Study On Transformation Of Citrate Synthase Gene And Phosphoenol Pyruvate Carboxylase Gene In Medicago Sativa L.

With the long history and the wide cultivation in the world, alfalfa (Medicago sativa L.) is perennial legume forage, known as "the king of grass". In China, alfalfa is cultured mainly in the northern region. While the existence of the toxicant of Aluminum in the acid soil restricted the cultivation of alfalfa extension in the southern region, therefore, it is important to breed new varieties of alfalfa with high tolerance to Aluminum in the south. In recent years, the developing of molecule biotechnology and the researches on the mechanism of aluminum tolerance in plant contribute to the nurturing of aluminum-tolerance alfalfa varieties.In this study, strain No.15 of alfalfa "YUMU No.l" and its transgenic strain (P-83) overexpressed phosphoenol pyruvate carboxylase (PEPC) gene were used as materials. The citrate synthase (CS) gene was transferred into these two materials by using the agrobacterium-mediated leaf-disk method, respectively. And then the transgenic plants with CS gene, PEPC gene and CS-PEPC genes were identified by PCR and RT-PCR. Enzyme activity, content of organic acid and secretion of organic acid in the transgenic plants were tested, and their ability to tolerate aluminum was also measured.The main results obtained in this study are as follows:1 Twenty-nine plants with resistance were obtained by transgenic operation. After PCR and RT-PCR identification,6 positive transgenic plants were detected, in which C-P-2,C-P-6,C-P-11,C-P-19 were transformed with CS-PEPC two genes, and CS-4,CS-9 were transformed with CS gene. Six transgenic plants identified by RT-PCR expressed target genes at transcriptional level. In those, it could be found that the transcription level of C-P-11 and C-P-2 was higher. In the result of the PEPC gene transcription, P-83 has the great expression compared to the other transgenic plants.2 The CS activity and PEPC activity in different transgenic plants leaves were tested. The result showed that the level of the CS activity of CS-PEPC transgenic plants was higher than CS transgenic plants and wild type, and the CS-PEPC transgenic plant C-P-2 shown the highest CS activity, up to 0.0225μmolNADH/min/mg protein, about 2.6 fold of the wild type. The level of the PEPC activity of PEPC transgenic plants was higher than CS-PEPC transgenic plants and wild type, and the PEPC transgenic plant P-83 shown the highest PEPC activity, up to 0.049μmolNAD/min/mg protein, about 2 fold of the wild type, and the following was CS-PEPC transgenic plant C-P-2, up to 0.0442μmol NAD/min/mg protein.3 The content of organic acid in leaves and root tips from different transgenic plants were measured. The results showed that there was more oxalate,malate and citrate in the leaves of transgenic plants than that of the wild type plants, and the levels of them in the CS-PEPC transgenic plants were more than CS transgenic plants, and the PEPC transgenic plants were shown lower than those of CS transgenic plants. The tests on the root tips showed that there was more oxalate in the CS-PEPC transgenic plants and CS transgenic plants than PEPC transgenic plants. There had no significant on the content of malate and citrate in the root tips among the three kinds of transgenic plants:CS-PEPC transgenic plants,CS transgenic plants and PEPC transgenic plants, and either of them were shown higher significantly compared to the wild type.4 The quantities of the organic acid secreted by root system were determined, and the result showed that there was no significant difference among transgenic plants and the wild type under no Al+,however when the plant were suffered with aluminum-toxicity, secretion of oxalate was shown higher significantly than the wild type, but there was no significant difference among transgenic plants. The roots of PEPC transgenic plants secreted more malate significantly than CS transgenic plant and CS-PEPC transgenic plants. The CS-PEPC transgenic plants secreted more citrate than CS transgenic plants. There was no secretion of malate and citrate in wild plants, and no citrate in PEPC transgenic plants under suffered with aluminum-toxicity.5 The experiment on aluminum-tolerance showed that there was no significant difference on root extension among transgenic plants and the wild type under no Al+, and the root tips could not be coloured by chromeazurol S. when the plant was suffered with aluminum-toxcity, the roots extension of CS-PEPC transgenic plants longer than PEPC transgenic plants, and the shortest was the wild type. The colour of root tips of CS transgenic plants were deeper than those of PEPC transgenic plants, and the colour of CS-PEPC transgenic plants root tips was shown light. The root tips of CS-PEPC transgenic plants C-P-2 was not coloured by chromeazurol S, shown no damage with aluminum-toxcity.The conclusion of this research was that:the CS gene and PEPC gene could be integrated into the genome of alfalfa and transcript, and increase the enzyme activity, add the organic acid content in leaves and root tips, enhance organic acid secretion from the roots. The CS-PEPC transgenic plants had more aluminum-tolerance ability than PEPC transgenic plants and CS transgenic plants. The CS-PEPC transgenic plant C-P-2 had shown the best aluminum-tolerance ability.