【作者】 张小军；

【导师】 赵政阳；

【作者基本信息】 西北农林科技大学 ， 果树学， 2013， 博士

【摘要】 苹果果皮颜色是苹果一重要的农艺性状，它直接影响着苹果的商品价值。因而有关苹果果实色泽调控机理的研究一直为诸多科研工作者所关注。在渭北黄土高原，‘澳洲青苹’苹果经套袋处理后果皮着鲜红色，这为研究苹果着色机理提供了重要试验材料。苹果果皮颜色主要是由花青素、叶绿素、类胡萝卜素和类黄酮等决定。本试验对解袋后‘澳洲青苹’果实的表皮色度、叶绿素、类胡萝卜素、花青苷和多酚类代谢物质的含量变化进行了研究，并对花青苷合成相关的主要结构基因及调节基因进行了表达模式分析；另外，克隆得到苹果类胡萝卜素合成途径中关键基因PSY、PDS和LCYB，以期探究‘澳洲青苹’果实的着色机理，为苹果果实色泽的遗传改良提供理论依据。本研究获得的主要结果如下：（1）‘澳洲青苹’苹果解袋后果皮色泽参数a*值升高，L*、b*、h°降低，C*先下降后升高；果皮表色（红色）迅速增加，覆盖底色，导致果实表面亮度降低，色泽饱和度增加。（2）利用HPLC技术对套袋处理‘澳洲青苹’红色果皮中的矢车菊素3-O-半乳糖苷和矢车菊素3-O-阿拉伯糖苷进行了测定，结果表明矢车菊素3-O-半乳糖苷对‘澳洲青苹’果皮红色起主要作用。对照果实中，几乎检测不到矢车菊素3-O-半乳糖苷和矢车菊素3-O-阿拉伯糖苷。同时，叶绿素与类胡萝卜素在套袋‘澳洲青苹’果皮中的含量却远低于对照组果实，表明套袋抑制了果皮叶绿素和类胡萝卜素的合成。（3）基因表达分析表明，解袋后‘澳洲青苹’果皮中MdF3H、MdDFR、MdANS和MdUFGT基因表达水平显著提高；与花青苷合成相关的转录因子MdMYB1、MdBHLH33和MdTTG1在解袋后0~4天表达水平也显著提高。对照组果实中这些基因的表达水平保持恒定且表达量很低。表明MdF3H、MdDFR、MdANS、MdUFGT、MdMYB1、MdBHLH33和MdTTG1等基因均参与‘澳洲青苹’果实着色的调控。（4）构建了‘澳洲青苹’着色期果皮SSH-cDNA文库，得到特异基因片段282条，其中156条（占55.3%）与数据库中已知功能基因有较高的同源性，53条（占18.8%）为功能未知基因，73条（占25.9%）比对没有同源性结果。这些ESTs的功能主要涉及色素合成、信号转导、能量和代谢、转录因子、抗病防卫等方面。这些ESTs已登录至GenBank，登录号为：JK750315-JK750413和JK973899-JK9734071。对SSH-cDNA文库中筛选到的与‘澳洲青苹’果皮着色及成熟相关的基因：八氢番茄红素合成酶（PSY）、WD40蛋白、半乳糖苷酶（GAL）、聚半乳糖醛酸酶（PG）和乙烯受体（ETR）进行表达分析，结果显示5个基因在‘澳洲青苹’果实着色过程均有表达，表明这些基因直接或间接参与了‘澳洲青苹’果实着色。（5）克隆得到苹果八氢番茄红素合成酶基因MdPSY，其cDNA序列全长1500bp，具有完整的开放阅读框（87～1280bp），共1194个碱基，编码398个氨基酸。运用DNAMAN5.0软件构建苹果PSY和其它24种植物的PSY系统进化树。结果显示，苹果PSY与草莓、碧桃、枇杷PSY蛋白同源性较高。实时定量结果显示MdPSY在苹果的不同组织中均有表达，为组成型表达。构建了pCAMBIA1302-PSY植物表达载体，运用农杆菌介导法将MdPSY基因转入‘中蔬5号’番茄中。结果显示转MdPSY基因番茄中9个类胡萝卜素合成相关基因在根、茎和叶中的相对表达量发生明显变化。（6）克隆得到苹果八氢番茄红素脱氢酶基因（MdPDS）和苹果番茄红素β-环化酶基因（MdLCYB）。MdPDS基因cDNA序列全长2005bp，具有完整的开放阅读框（121~1845bp），共1725个碱基，编码575个氨基酸。用DNAMAN5.0软件分析苹果PDS蛋白与其他28种植物PDS蛋白的系统进化树，发现苹果PDS与碧桃、杏和草莓的PDS蛋白亲缘关系比较近。MdLCYB基因序列全长1837bp，具有完整的开放阅读框（314~1828bp），共1515个碱基，编码505个氨基酸。苹果LCYB与碧桃和枇杷的LCYB蛋白亲缘关系比较近，具有较高的同源性。两个基因在苹果不同组织中均有不同程度的表达。更多还原

【Abstract】 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.更多还原