Molecular Cloning And Expression Of Carotenogenic Genes Which Responses To Blue Light In Peach Fruit

Peach(Prunus persica L.) is an economically important fruit crop native to China with long cultivated history. Fully ripe peach fruits rich in complicated composition of secondary metabolites, such as phenolic acid, flavonol, flavan-3-ol, anthocyanin and carotenoids. Among them, carotenoids have both great contributions to fruit antioxidants and yellow color pigment. In this paper, the effects of maturity stages and postharvest treatments(blue light) on carotenoids accumulation and the expression of key biosynthesis genes(Pp FPPS, Pp GGPPS, Pp PSY, Pp PDS, Pp ZDS, Ppb LCY and Ppb HY) in developmental tissue and fruits with different peach varieties were investigated, which could provide theoretical basis for regulation of carotenoids biosynthesis and fruit coloring. Furthermore, in order to reveal the regulatory mechanism of blue light on carotenoids biosynthesis in postharvest peach fruits, the full-length c DNAs of two blue light photoreceptor genes(phototropin and cryptochrome) were isolated and the expression of these genes was investigated. The results were as follows:(1) The full length c DNA of seven carotenogenic genes(Pp FPPS, Pp GGPPS, Pp PSY, Pp PDS, Pp ZDS, Ppb LCY and Ppb HY) was obtained by using RT-PCR and RACE amplification from peach fruit. Sequencing and bioinformatics analyses indicated that two typical aspartate-rich motif can be found in Pp FPPS and Pp GGPPS, which called as the first aspartate-rich motif(FARM) and the second aspartate-rich motif(SARM). In addition, a squalene synthases domain and a phytoene synthases domain were located in the same highly conserved regions in Pp PSY protein and other plant. The deduced amino acid sequence of Pp PDS and Pp ZDS both contained functional domains, a dinucleotide-binding domain, and could be distinguished as NAD binding 8 super families. Ppb HY have two conserved domains with rich histidine content such as ‘HXXXXH’ and ‘HXXHH’, where X refers to any amino acid.(2) Tender bud exhibited significant higher levels of β-carotene, lutein, and zeaxanthin both in ‘Jinli’ and ‘Hujing’ peach varieties as compared to other organs, such as flower bud, flower, softcore, and hardcore fruits. A significantly higher level of carotenogenic genes transcripts in ‘Jinli’ fruit was found as compared to ‘Hujing’ fruit. Also, β-carotene and lutein are the prominent carotenoids in ‘Jinli’ fruit at different maturity stages. Meanwhile, the expression of carotenogenic genes in fruit mesocarp is accordant to the levels of total carotenoids, β-carotene, and lutein. After fruit enlarging period, the significantly higher level of Pp FPPS and Pp GGPPS transcripts in fruit exocarp was observed as compared to fruit flesh. Meanwhile, the expression of Pp PDS in fruit was positively correlated with the accumulation of β-carotene, lutein, β-cryptoxanthin, zeaxanthin, and total carotenoids, and the changes of β-carotene and lutein contents also positively correlated with the expression levels of Ppb HY. These results suggest that Pp FPPS, Pp GGPPS, Pp PDS, and Ppb HY are the key structural genes regulating carotenoids biosynthesis in postharvest peach fruit(3) 1-Methylcyclopropene treatment(10μL/L) significantly reduce transcripts of carotenogenic genes in postharvest peach fruit. However, the inhibitory effect could be effectively relieved by blue light treatment at 40μmol /m2 s. Except for Pp FPPS, the blue light treatment increased expression of carotenogenic genes involved in carotenoids biosynthesis, which as associated with the enhanced accumulation of carotenoids in both ‘Jinli’ and ‘Hujing’ peach fruit after 10 days of storage.(4) The full-length c DNAs of two blue light photoreceptor genes(phototropin and cryptochrome) were isolated from peach fruit by both 5’and 3’c DNA ends amplification using gene-specific primers. Sequencing and bioinformatics analyses indicated that phototropin contain two light, oxygen, and voltage(LOV) domains, which are related to PER-ARNT-SIM(PAS) domains, and are present at the N- and C-termini of phototropin. Pp CRY2 proteins contained typical N-terminal DNA photolyase domain and the PHR domain, which required to bind two chromophores, a light-harvesting cofactor(MTHF) and a catalytic cofactor(FAD). A significantly higher level of Pp PHOT1 and Pp CRY2 transcripts in blue light-treated fruit was found as compared to control fruit. In addition, the expression of Pp PHOT1 and Pp CRY2 in fruit treated with 1-MCP and then blue light was significant positive correlated with the expression of Pp GGPPS, Pp PSY, Ppb LCY, and Ppb HY. Pp PHOT1 and Pp CRY2 are the important response factors in sensing blue light, and mediating carotenoids biosynthesis in postharvest ‘Jinli’ peach fruits which treated with 1-MCP and then blue light.