鸭梨PAL基因的反义遗传转化及表达分析

Antisense genetic transformation and expression analysis of PAL gene in Yali Pear(Pyrus bretschneideri)

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Gen Bank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p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。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。

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 Gen Bank,a pair of primers were designed,and a 496 bp partial PAL gene fragment was amplified by RT-PCR. The PAL antisense expression vector p BI121-As PAL was constructed by reverse inserting PAL gene fragment into p BI121 MCS region. The p BI121-As PAL was transduced into Agrobacterium EHA105 by electro transformation method. Then,p BI121-As PAL 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.