鸭梨PAL基因的反义遗传转化及表达分析
摘 要：苯丙氨酸解氨酶（phenylalanin ammonia-lyase，PAL，EC4.3.1.5）是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶，其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量，该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明：（1）采用RT-PCR技术，利用根据GenBank中西洋梨PAL基因序列设计特异性引物，扩增得到496 bp的鸭梨PAL基因片段。（2）将扩增片段反向插入载体pBI121的MCS区域，构建植物PAL基因反义表达载体pBI121-AsPAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中，并制备出农杆菌工程菌液。（3）利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化，得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中，实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低，为非转基因苗的65%～75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株，为改善鸭梨果实品质、改良品种奠定了基础。
关键词：鸭梨， 苯丙氨酸解氨酶， 反义RNA， 遗传转化， 基因表达
中图分类号：Q943.2， S772.3， S661.2
文献标识码：A
文章编号：1000-3142（2018）04-0492-09
Abstract：Phenylalanine ammonia-lyase （PAL，EC4.3.1.5） catalyzes the first step from precursors to synthesizing lignin in phenylpropanoid pathway. Lignin accumulation is correlated with stone cell differentiation and fruit quality. In order to reduce the content of PAL， the antisense PAL gene transformed into Pyrus bretschneideri was used to reduce the expression of endogenous PAL gene in P. bretschneideri. According to pear PAL gene sequences in GenBank， a pair of primers were designed， and a 496 bp partial PAL gene fragment was amplified by RT-PCR. The PAL antisense expression vector pBI121-AsPAL was constructed by reverse inserting PAL gene fragment into pBI121 MCS region. The pBI121-AsPAL was transduced into Agrobacterium EHA105 by electro transformation method. Then， pBI121-AsPAL was transformed into tissue cultured P. bretschneideri plants mediated by Agrobacterium. Twenty-three transgenic lines harboring the anti-sense PAL fragment were verified by PCR. The Real-time PCR results showed that the expression level of endogenous PAL gene in transgenic plants was lower than that of non transgenic pear，with amount of non transgenic pear 65%-75%. The results showed that with transformation mediated by Agrobacterium， the expression of endogenous PAL gene was specifically inhibited in antisense RNA transgenic P. bretschneideri seedlings. Therefore， this study provides reference for improvement of the pear fruit quality and species.
Key words：Pyrus bretschneideri， phenylalanine ammonia-lyase， antisense RNA， genetic transformation， gene expression
鴨梨（Pyrus bretschneideri）作为中国的白梨品种，味甜多汁，香气浓郁，营养丰富。梨果肉中的石细胞团严重影响了梨果实的食用品质（田路明等，2011；吕芮宏等，2011）。木质素作为石细胞的主要成分，其合成是从苯丙氨酸解氨酶（phenylalanin ammonia-lyase，PAL）催化L-苯丙氨酸解氨生成反式肉桂酸开始的（Kumar & Ellis，2001）。梨果实中的PAL首先参与木质素单体的合成，然后在过氧化物酶的作用下聚合成为木质素，进而为石细胞的形成提供原料（Vanholme et al，2010）。因此，PAL为木质素合成的第一个关键酶（Huang et al，2010），酶蛋白的合成与沉积以及酶蛋白的活性与石细胞的发育密切相关。