The Effects Of Virus-induced PSY And CYCB Gene Silencing On Carotenoid Accumulation In Eriobotrya Japonica Lindl.

In the study,peel and pulp at different developmental stages in loquat(Eriobotrya japonica Lindl.‘Dawuxing' and “Chuannong 1-5-9”)were used for experimental materials.The PSY and CYCB which were key enzyme genes in carotenoid synthesis pathway were cloned by homology cloning,bioinformatics methods were used to analyze obtained cDNA sequence and putative amino acid sequence,real-time PCR was used to study the expression of PSY and CYCB genes in different developmental stages.Virus-induced gene silencing(VIGS)was used to inhibit the gene expression and real-time PCR were used to analyze the related genes expression in carotenoid metabolic pathways after silencing,in order to investigate the function of PSY and CYCB genes in carotenoid accumulation pathway in loquat fruit.The results of the study were as follows:(1)The full-length cDNA sequences of PSY and CYCB were obtained by homology cloning,which had intact open reading frames of 1 191 bp and 1 534 bp,respectively,and encoding 396 and 496 amino acids,respectively.The sequences alignment of PSY/CYCB gene between different variety(line)showed that the sequences had high similarities,and there was only one amino acid difference.Homology analysis indicated that the deduced PSY/CYCB protein was highly homologous to other PSY/CYCB proteins respectively,which may have similar physiological and morphological characteristics.(2)Analysis of real-time PCR showed that the expression of PSY and CYCB genes both increased significantly,which was consistent with the accumulation of carotenoid after fruit colour-changed period.The result showed that PSY and CYCB genes were closely related to the accumulation of carotenoid.(3)After interfering the genes expression of PSY and CYCB respectively,the infected parts of negative control fruits were yellow,while the silencing fruits were green.TRV molecules were detected in the pulp of negative control and silencing fruits.After silencing the PSY gene,the PSY gene expression decreased by 55.6%,and the total carotenoid content decreased by 46.8%,and other related genes synergistically responsed to the downregulation of PSY gene expression except for CRTISO and LCYB genes.After silencing the CYCB gene,the CYCB gene expression decreased by 35.2%,and the total carotenoid content decreased by 44.1%,and other related genes synergistically responsed to the downregulation of CYCB gene expression except for the PDS ZDS and BCH genes.The result indicated that PSY and CYCB genes both positively regulated the carotenoid accumulation in loquat fruit,and moreover most genes in carotenoid metabolic pathways synergistically responsed to the down-regulation of PSY and CYCB genes expression.