Expression Analysis,Subcellular Localization And Transformation Of GmPLC Genes From Glycine Max

The growth and metabolism of plants are dependent on a number of factors, among which the most significant two are the genetic and environmental factors. The salinization of soil does a large influence on agricultural development. Thus the study on mechanism of tolerance for salinization of plants is of great importance.Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate(PIP2) to generate the second messengers, IP3 and DAG. IP3 urges the release of Ca2+ which plays an important role in transporting Na+,K+,Cl-. These particles are the leading factors of poisoning inside plant cells upon salt treatment.In this study the expressions of a series of Gm PLCs genes of soybean in leaves and roots upon different stress treatments as long as the investigation of sub cellular localization and experiment on prokaryotic expression have been analyzed. The botanical over-expression carrier p BASTA-Gm PLC2 has been constructed. The target genes have been agrobacterium mediated transplanted to wild Arabidopsis, which has made foundation for further investigations on principles of botanical resistance against stress controlled by Gm PLC2.The main results are as follows:1. The expression of Gm PLCs in the leaves and roots of soybean upon different stress treatments have been via real-time qualitative fluorescence PCR method analyzed. The results suggest that upon salt-alkali, salt, alkali, PEG and ABA treatments the expression amounts of Gm PLCs in soybean leaves in most cases tend to increase and then decrease. The expression amounts of Gm PLCs in soybean roots have showed the same tendency.2. By DNA recombination the GmPLC2 was constructed into the carrier pCAMBIA1302. The Gm PLC2 was transmitted via agrobacterium injection into mesophyll cells of wild tobacco. It was confirmed by observation through confocal microscopy that Gm PLC2 located on the membrane.3. The Gm PLC2 was constructed into the botanical over-expression carrier p BASTA via genetic engineering. Through floral dip method was Gm PLC2 transplanted into wild Arabidopsis and 14 transgenic plants of T1 generation were obtained.4. The separation ratio appraisal of transgenic Arabidopsis plants of T2 generation has been carried out. It was shown in 3 lines that the separation ratio was 3:1, which is in approval of Mendel genetic law. And it was confirmed that these 3 lines were single copy transgenic lines. These lines provide botanical materials for further appraisal of gene functions.5. The GmPLC2 was transplanted to the prokaryotic expression carrier pET-28a(+) and has successfully expressed in BL21(DE3) by induction. The optimal induction condition was 28 ℃ IPTG 500 m M. The induction time was 4 hours. The Western blotting hybridization analysis of protein products of Gm PLC2 was carried out, which makes a good foundationfor further appraisal of Gm PLC2 protein functions.