山黧豆CASase基因的克隆及RNAi载体的构建

摘要

β-腈基丙氨酸合成酶(CASase)是调控山黧豆(Lathyrus sativus)内源毒素β-ODAP合成的关键酶.本研究以山黧豆幼根RNA为模板,利用RT-PCR扩增了505 bp的山黧豆CASase基因序列;通过Gateway BP反应将扩增片段连接到入门载体构建pENTR-CASase.经测序验证后,将目的片段插入到植物表达载体构建pSGRNAi-CASase,并将其转化到农杆菌GV3101中;为进一步的遗传转化奠定了基础.%CASase is thought to be the first key enzyme in β-ODAP biosynthetic pathways of Lathyrus sativus.To investigate the function of CASase gene and breed novel lines with low β-ODAP and high sulfurcontaining amino acids,a 505 bp fragment of CASase gene was cloned from radicle RNA of L.sativus through RT-PCR.And then,an entry clone vector pENTR-CASase was constructed through Gateway BP cloning.An RNAi transformation vector pSGRNAi-CASase was constructed.Finally,the RNAi vector was transformed into Agrobacterium tumefaciens strain GV3101,which laid a foundation for obtaining transgenic plants with CASase gene silenced.