Study On The Metabolism Characteristics Of AsA During The Fruit Development Of Actinidia Eriantha ’Ganmi 6’ Based On Transcriptome

Actinidia eriantha is a unique germplasm resource in China,the content of Ascorbic acid(AsA)in fruit is very high.It is a valuable material to study the metabolism and regulation of AsA in plants.At present,the pathway of AsA biosynthesis in plants has been understood,but the mechanism of AsA metabolism and regulation is not clear.In this study,the content of AsA and dehydroascorbate(DHA),the activity of enzyme related to metabolism of AsA in the fruit of kiwifruit ‘Ganmi 6’ during its growth and development were determined and analyzed by high performance liquid chromatography(HPLC)and ultraviolet spectrophotometry.The transcriptome sequencing of DAF95,DAF110 and DAF125 fruits was studied.The relative expression of AsA metabolism related candidate genes was detected by real-time fluorescence quantitative PCR,and the transcriptome sequencing results were verified,with a view to discovering key candidate genes in AsA metabolic pathway of kiwifruit.The main results are as follows:(1)In this study,it was found that AsA mainly occurred in the young fruit stage(DAF20-DAF35)during the fruit growth and development of kiwifruit ‘Ganmi 6’.There was a significant positive correlation between the change trend of activity of L-galactose dehydrogenase(Gal DH),L-galactono-1,4-lactone dehydrogenase(Gal LDH),monodehydroascorbate reductase(MDHAR)and dehydroascorbate reductase(DHAR)and the change of AsA content.It was suggested that Gal DH and Gal LDH were the key enzymes in AsA biosynthesis,and MDHAR and DHAR were the key enzymes in AsA regeneration cycle.In addition,the activities of D-galacturonic acid reductase(Gal UR)and L-ascorbate oxidase(AAO)and L-ascorbate peroxidase(APX),which are involved in AsA synthesis,have no significant effect on AsA accumulation during the fruit development of ‘Ganmi 6’.(2)The results of transcriptome sequencing showed that the average output of each sample was 7.18 Gb,Q20 was 98.43%,Q30 was 95.10%,GC content was 47.73%,and the sequencing results were reliable.After splicing and assembling,98656 unigenes sequences with an average length of 932 bp,50184 unigenes have been annotated in four databases:Nr,Swissport,KOG and KEGG.The annotation rate has reached 50.87%,49476 unigenes have been annotated to Nr database,34041 unigenes have been annotated to Swissport database,28919 unigenes have been annotated to KOG database,22900 unigenes have been annotated to KEGG database.23 candidate genes related to AsA biosynthesis and degradation were screened in pathway analysis.(3)In the further verification of transcriptome sequencing results,the relative expression analysis results of 23 candidate genes were basically consistent with that of transcriptome sequencing.At the same time,the expression level of GME,GGP1,Gal DH,Gal LDH in the L-galactose pathway,Gal UR1 in the D-galacturonic acid pathway and MDHAR5,DHAR2,AAO in the AsA recycling pathway were significantly correlated with AsA content.The activities of Gal DH,Gal LDH,MDHAR and DHAR in fruits are mainly regulated at the transcription level by Gal DH,Gal LDH,MDHAR5 and DHAR2.The results showed that the high content of AsA was maintained by L-galactose pathway,D-galacturonic acid pathway and AsA regeneration cycle pathway.