions flowed a "weak-strong-weak" trend,which was similar to the changes of APX activity.With the fruit ripening,the activity of APX could not be detected,but the accumulation of AsA reached to the highest level.During the pastharvest storage of fruit,the total content of AsA decreased slowly,and the decrease rate was slower in 4℃than in 20℃.After 64 days of storage in 4℃,the content of AsA reduced by about 27%.During the storage in 20℃,the content of reduced AsA increased in the first 2 days,and then decreased.However,the enzymatic activities of AAO and APX were not tested at two different temperatures in the fruits,and the cAPX gene was also weakly expressed.In addition,the expression of cAPX gene was stronger in 20℃than that in 4℃,which was in accordance with the decomposition rate of AsA.During the process of mechanical wounding treatment,the content of AsA flowed a "slow-fast-slow" declining trend,but the content of DHA continued to increase.During this process, the cAPX gene expression increased gradually,although there were no activities of APX,AAO detected.There were significantly negative relationships between the content of AsA and the production rate of O₂^.-,the content of MDA and H₂O₂(r=-0.9127**,-0.7472* and -0.7866*, respectively).In addition,during this process,the activities of SOD were becoming stronger,but the activities of POD and CAT were not detected.In conclusion,transitory and low activites of AAO and APX in the fruit were detected in the initial stage of development,suggesting that little AsA was degraded during the whole development. This data elucidated partly the mechamism for high accumulation of AsA in the fruit.During the whole development period,the gene expression of cAPX could be tested,and this expression mode was basically in accordance with the activity of APX.During the pastharvest storage of fruit,the cAPX gene were weakly expressed,and the expression of cAPX gene was higher in 20℃than that in 4℃,which was in accordance with the decomposition rate of AsA.During the process of mechanical wound,the gene expression mode of cAPX was on the contrary to AsA levels.