鸭梨果胶甲酯酶基因反义表达载体的构建及转化  

The Construction of Antisense Expression Vector of Pectin Methylesterase Gene and its Transfer to Yali Pear( Pyrus bretschneideri Rehd. )

作　　者：李会宣[1] 许冬倩[1] 齐靖[1] 闫洪波[1] 李桂琴[1] 

机构地区：[1]河北经贸大学生物科学与工程学院,河北石家庄050061

出　　处：《核农学报》2014年第4期597-604,共8页Journal of Nuclear Agricultural Sciences

基　　金：河北省自然科学基金(C2010001491)

摘　　要：果胶酯酶(pectin methylesterase,PME)催化细胞壁中多聚半乳糖醛酸的脱甲酯化,可以降解植物细胞壁中的果胶多糖而与果实软化有关。本研究根据白梨果胶酯酶基因PcPME4的序列,设计一对含有特定酶切位点的特异性引物,以鸭梨(Pyrus bretschneideri Rehd.)cDNA为模板通过PCR扩增得到一段294bp的PME基因片段。将片段反向插入植物表达载体pBI121的CaMV35S启动子和NOS终止子之间,经限制性酶切分析和测序分析证明成功构建PME基因的反义表达载体pBI121-AsPME。将pBI121-AsPME电转化农杆菌EHA105,利用含pBI121-AsPME的农杆菌工程菌液侵染鸭梨外植体后于抗性培养基中诱导愈伤组织,获得的抗性愈伤组织经PCR初步检测含有反向PME基因片段,荧光定量PCR检测结果显示受侵染的愈伤组织中PME基因表达量降低,表明反向插入的PME基因片段起到了抑制内源基因表达的效果。Pectin methylesterases catalyse the demethylesterification of cell wall polygalacturonans. The degradation in cell wall polysaccharides is correlated with fruit softening. In this paper, specific primers with pairs of restriction endo- nuclease sites for Yali pear pectin methylesterase gene were designed according to the PcPME4 sequence in Pyrus com- munis subsp. Caucasica. A 294bp fragment of PME gene was amplified using Yali pear cDNA as template. The anti- sense expression vector pBI121 -AsPME was constructed by inserting the PME gene fragment into expression vector pBI121 and verified by restriction endonuclease digestion reaction. Then the pBI121 -AsPME was transferred to agrobaeterium EHA105 and positive colonies were verifiey by restriction endonuclesas digestion reaction and agrobacteri- um recovery test. The Yali pear leaf explants were infected with engineering agrobacterium transformed by pBI121 -As- PME, then cultivated on selective medium for callus induction. The resistance callus were verified by PCR to select pos- itive ones. The PME expression levels in positive callus were quantified by Real-time PCR and the results showed the level was lower in positive than in control callus, moreover, the change maintained with the time extension. The results showed that antisense PME gene fragment inhibited the expression of endogenous PME gene.

关 键 词：鸭梨 果胶酯酶 反义表达载体 转化 

分 类 号：S661.2[农业科学—果树学]