Functional Analysis Of Salt-responsive Gene GmMYB84&GmPM30and Constrction Of A Marker-free Transformation Vector

The stress tolerance of the plant is controlled by many genes, these genes constitute a complex regulatory network. Therefore, it is necessary to analysis the whole genome transcriptional profile of plant under abiotic stress, then get a more comprehensive understanding of plant stress resistance mechanisms.In our lab, we use gene chip technology to establish a gene expression profiling of soybean induced by salt. Comparing the differences of expression of genes between wild-type soybean and salt induced one, we explored some candidate genes which participate in the salt stress. We cloned a MYB gene and a LEA gene from the candidate genes, made further functional studies. Also, we build a marker-free transforming system by using double T-DNAs vector for genetic transformation. The main contents and results including:Part1PCR amplification of GmMYB84and preliminary functional analysis1. The microarray data verification of GmMYB84RT-PCR analysis showed that the expression level of GmMYB84gene increased gradually under salt stress as time went by.2. Structure, properties and phylogenetic analysis of MYB protein The protein encoded by GmMYB84gene is a typical R2R3-MYB protein, it has two MYB domains, subordinating to the20subgroup of the R2R3-MYB protein. The protein is a hydrophilic one.3. Transcriptional activation analysis of GmMYB84This gene has transcriptional activation activity.4. Analysis of gene expression patterns of GmMYB841) Tissue-specific analysis. GmMYB84is highly expressed in soybean roots and flowers, lower expressed in stems, leaves, and seeds.2) The GmMYB84expression pattern analysis under abiotic stress and hormone treatment revealed that GmMYB84significantly induced by salt stress and ABA, and not induced by drought and cold stress. 5. GmMYB84Cloning and Arabidopsis Transformation Clone the full-length CDS of GmMYB84, construct the plant expression vector and then transform into Arabidopsis, finally get the pure line overexpression plant.6. Performance of GmMYB84transgenic lines under salt stress1) Comparing the seed germination rate under different salt concentrations indicts that the germination rate of the overexpression plants gradually higher than the wild type as the salt concentration increased.2) Comparing the root length of the overexpression line and the wide type indicts that, the root length of the overexpression line are longer than the wide type under150mM and200mM NaCl conditions.3) Phenotypic comparison of the overexpression line and the wide type under the condition of increasing concentrations of NaCl. The inflorescence height of the overexpression plants are higher than the wild type.4) The high salt stress treatment results showed that the survival rates of the overexpression plants is higher than the wild type.Part2PCR amplification of GmPM30and preliminary functional analysis7. The microarray data verification of GmPM30RT-PCR analysis showed that the expression level of GmPM30gene increased gradually under salt stress as time went by.8. Structure, properties and phylogenetic analysis of LEA protein The protein encoded by GmPM30gene is a hydrophilic protein, subordinating to the group4Late Embryogenesis Abundant Proteins.9. Analysis of gene expression patterns of GmPM301) Tissue-specific analysis. GmPM30is highly expressed in soybean roots, flowers and seeds, almost no expression in the stems and leaves.2) The GmPM30expression pattern analysis under abiotic stress and hormone treatment revealed that GmPM30significantly induced by salt,drought,cold stress and ABA.10. GmPM30Cloning and Arabidopsis Transformation Clone the full-length CDS of GmPM30, construct the plant expression vector and then transform into Arabidopsis, finally get the pure line overexpression plant.11. Performance of GmPM30transgenic lines under salt stress1) Comparing the seed germination rate under different salt concentrations indicts that the germination rate of the overexpression plants gradually higher than the wild type as the salt concentration increased.2) Comparing the root length of the overexpression line and the wide type indicts that, the root length of the overexpression line are longer than the wide type under150mM and200mM NaCl conditions.3) The high salt stress treatment results showed that the survival rates of the overexpression plants is higher than the wild type.12. Performance of GmPM30transgenic lines under drought stress1) The survival rate of the overexpression plants is higher than the wild type under drought stress.2) Water loss in the overexpression plants is lower than the wild type.13. Performance of GmPM30transgenic lines under drought stress GmPM30transgenic lines did not show obvious tolerance of cold stress than the wild type.Part3Construct a marker-free transforming system14. Construct a marker-free transforming system Construct a marker-free transforming system by using double T-DNAs vector for soybean genetic transformation. We can get the marker-free transgenic plants after sexual generation separation.