Study On Molecular And Photosynthetic Characteristics Of Transgenic Soybean Overexpressing A Maize C₄-type PEPC Gene

Soybean is one of the major source of oil and vegetable protein,and it is also an important industrial raw material source.China is a country with large population,and there is an increasing demand for soybean which depends on import recently.Therefore,soybean production is particularly important to the entire national economy.Recently,using genetic engineering to improve plant photosynthesis has become a research hotspot to improve the crop yield,but the current research progress in this direction on soybean is relatively slow.The photosynthetic efficiency in C₄ plant（such as Maize）is high,and they have the obvious growth advantages under high temperature,drought and other stress conditions.This study attempts to transform maize C₄-type PEPC gene into soybean,and to get stable genetic homozygous lines with improved photosynthetic efficiency,which would provide the theoretical basis and important material support for genetic engineering breeding of soybean plants with higher photosynthetic efficiency.In this study,C₄-type PEPC gene of maize in pCAMBIA3301-PEPC vector（containing the Bar gene as selection marker）was transferred to six soybean varieties by using the Agrobacterium-mediated soybean cotyledonary node transformation.The herbicide glufosinate was as the selection agent with the following concentrations:3 mg·L-1 at shoot induction stage,4 mg·L-1 at shoot elongation stage,and 0 mg·L-1 at the rooting stage.Total of 13 batches of 2542 explants were transformed,and finally got 68 plants with glufosinate resistance.We tested the optimal method to identify positive transgenic plants.We set different concentration of glufosinate by using Jack purple soybean variety as experimental material,The results showed that after 3 days of the testing soybean leaves with 125 mg·L-1 glufosinate,positive transgenic plants had no obvious damage,while the negative became yellow and even wilted.Therefore,we used this method later to identify positive transgenic plants.Four positive C₄-type PEPC transgenic T0 lines were identified,by using herbicide glufosinate painting,Bar protein strip and PCR amplification.Our results showed that the positive plants identified by painting leaves with the herbicide glufosinate also showed positive using Bar test strip and PCR amplification,which suggested these three methods could be used to identify the positive transgenic plants when using Bar gene as the selection marker.Only 4 out of 68 plants showed glufosinate resistance,which might due to the low selection concentration of glufosinate during tissue culture.The PCR results showed that T0-1,T0-2 and four T1 progenies of T0-1 were positive.And these were further analyzed by Southern blot,which showed that T0-1 and its T1 progenies had two copies of Bar gene,while T0-2 did not have hybridized band.The PCR assays for 30 T1 progenies of T0-2 were also negative,which suggested that T0-2 might be a false positive.The semi RT-PCR and qRT-PCR assays of maize PEPC gene using the RNA from leaves of the 4 T1 positive progenies of T0-1（T1-1-1、T1-1-2、T1-1-3 和 T1-1-5）showed that PEPC gene was expressed at different levels.Finally,we determined the photosynthetic rate of flowering stem third leaf of the 4 lines and control plants.And the photosynthetic rates of T1-1-1,T1-1-2,T1-1-3,and T1-1-5 were increased by 10%,6%,4%and 23%,respectively,while the differences between T1-1-5 and the control were extremely significant.In summary,this study transfered the maize C₄ type PEPC gene into soybean via Agrobacterium-mediated transformation.Southern blot indicated that PEPC gene had been integrated into the soybean genome,and expressed at different levels in different transgenic lines.The photosynthetic efficiencies of the maize C₄-type PEPC transgenic soybean were improved and their yield should be tested in the future.