Functional Analysis Of Stress Resistance Soybean Gene GmZnF1&GmPK6

The stress resistance process of the plant is regulated by many genes; these genes constitute a complicated regulatory network. Therefore, it is important to make an analysis of the genome-range transcriptional profile of plant under abiotic stress to get a more comprehensive understanding of plant stress resistance mechanisms.In our lab, we used gene chip technology to establish a gene expression profiling of soybean induced by salt. By comparing the differences of genes expression obetween wild-type soybean and salt induced one, we explored some candidate genes which responsed to the the salt stress. In this article, a GmZnF1gene and a GmPK6gene were cloned with further functional studies. The main contents and results of the dissertation including:Part1Cloning and preliminary functional analysis of GmZnFl1. The microarray data validation of GmZnF1RT-PCR analysis showed that the expression level of GmZnF1gene increased gradually under salt stress as time going.2. Structure, properties and phylogenetic analysis of GmZnF1protein The protein encoded by GmZnF1gene is a typical C2H2RING-Finger protein; it had a low level complexity domain and a DPBB-1-like domain. GmZnF1had similarity to E3ubiquitin ligase (99.0%).3. Transcriptional activation analysis of GmZnF1It has been confirmed that GmZnF1gene has transcriptional activation activity.4. Analysis of gene expression patterns of GmZnF11) Tissue-specific analysis. Gene GmZnF1is highly expressed in soybean roots, stem and flowers, and expressed in leaves and seeds at a relatively low level.2) The GmZnF1expression pattern analysis under both abiotic stress and ABA hormone treatment showed that GmZnF1significantly induced by ABA, salt stress, drought and cold stress, especially by salt and drought. 5. GmZnF1Cloning and Arabidopsis Transformation Cloned the full-length CDS of GmZnFl, constructed the plant over expression vector and then transformed it into Arabidopsis, and finally harvested the pure lines.6. Phenotypes of GmZnF1transgenic lines under salt stress1) By comparing the seed germination rates in different salt concentrations, the results indicated that the germination rates of the overexpression group was increasingly higher than that in the wild type group with the increase of the salt concentration.2) By comparing the primary root lengths of the overexpression lines and the wide type’s, it showed that the root lengths of the overexpression lines were significantly longer than the wide type ones in150mM and200mM NaCl conditions.7. Soybean transformation We transformed the soybean tissues by using the agro bacterium containing the35S::GmZnFl expressing vector, and harvested the T1generation seeds. We detected the T1plants by both PCR and Southern Blot. The results showed the300of T1plants containing6positive with a ratio of2.0%, and the positive ones are all single-gene inserted.8. Physiological index of35S::GmZnFl Under the treatments of2.0μM ABA,200mM NaCl,250mM Mannitol and4.0℃cold stress, the indexes of SOD, POD, CAT, MDA and Proline are higher than that in the control group, suggesting that the transgenic lines have resistance to those adversity, with more stable cellular structure and membrane system.Part2Cloning and preliminary functional analysis of GmPK69. The microarray data validation of GmPK6RT-PCR analysis showed that the transcription level of GmPK6increased gradually with the salt stress time.10. Structure, properties and evolutionary analysis of GmPK6protein The protein encoded by GmPK6gene was predicted to be a protein kinase, subordinating to the LAMMER protein kinase subfamily. The results showed that GmPK6had similarity to Glycine max PKC-like, Arabidopsis thaliana AFC2, Arabidopsis thaliana FUS3and tobacco PK12(～100.0%).11. Analysis of gene expression patterns of GmPK61) Tissue-specific analysis. GmPK6was highly expressed in soybean roots, stem and leaves, but expressed in the flowers and seeds at a relatively low level.2) The GmPK6expression pattern analysis under abiotic stress and hormone treatments revealed that GmPK6was significantly induced by salt, drought, cold stress and ABA.12. GmPK6cloning and Arabidopsis Transformation We cloned the full-length CDS of GmPK6, constructed the plant over expression vector, then transformed it into Arabidopsis and finally obtained the homozygous lines of over expression plants.13. Phenotypes of GmPK6transgenic lines under salt stress1) By comparing the seed germination rates under gradient salt concentrations, it suggested that the germination rates of the overexpression plants were significantly higher than that in the wild type with the increasing salt concentration.2) By comparing the primary root lengths of the overexpression lines, and the wide type, it showed that the root lengths of the overexpression lines were longer than that in the wide type in150mM and200mM NaCl treatments.14. Phenotype of35S::GmPK6transgenic lines under drought stress Water loss rates in the overexpression plants were lower than that in the wild type, indicating the transgenic lines holding more water molecular in vivo.15. Phenotypes of GmPK6transgenic lines under cold stress The35S::GmPK6transgenic lines appeared obvious more resistance of cold stress than the wild type.16. Soybean transformation We transformed the soybean tissues by using the agro bacterium containing the 35S::GmZnF1expressing vector, and harvested the T1generation seeds. We detected the T1plants by both PCR and Southern Blot. The results showed there were six positive plants out of300T1generation plants, with a positive ratio of2.0%. The positive ones are detected to be single gene inserted.17. Physiological index of35S::GmPK6Under the treatments of2.0μM ABA,200mM NaCl,250mM Mannitol and4.0℃cold stress, the indexes of SOD, POD, CAT, MDA and Proline are higher than that in the control group, suggesting that the transgenic lines have resistance to those adversity, with more stable cellular structure and membrane system.