Cloning And Functional Analysis Of P5CS From Apocynum Venetum L And Populus Euphratica Oliv

The plants growing in the desert fringe, have the powerful capacity of drought resistance, salt and alkali tolerance , wind-prevention, sand-fixation, soil improvement, and the ability to manage the desert. In this study, Apocynum venetum L, Populus euphratica Oliv and Glycyeehiza uralensis Fisch which we collected near the desert of Luobupo in Xinjiang have the strong tolerance to drought stress. Under the conditions of light drought stress, the plants resist the adversity mainly by osmoregulation. Proline is one of the most widely distributed endougenous osmoregulation sources of plants. As the key enzyme in glutamate pathway,Δ1-pyrroline-5-carboxylate synthetase (P5CS) catalytic the synthesis of P5C from glutamate, and it is the rate-limiting enzyme in that process. Therefor some P5CS encoding genes was cloned by the way of homologous cloning and RACE in this study. The results are as follows:1). Improvement of the method of psammophytes RNA ectraction, the homologous fragment of Apocynum venetum L, Populus euphratica Oliv, Proline P5CS gene was obtained using homologous cloning2). The homologous fragments of Apocynum venetum L and Populus euphratica Oliv were obtained using homologous cloning.. The whole cDNA sequence of Apocynum venetum L and Populus euphratica Oliv genes lbmp5 cand hyp5c were obtained. In comparison with the sequences of previously isolated P5CS gene by BLAST search, the Apocynum venetum L P5CS gene showed 75.40% identity to that gene from Solanum lycopersicum, Actinidia deliciosa, Aegiceras corniculatum, Glycine max and Medicago truncatula. The Populus euphratica Oliv P5CS gene showed 77.63% identity to that gene from Vitis vinifera, Vigna aconitifolia, Glycine max, Solanum lycopersicum.3). According to the characteristics of P5CS genes of Apocynum venetum L and Populus euphratica Oliv, the plant expression vector p-lbmp5c and p-hyp5c were constructed using pBI121 and transformed into the tobacco, seven transgenic tobaccos of p-lbmp5c was obtained by screening of Kanamycin resistance , PCR and RT-PCR.4). 4. Equation of linear regression was acquired by drawing standard curve of proline content and using spectrophotometry to test the content of dissociate praline in plants , the praline content of positive transgenic plants was determined. Under normal growth conditions, the praline content in transgenic tobacco is 9 times of the contont in Control plants. Under conditions of adversity, the praline content of both transgenic tobacco and the control are significantly improved, but the content of transgenic tobacco is 1.5 times higher than the control.