Study On The Physiological Response Mechanism Of Transgenic Alfalfa With GsPPCK1/GsPPCK3

Alfalfa(Medicago sativa L.), belonging to the legume Medicago spp, which is a perennial legume, is a high protein content, nutrient rich perennial, with strong resistance, wide adaptability and other characteristics, is a very important mode of crop improvement and development of alkaline.Using genetic engineering methods to cultivate resistant transgenic alfalfa, make full use of saline alkali land, has important application value and broad prosp ects for development. Previous studies found that wild soybean sources of alkali stress responsive genes GsPPCK1 /GsPPCK3 and improve its overexpression transgenic alfalfa plants resistance to alkali, but its alkali resistant physiological mechanism and molecular mechanism is still not clear.This study will on the basis of previous studies, using physiological, biochemical and molecular biological methods, of transgenic alfalfa were cutting propagation, and 200 mmol / L Na HCO3, through alkali resistance, photosynthesis and the study on the correlation between, in order to clear ppck protein enhances the alfalfa alkaline tolerance physiological mechanism, clarify ppck protein improve the molecular mechanism of alkali resistance of alfalfa. The following results have been obtained:(1) Alkali resistance to GsPPCK1/3 gene in alfalfa1) Received 1 copies of GsPPCK1 gene enhanced alkali resistant alfalfa Longmu 806 newmaterials. Transgenic line GsPPCK1-5 was able to withstand continuous 9d with 200 mmol/Lhigh alkali stress.2) Received 1 copies of GsPPCK3 gene enhanced alkali resistant alfalfa Longmu 806 newmaterials. Transgenic lines GsPPCK3-8 were able to withstand continuous 9d with 200mmol/L high alkali stress.(2) Determination of photosynthesis indexes of transgenic alfalfa Leaves under alkali stress1) Under alkali stress, transgenic alfalfa in the phosphoenolpyruvate, malic acid 、 oxaloacetic acid 、citric acid and organic acid were significantly higher than those of the control plants, that GsPPCK1/3 gene of the excess expression participates in the process of C4 metabo lism, and ultimately improve the accumulation of organic acids in the cell, effectively alleviate the damage of intracellular alkaline environment of alfalfa, thereby improving the alkali resistance of transgenic alfalfa.2) In the alkali stress, P-H+-ATPase gene and V-H+-ATPase gene were obviously up-regulated expression in transgenic lines. This indicates that the GsPPCK1/3 gene of the excess expression increased the expression level of cell membrane P-H+-ATPase and vacuolar V-H+-ATPase, by adjusting the p H of the cytoplasm and to adapt to alkali stress.(3) Overexpression of GsPPCK1/3 of transgenic alfalfa under alkali stress on promote the photosynthetic efficiency1) Under alkali stress, the photosynthetic indexes of transgenic alfalfa were obviously higher than that of wild type control, which showed that the photosynthetic efficiency of transgenic alfalfa can alleviate the damage of alkali stress on plants.2) C3 and C4 in transgenic alfalfa photosynthetic enzyme activity increased, the cascade effect of PPCK-PEPC Pro the photosynthetic enzyme activity increased, more effectively improve the transgenic alfalfa under alkali stress on Photosynthetic efficiency.Through the above results, we can conclude that genes and GsPPCK1/3 through the accumulation of organic acids, pH value regulation and improving the photosynthetic efficiency of the two together as a physiological response to alkali stress. The gene overexpression and to improve plant resistance to alkali has a positive role in promoting.