【作者】 张士鸿；

【导师】 蔡永萍；

【作者基本信息】 安徽农业大学 ， 植物学， 2010， 硕士

【摘要】 砀山酥梨（Pyrus bretschneideri cv.）原产安徽省砀山县,是我国梨的主栽品种和主要出口品种之一。但是由于近年来管理不善、品种退化等原因,使得石细胞含量明显增加,肉质变粗、多渣,口感变差,严重降低了砀山酥梨的品质及其经济价值。因此,通过生物学等手段降低石细胞含量,提高砀山酥梨品质,以增强砀山酥梨市场竞争力成为亟待解决的问题。本实验以砀山酥梨果实为材料,研究砀山酥梨果实发育过程中石细胞含量、木质素含量及木质素合成关键酶苯丙氨酸解氨酶（PAL）活性变化,找到梨石细胞发育与PAL的关系。并对PAL的酶学特性进行研究,以期了解梨PAL的性质,同时克隆PAL基因,为从生理生化及分子生物学水平调控砀山酥梨石细胞发育提供理论基础,主要研究结果如下:1、通过对五种梨果实切片观察发现砀山酥梨、贡梨和丰水梨石细胞团较大,分布较密集;砀山酥梨石细胞团最大,分布最密集;库尔勒香梨石细胞团较小,分布较疏散。苹果梨,石细胞团最小,分布最疏散。2、在砀山酥梨果实整个发育过程中,石细胞含量于花后51天出现高峰,高峰值为17.83%;木质素含量于花后47天和花后63出现两个高峰,峰值分别为6.47%和7.00%;PAL活性最大值出现在花后27天,最大值为85.53∪/g·h。三者高峰期的出现在时间上表现出一定的时序性,PAL活性的高峰期最先出现,然后木质素含量出现高峰期,石细胞含量的高峰期最后出现。它们三者的变化趋势基本相同,总体上均呈先升高后下降的趋势,且三者之间均呈显著正相关。3、砀山酥梨果实PAL的最适pH为9.2;在pH9.0左右时稳定性较高;用pH4.0和pH10.0的缓冲液处理均导致酶分子结构发生变化,活性降低;PAL最适反应温度为60℃左右,较耐热;当反应时间在180min内PAL的活性与反应时间呈线性关系;PAL对L-苯丙氨酸有较高的专一性,对L-酪氨酸也表现出一定的活性;最适底物浓度为0.0004～0.0012mol/L;砀山酥梨果实PAL的Km值有两个,Km₁为0.7500mmol/L,Km₂为1.0690 mmol/L。4、克隆得到砀山酥梨果实PAL基因cDNA片段长度为585bp,推断编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90%以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。更多还原

【Abstract】 Pyrus bretschneideri cv.originates in Dangshan, Anhui Province, which is one of the pear products for main cultivating and exporting. However, due to poor management, species degradation and many other factors in recent years, the stone cell content is significantly increased in the pear. What’s more, the flesh becomes thicker, more residue and taste variation than before, which severely decline the quality of Pyrus bretschneideri cv. and economic value. At the present time, reducing the stone cell content by utilizing biotechnology to improve the quality and enhance the market competitiveness of Pyrus bretschneideri cv. become an urgent question. In this experiment, we use Pyrus bretschneideri cv. fruit as research sample. We try to analyze the fluctuation of stone cell content, lignin content and the PAL activity, a critical enzyme to produce lignin, in the process of fruit development; and dissect the relationship between fruit stone cell development and PAL activity. In addition, in order to understand the nature of PAL, the characterization of the PAL was studied; and PAL gene was cloned at the same time. All of these provided a theoretical foundation for regulating the stone cell development from the level of physiological, biochemical and molecular biology. The main research results are as follows:1. Slices observation of five types of pear fruit about the shone cell group and instensive situation of structure, the order is: Pyrus bretschneideri cv., Fung pear, Korla fragrant pear, Gong pear and apple pear. Among these pears, Pyrus bretschneideri cv. showed the largest stone cell group and the most densely distributed, Gong pear and Fung pear were secondary, Korla fragrant pear show the smaller stone cell group and distribute comparably scattered, and Apple pear show the smallest stone cell group and the most scattered distributed.2. In the whole process of Pyrus bretschneideri cv. fruit development, stone cell content peaked at the stage of DPA51, the peak value is 17.83%; lignin content appeared two peak at the stage of DPA47 and DPA63, the peak value are 6.47% and 7.00% respectively; PAL activity peaked at the stage of DPA27, the value is 85.53U/g·h. The emergences of these three peaks showed some sequential relationship. PAL activity was first to reach the peak, then lignin content, the final one was stone cell content. Their trends were basically same, generally was first increased then declined. Besides, all of them showed significantly positive correlation.3. The most suitable pH for Pyrus bretschneideri cv. fruit PAL activity was 9.2. The PAL activity was comparably stable at around pH9.0. The PAL activity depressed remarkably both at pH4.0 and pH10.0 buffer due to the conformational destruction. The most suitable reaction temperature was about 60℃.The PAL activity and reaction time present linear relationship when the reaction time within 180min. In addition, PAL has a high specificity on L-phenylalanine, and also shows some activity on L-Tyrosine. The most suitable substrate concentration of PAL is 0.0004~0.0012 mol/L. Dangshan pear fruit PAL has two Km values, Km1 is 0.7500mmol/L and Km2 is 1.0690mmol/L.4. The PAL gene was cloned from the fruit of Pyrus bretschneideri cv.by RT-PCR. The sequence of the cDNA fragment was 582 base pairs and encoded a 195 amino acid protein. And it was found to have 90% homology with those of other plants PAL genes via multiple alignments. The catalytic active sites in PAL protein of other plants were also found in the deduced PAL protein. Phylogenetic tree analysis revealed that the PAL amino acid of the Pyrus bretschneideri cv. had closer relationship with the PAL amino acid of the Pyrunus avium. than other plants.更多还原