农杆菌介导PSAG12-IPT基因转化辣椒的研究

Agrobacterium tumefaciens-Mediated Genetic Transformation of Hot Pepper with PSAG12-IPT

异戊烯基转移酶(Isopentenyl-Transferasas,IPT),也称作细胞分裂素合成酶,是植物中细胞分裂素合成的限速酶。IPT在转基因植株中超表达后,会使叶片中的细胞分裂素含量增加,从而延缓叶片衰老。但过高对植物的生长和育性都是有害的。如果将衰老特异性启动子(PSAG)与IPT基因融合,在它的驱动下表达,只有在叶片衰老时才表达合成分裂素,既能延缓衰老又不影响植物的生长发育。以p CAMBIA1301-PMI表达载体为基础载体,用衰老特异性启动子驱动目的基因IPT,用辣椒Flamingobill外植体作受体,采用农杆菌介导的方法转化辣椒,并利用甘露糖筛选体系对辣椒转化体进行筛选,对转基因植株进行分子生物学检测和抗衰老检测。结果表明,共获得82株抗性植株,PCR检测的阳性率约为50%。而在对T1代的PCR检测表明该基因能稳定遗传给下一代。这些经转化的植株具有叶片衰老延缓及植株生命周期延长等现象,花的衰老也有所延缓。T1的株高和侧芽萌发与对照相比无明显差异。

Isopentenyl transferase （IPT）, also known as cytokinen synthetase, is a key enzyme during the synthesis of cytokinin. Over-expression of the IPT gene in transgenic plants leads to higher foliar cytokinin concentrations to delay leaf senescence. However, the high cytokinin levels are largely detrimental to growth and fertility of plants. A highly senescence-specific promoter （PSAG12） from an Arabidopsis gene SAG12 encoding a cysteine protease is fused to the IPT gene from the Ti plasmid of A. tumefaciens. The fused PSAG12-IUF gene is activated only at the onset of senescence in the lower mature leaves. This results in cytokinin biosynthesis in the leaves, which inhibits leaf senescence and consequently represses further expression of the promoter, thus preventing cytokinin over-production. In this study, the pCAMBIA1301-PMI was used as basic vector. The flamingo-bill explants （seedling with only one cotyledon, stem and root, without meristem ） were used as receptor and PMI （phosphomannose isomerase ）gene was used as selective gene to avoid the ecological risk. The transfer was mediated by Agrobaeterium tumefaciens. Eighty-two transgenic plants were obtained. PCR confirmed that positive rate was 50% in To plants. These transgenic plants delayed the leaf and flower senescence and prolonged the whole life of these plants. No difference was observed in height and lateral bud between T1 plant and the control.