Preliminary Study On The Physiological And Molecular Mechanism Of Ascorbic Acid Synthesis Of Black Currant

This experiment was conducted at the Northeast Black Currant Germplasm Resource Repositories and the Small Berry Germplasm Resource Laboratory at the Northeast Agricultural University from 2015 to 2017.Three different varieties of black currant were used as materials to analyze the dynamic changes of related enzyme activities in the anabolic process of ascorbic acid(AsA)in leaves and fruits.They were Adelinia,Brodtrop and Heifeng.The gene expression level of the key enzyme of L-galactose-1,4-lactone dehydrogenase(GalLDH)during AsA synthesis in different stages of fruit development of black currant reveals the accumulation of AsA in black currant fruit and the results were analyzed by real-time PCR.The mechanism lays the foundation for providing scientific basis for the selection of special varieties with high AsA content.The main results as follows:(1)There were differences in AsA content in different varieties of black currant.Among the selected varieties,Adelinia was the highest,followed by Brodtrop,and Heifeng was lower.The content of T-AsA in fruits reached the highest in young fruit,and the content of T-AsA dropped sharply during the fruit’s enlargement.It decreased to the lowest at the stage of fruit ripening,and its content were 25.82 μmol/gFW,21.08 μmol/gFW and 14.61 μmol/gFW,respectively.They were 25.19%,35.25%,and 33.25% of each period of young fruit.(2)Though a variety had high content of the AsA in leaves and fruits,the contents of AsA in leaves was significantly higher than that of fruits.The content of AsA in leaves was low at the initial stage,then rose rapidly with the growth and development of the leaves,and the AsA content peaked at the stage of leaf maturation.At this time,the AsA contents of Adelinia,Brodtrop and Heifeng AsA was 98.33 μmol/gFW,68.17 μmol/gFW,and 44.94 μmol/gFW respectively.They were 6.23,6.02 and 5.09 times higher than the stage of young leaf.(3)The T-GSH levels of fruit in the three varieties have different trends.The contents of T-GSH and GSH in Adelinia showed a trend of rising first,then falling,rising.The T-GSH content in young fruit was 0.18 μmol/gFW,at the lowest level,as the fruit ingression increased,it reached a peak at the fruit’s half-turn period sharply,which were 0.64 μmol/gFW,and then declined slightly during the color change period,and the content of T-GSH rose during the fruit ripening period.The change of T-GSH content in the fruit of Brodtrop and Heifeng were similar to that in Adelinia,and it was presumed that may be due to the difference in varieties.(4)There was no significant difference in T-GSH content among the three cultivars,but there were significant differences in GSH levels among the leaves of different leaf ages.The content of T-GSH and GSH in leaves gradually increased and then decreased with the growth of the leaves,reaching the maximum at the time of leaf expansion.The total change of leaf GSSG content was not significant,it was the lowest in the leaf stage of senescence.(5)The synthesis pathways and activities of enzymes related to the regeneration pathways in fruit and leaf of three cultivars were determined by UV spectrophotometry.The results showed that different metabolic enzymes showed different trends in the growth period of fruits and leaves.The changes of GalLDH,DHAR and MHDAR activities were basically consistent with the changes of AsA content,and GalLDH,DHAR and AsA showed extremely significant positive correlation.(6)The genomic sequence of GalLDH was obtained from the transcriptome and named RnGLDH.The cDNA fragment of RnGLDH was 2293 bp in length and encoded 262 amino acids.BLAST analysis showed that the similarity between nucleotide sequence of RnGLDH gene and other known plant nucleotide sequences was more than 78%.Among them,GalLDH gene had the highest similarity with grape,the sequence identity rate was 92%,and it contained a conserved FAD domain.Subcellular localization is predicted to be located in the vacuole.(7)The gene expression levels of GalLDH,a key enzyme gene for AsA biosynthesis,were analyzed using real-time PCR.The results showed that the expression level of GalLDH gene was the highest in young fruit of Adelinia and Brodtrop,and it was the lowest in fruit ripening stage.The expression pattern of GalLDH gene was highly consistent with the change trend of AsA content during the fruit development of Adelinia and Brodtrop.The results showed that the GalLDH gene was significantly associated with the accumulation of AsA in black currant,and the difference in AsA content between varieties might be mainly regulated by GalLDH.The expression level of GalLDH gene in Heifeng fruit was slightly different from its activity.