| Flesh color is important economical traits for fruit quality germplasm evaluation and improvement of Actinidia chinensis. Genes, controlling color, play an important role throughout the growing in Actinidia chinensis, especially color-turning phase during the development stage of maturity.To elucidate the mechanism of the development of kiwifruit flesh color from horizontal, in this paper, we used yellow flesh kiwifruit as material to explore the dynamic changes of pigment in the various periods of full bloom by real-time quantitative PCR technology, carotenoid synthesis-related gene expression and cloning,which can lay a good foundation for molecular breeding and germplasm of kiwifruit. The main findings are as follows:1.Taking yellow flesh kiwifruit‘Jinfeng’as material, after measuring and analying pigment content of 14 periods kiwifruit the premise, we selected eight major turn color to extract RNA.Using real-time quantitative PCR technique, 11 differential genes related to carotenoid synthesis were carried out expression analysis.besides, doing correlation analysis with the pigment and cluster analysis, the results show: Eleven genes of the related carotenoid synthesis were found to be expressed in the mesocarp, but their expression levels varied. Correlation analysis showed a significantly positive correlation existed between the carotenoid contents with gene Unigenes11266, Unigenes23885, Unigenes2673(P < 0.05) expression; the cluster analysis showed that Unigenes23885 and Unigenes11266, gene CL1511 and CL10798 were clustered together and belong to the same category, but different from other genes as they had the same expression pattern in which it was significantly different from other genes; based on all the above results Unigenes11266 and Unigenes23885 might be the key genes during the carotenoid synthesis in yellow flesh kiwifruit,and the genes CL10467, Unigene12775, CL10798, CL1511 and Unigene2673 had a closer relationship carotenoid synthesis,compared with other genes.2. By cloning technology to code Unigenes11266 encoding lycopene β-cyclase. CL10467, CL1511 in p450 gene family in yellow flesh kiwifruit ?Jinfeng?, named them AcLCYB-1, AcP450-1 and AcP450-2, the results are as follows:(1) Unigenes11266 had a 1482 bp open reading frame and encode 493 amino acids; the protein sequence had the similarity of 99% with protein Achn347121, which in draft genome of the kiwifruit(Actinidia chinensis), and there was just a difference in one amino acid sites; the amino acid sequence comprise Lcyb characteristic sequence of some plants, containing some FLYAMP sequence and there was FAD/NAD(P) binding region;Phylogenetic analysis showed that the gene has close distance with Actinidia chinensis, Actinidia deliciosa, who belong to Actinidia, and grapes and strawberries, belonging to berry, while far from the pepper, tobacco, Arabidopsis.(2) CL1511 had a 1449 bp open reading frame and encode 482 amino acids; the protein sequence had the similarity of 99% with protein Achn313551 from draft genome of the kiwifruit(Actinidia chinensis), intermediate portion 33 more amino acids, besides, there were three additional amino acids are not the same; the amino acid sequence was in line with the basic characteristics of P450 family, such as characteristic heme binding site sequence, spiral K zone, spiral I area; Phylogenetic analysis showed that the gene had close relationship with cocoa, Arabidopsis, Salvia, but was distant with grapes.(3) CL10467 had a 1458 bp open reading frame and encode 485 amino acids; the protein sequence had the similarity of 100% with protein Achn223051,which in draft genome of the kiwifruit(Actinidia chinensis), the amino acid sequence contained characteristic heme binding site sequence and spiral K zone, which was the basic characteristics of P450 family; Phylogenetic analysis showed that the gene had close distance with the potato, grape, castor, while with the cocoa, Arabidopsis, was very far. |
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