Cloning And Expression Analysis Of Genes That Were Responsible For Anthocyanin Biosynthesis And Transportation In Red-fleshed Kiwifruit

‘Hongyang’(Actinidia chinesis cv ‘Hongyang’) is one of the most popularkiwifruit cultivars for its beautiful color in the cross section and its unique flavor. Redappearance is the most important quality for ‘Hongyang’, and is the major attractionto consumers, however, the coloration levels of the inner pericarp changes much whenthis cultivar growed at different region. In order to elucidate the molecular mechanismof instability of red color in ‘Hongyang’ production, herein,‘Hongyang’ fruit indifferent developmental phase from one commercial kiwifruit orchard located atmiddle altitudes(750meters) in Cangxi county of Sichuan and kiwifruit germplasmrepository at Wuhan Botanical Garden, Chinese Academy of Sciences were chosen forexperimental material. In addition, fruits with different flesh color named ‘Chuhong’,‘Jinnong2’,‘Lushnaxiang’,‘Xiangma6’,‘Jinkui’ from Wuhan Botanical Garden,Chinese Academy of Sciences were added. The relationship between the anthocyaninand the fruit colour was analyzed, as well as the expression patterns of anthocyaninbiosynthesis genes. The main results are listed as follows:A novel UFGT(UDP glueose-flavonoid3-O-glucosyltransferase) gene and anovel MYB(myeloblastosis) gene were isolated from Actinidia chinesis cv‘Hongyang’ by reverse transcription-polymerase chain reaction (RT-PCR) and rapidamplification of cDNA ends (RACE). Sequence analysis indicated that both of themhad a complete open reading frame (ORF), thereinto, the full-length cDNA of UFGTis1802bp, it has an open reading frame (ORF) with1533nucleotides encoding aprotein of510amino acids, and the full-length cDNA of MYB is962bp, it has an openreading frame (ORF) with666nucleotides encoding a protein of221amino acids.Homology analysis showed they had a high degree of similarity with UFGT or MYBsequences from other plant species related to the elevation of anthocyaninpigmentation, respectively, suggest that they may participate and play an importantrole in anthocyanin biosynthesis.The expression patterns of anthocyanin biosynthesis genes, such as CHS (chalcone synthase), CHI(Chalcone isomeras), F3H(flavanone3-hydroxylase),DFR(dihydroflavonol4-reductase), LDOX(Leucoanthocyanidin dioxygenase), UFGT,MYB and MRP (multidrug resistance–associated proteins) in ‘hongyang’ fruitscultivated in different regions, in different tissues of ‘hongyang’ and in different kiwifruitcutivars with different flesh color were analyzed by real-time PCR. The main resultsare showed as follows:In ‘hongyang’ fruits that growed at different region, with the exception of UFGTthat had a higher transcriptional level in fruits cultivated in Wuhan, the expression ofother genes in anthocyanin biosynthesis pathway were consistent with the degree ofanthocyanin concentration, higher expression level was found in fruits cultivated atmiddle altitudes in Cangxi county of Sichuan when lower expression level was foundin fruits cultivated in Wuhan. In different tissues of ‘hongyang’, we found that theexpression of the early genes in anthocyanin biosynthesis pathway, such as CHS, CHI,F3H, DFR and LDOX were notable in leaf but weak in flesh; the expression of thelate anthocyanin biosynthetic pathway gene UFGT was notable only in the fruit flesh,whereas in other tissues, it was weak. These results suggest that the expression ofthese genes have different tissue specificity. The expression of MYB and MRP werehigh in tissues with visible red color, such as leaf and fruit flesh, this was consistentwith the degree of anthocyanin concentration. In addition, six cultivars were chosenfor comparative analysis, the expression of UFGT was notable only in the red-fleshcultivars, displayed obvious varieties specificity; other genes in anthocyanin synthesispathway had notable expression in different cultivars, displayed less obvious varietiesspecificity, suggest that they may play multiple roles in kiwifruit, such as facilitatingthe accumulation of anthocyanin, or paticipate in other competitive branches in theanthocyanin pathway. So further study is needed in the future to elucidate the functionof these genes. The expression of MRP was notable both in red and non-red cultivars,presumably, in addition to participate in anthocyanin transportation, MRP may also doother jobs, such as participate in the trans-membrane of other secondary metabolites.