Research On Transgenic Brassica Napus With C₄-type PEPC Gene

Higher plants can be devided into C3, C4and CAM types according to their specific carbon assimilation mechanism. In the long evolutionary process, C4plant acquired unique photosynthetic genes, which ascribe them more efficient photosynthesis compared with C3plants under the condition of high photo intensity, high temperature, and high oxygen partial pressure. Transducing with C4photosynthetic genes into C3plants to increase production has been a hot spot in major crops breeding. This study employed two Agrobacterium mediated gene transduction process, hypocotyl disseminated infection and floral-dip,in order to integrate C4-type photosynthetic PEPC gene into C3crop Brassica napus. Meanwhile key factors that influence gene transduction efficiency and the photosynthetic characteristics of transgenic plants were evaluated. The results showed that after2d preculture, the callus inducted reached maximum rate and the quality was best in preculture medium of1.5mg/L2,4-D+0.2mg/L6-BA and culture medium of4mg/L6-BA+0.lmg/L NAA, together with5mg/L AgNO3. When GA3was added, the callus tend to form white compact tissue and to be difficult to differentiate, while high concertration of NAA and6-BA induce root formation but inhibit bud differantiation. Immersed in bacterium medium of0.3-0.5OD600value for30s, with kanamycin concerntration raised from5mg/L to10mg/L, antibiotic green callus reach highest induction rate. The callus were subcultured and transferred in differentiation medium, then it has been grown into antibiotic seedling. These seedling were subsequently transferred into root-forming medium, and6possible transgenic plants were got. Molecular markers analysis showed that2of them were transgene plants.In floral-dip process, under suitable conditions, rapes bud immersed into bacterium supplemented with0.05%surfactant Silwet-77for a week to harvest maximum transgenic offspring. When Silwet-77concerntration reached0.1%, offsprings reproduction ability decreased, and no transgenic descendent harvested. The seeds of transgenic plants were collected, and selected for kanamycin resistant by soaking or leaf spray process as respect to grown up in field.At350mg/L kanamycin sprayed onto leaf for3-4d, which shows best appropiate,5positive plants were identified a.The transgenic plants aquired with the two different method of PEPC enzyme activity and net photophythetic rates assays was analyzed. The results showed that compared to CK, the PEPC activity of transgenic plants increased17folds, and net photophythetic rates enhanced15.5%, and intercellular CO2concerntration decreased35%. These result proved that maize C4-PEPC gene has been efficiently expressed in transgenic Brassica napus.