Construction Of C₄ Phosphoenolpyruvate Carboxylase Gene From Sugarcane And Genetic Transformation Of Soybean(Glycine Max (L.) Merrill)

Three plant expression vectors were constructed by inserting the C4 phosphoenolpyruvate carboxylase gene which includes all introns; partial 5'flanking and partial 3'ending sequence with 6.8 Kb from sugarcane, into pBI121 getting pBIpepc with a one copy CaMV35S as promoter and nptll as selectable marker gene; into pCAMBIA1301 getting pLApepc with no promoter and hyg as selectable marker gene , into pCAMBIA1301 getting pCBIpepc with a two copy CaMV35S as promoter and hyg as selectable marker gene. Then, they were introduced into Agrobacterium tumefaciens strain LBA4404 via the freeze-melting. The protocol of transformation for soybean(Glycine max (L.) Merrill) cotyledonary node has been optimized. Soybean's cotyledonary node which was cultured with 1 .Omg/L BA and low temperature for a few days was transformed by Sonication-Assisted Agrobacterium tumefaciens-mediated method. The transformed cotyledonary node and shoots were screened under the selective pressure of Kan or Hyg. The transformed plants of pBIpepc from Agrobacterium tumefaciens-mediated and pollen-tube were detected by PCR and dot Southern bolting. In order to compare the expression efficiency of different types of promoters for sugarcane C4 phosphoenolpyruvate carboxylase gene in transgenic €3 plants, Three plant expression vectors were transformed into tobacco by Agrobacterium tumefaciens . The results were as follows:1.The direction of sugarcane C4 phosphoenolpyruvate carboxylase gene was identified by partial 5'flanking and 3'ending sequence through the Blast n analysis.2.Three plant expression vectors pLApepc, pBIpepc and pCBIpepc were constructed by inserted the C4 phosphoenolpyruvate carboxylase gene from sugarcane into pBI121 and pCAMBIA 1301. The selectable marker gene of pBIpepc was nptll and the selectable marker gene of pLApepc and pCBIpepc was hyg.3.The main factors of transformation mediated by Agrobacterium tumefaciens were investigated and the physiological character of explant and plant expression vector were very important fortransformation mediated by Agrobacterium tumefaciens.4.An efficient protocol for plant regeneration from soybean cotyledonary node and for soybeancotyledonary node transformation mediated by Agrobacterium tumefaciens has been established.Some factors may be important for the success:(l) cotyledonary node were induced ascompetent explants such as 6-BA and culturing in low temperature after seed germination inaerated MSB media , (2) Several transformation conditions were combined to improve theactivation of vir genes and T-DNA transfer, such as methods of incising soybean cotyledonarynode; Acetosyringone concentrations; co-culture time; infection time and acid infectioncondition and low co- culture temperature.S.Transgenic soybean plants were recovered from co-cultrue of cotyledonary node withAgrobacterium tumefaciens I pBIpepc and transformants were obtained after long Kan resistanceselection, 8 of them have been detected by PCR and dot Southern bloting, and the DNA of 2plants showed positive.6. Tobacco transformants were obtained for pLApepc; pBIpepc; pCBIpepc and transformantshave been detected by GUS.The pBIpepc transformants have been detected by PCR and dotSouthern bloting.7.The slow growth in a few transformants of soybean and tobacco has been observed .