Cloningin And Expression Analysis Of Color Related Genes In ’Granny Smith’ Apple After Bagging Treatment

Fruit color is an important agronomic trait, which significantly influences the marketvalue of apple, so many scientists pay attention to study the regulation mechanism of applecoloration. In the Loess Plateau region of China,‘Granny Smith’ apples developed red peelafter the removal of a bagging treatment. This could be an important experimental materialfor the research of apple coloring mechanism. Apple skin color is thought to be determinedby the interaction of anthocyanin molecules, with other compounds, such as chlorophyll andcarotenoids. In order to clarify the reason for the color formation of this cultivar, fruit color,the contents of chlorophylls, carotenoids, anthocyanins, phenolic compounds were analysedin the coloring process of ‘Granny Smith’ apples after bag removal. The expression ofanthocyanin biosynthetic and regulatory genes was also detected, and the key gene PSY, PDSand LCYB which determined the pathway carotenoid synthesis of ‘Granny Smith’ apple werecloned. The main results in this study as follow:(1) The a*values increased, L*, b*and h°values decreased and C*values dropped atfirst and rose later after bag removal with ‘Granny Smith’ apple. The surface color (red color)increased rapidly, and tended to cover the ground color; brightness of fruit surface decreasedand color saturation increased.(2) Two anthocyanins, cy3-gal and cy3-ara, were detected in the red ‘Granny Smith’apple peels by using HPLC, and the cy3-gal was chiefly responsible for the red color of‘Granny Smith’ peels. While cy3-gal and cy3-ara were barely detected in the non-baggedapples. In the meanwhile, the concentrations of chlorophylls and carotenoids in the skin of‘Granny Smith’ apples treated with bagging were significantly decreased.(3) After bag removal, the expression levels of MdF3H, MdDFR, MdANS and MdUFGTwere increasing gradually. In addition, the expressions of MdMYB1, MdBHLH33andMdTTG1increased sharply at0-4DABR. While the expression of those genes in the controlgroup basically remained stable and lower. This indicated that MdF3H, MdDFR, MdANS,MdUFGT, MdMYB1, MdBHLH33and MdTTG1were the key factors that could promote theaccumulation of anthocyanin on ‘Granny Smith’ apples.(4)The ‘Granny Smith’ skin subtracted cDNA library was constructed,282unique geneswere obtained. Among the obtained fragments,156(55.3%) ESTs were found to have homologous genes with known functions,53(18.8%) ESTs with unknown functions, and73(25.9%) ESTs with no homologous genes. Results showed that the differentially expressedgenes are involved in pigments biosynthesis, signal transduction, energy and metabolism,transcription factors and disease resistance, and so on. All of the ESTs had been submitted toGenBank, and the accession number is JK750315-JK750413and JK973899-JK9734071. Fivegenes encoding phytoene synthase (PSY), WD40repeat protein, polygalacturonase (PG),galactosidase (GAL), and ethylene receptor (ETR) were selected, and their expression wasconfirmed in ‘Granny Smith’ through Real-time PCR, and the results showed that these genesare directly or indirectly involved in ‘Granny Smith’ coloration during fruit maturation.(5)The full-length cDNA sequence of apple PSY gene was cloned, the complete cDNAsequence contains1500bp, with a complete open reading frame of1194base pairs(87~1280bp), encoding398amino acids. The plant PSY protein system evolution wasanalysis by DNAMAN5.0software, discovering that the PSY protein relationship betweenapple, strawberry, flowering peach and loquat were close. The expression levels of MdPSY indifferent apple tissues were different. Plant expression vector pCAMBIA1302-PSY wasconstructed, and introduced into the tomato plants via Agrobacterium-mediatedtransformation. The results showed that the expression levels of9carotenoid synthesis relatedgenes of tomato evidently increase or decrease in roots, stems and leaves of transgenictomato.(6)The full-length cDNA sequence of apple PDS and LCYB genes were cloned. Thecomplete cDNA sequence of MdPDS contains2005bp, with a complete open reading frameof1725base pairs(121~1845bp), encoding575amino acids. The plant PDS protein systemevolution was analysis by DNAMAN5.0software, discovering that with the PDS proteinrelationship between apple, strawberry, flowering peach and apricot protein were relativelycloser. The full-length cDNA sequence of apple LCYB gene contains1837bp, with acomplete open reading frame of1515base pairs(314~1828bp), encoding505amino acids.The LCYB protein relationship between apple, flowering peach and loquat protein wererelatively closer. The expression levels of MdPDS and MdLCYB in different apple tissueswere different.