Cloning Of Cucumber Auxin Receptor Homologous Gene And Its Genetic Transformation

Cucumber (Cucumis sativus L.) is one of the important horticultural and economic crops, which is also a good material for plant molecular biology research for its short vegetative period and autonomous flowering without special photoperiodic induction. The whole genome sequences of cucumber had been reported in2009(Huang et al.,2009), but there’s still a lot remains unknown. We are interested and focused on auxin receptors in cucumber, which there’s no report about its cloning, expression pattern, regulatory mechanism and biological functions yet. We did experiments based on these and get some results as follows:1. The auxin receptor homologous genes of CsTIR and CsAFB had been cloned in cucumber. We constructed over-expression vectors of35S:CsTIR and35S:CsAFB and RNA interference vectors of CsTIR-RNAAi and CsAFB-RNAi.2. The effeciency of RNAi vectors had been tested by using transient transformation in tobacco leaves. Injection of both over-expression and RNAi vectors led to target gene inhibition as expected.3. An efficient regeneration system of cucumber in vitro had been established:using6-day-old cucumber cotyledon nodes as explants, a lot of adventitious buds after25days’culture on MS+0.5mg/L6-BA+2.0mg/L AgNO3medium can be achieved.4. The concentration of hygromycin for transgenic cucumber buds had been tested and set at10mg/L finally.5. The expression pattern of cucumber endogenous CsTIR and CsAFB genes had been analysed by semi-quantitative RT-PCR. In10-day-old seedling, the expression of both CsTIR and CsAFB are higher in roots and leaves than in stem. In adult cucumber plants, expression of both CsTIR and CsAFB can be detected in every part, while CsTIR gene prefers to accumulate a bit more in stem, leaf and tendril than in flowers.6. Arabidopsis with35S:CsTIR and35S:CsAFB genes had been transformed and obtained T2and T1tansgenic seedlings with hygromycin resistance, respectively.