The Transcriptional Expression Pattern And Genetic Transformation Of GmFERL And GmNEK4L Under Aluminum Stress In Soybean

Aluminum(Al) toxicity is one of the most important factors which limiting plant growth and crop yield in acid soils. Al-induced organic acids secretion from plant roots is the most important Al-resistant mechanism as so far. In the last decade, researchers have identified a series of genes related to the Al induced organic acid secretion in wheat, Arabidopsis, rice and other plants spesiess, such as the transcription factor STOP1 of C2H2 zinc finger family and citrate transporter gene in MATE family. The Al induced organic acid efflux mechanism need further clarifying. Protein kinase plays a vital role in the sensing signal and the internal signal transduction. Besides, physiology and pharmacology experiments also show that protein kinase involved in the aluminum-induced secretion of organic acids in plant roots. Therefore, function analysis of protein kinase might further reveal the Al induced organic acids secretion mechanism.Al induced citric acid secretions is an important Al resistant mechanism in soybean. Our previous the gene chip analysis showed that, a series of serine / threonine protein kinase gene transcript abundance increases under Al stress. Two genes Gm FERL and Gm NEK4 L were selected to study their role in Al resistance mechanism in soybean. Soybean Al-resistant varieties Jiyu 70 and Al- sensitive varieties Jiyu 62 were selected as plant material. Qunatitative real time PCR were used to study the transcriptional expression of Gm FERL and Gm NEK4 L under Al stress in soybean. The full length of CDS were cloned and introduced into different vector to study their, overexpressed in Arabidopsis and hairy roots to study their function. The results are as follows:1. Time course experiments showed that Gm FERL and Gm NEK4 L transcripts constitutively expressed in soybean root tip, increased with 30μmol Al Cl3 after 2h, reached the peak at 4h, declined at the following Al treatment duration. At the 2h and 4h treatment duration, the expression of two genes in the Al-resistant varieties Jiyu 70 was higher than Al-sensitive varieties Jiyu62. Compared with the control, under the treatment of Al, the transcriptional expression of Gm FERL and Gm NEK4 L were significantly increased in the apical 0-1 cm. The transcriptional expression of Gm FERL in the apical 0-1 cm and 1-2 cm was higher than the 2-3cm, but the transcriptional expression of Gm NEK4 L in the apical 0-1 cm was lower than the 1-2cm and 2-3cm.Tissue expression analysis found that Gm FERL and Gm NEK4 L was expressed in the whole plant. The transcriptional expression of Gm FERL in the pods was higher than the root tip. The transcriptional expression of Gm NEK4 L in the flowers and pods was similar with the root tip.In the specific experimental analysis found that, in addition to aluminum, Cu can also induced the transcriptional expression of Gm FERL and Gm NEK4 L in soybean root tip. But under the treatment of Cd, La and iron deficiency there was no significant effect on the expression of Gm FERL and Gm NEK4 L. Under the Na Cl stress, the expression of Gm FERL and Gm NEK4 L increased within 8 hours, decreased at 12 hours, after that showed a downward trend. Under the treatment of ABA, the expression of Gm NEK4 L gradually increased from 0 to 24 hours, but Gm FERL was no obvious response.2. With the 35 S Ca MV promoter, Gm FERL and Gm NEK4 L was constructed into the vector p ENSG-N-GFP by Gateway system, and transformed into protoplast extracted from Arabidopsis thaliana leaf. Observed by microscopic, the results of subcellular localization determined that Gm FERL and Gm NEK4 L proteins were located on the cell nucleus.3. Gm FERL and Gm NEK4 L was constructed into the plant vector p EGAD-3XHA-LUC which has a label of LUC, mediated by Agrobacterium Agl0 and dipped flowers infected Arabidopsis. Obtain the over-expressing T2 generation by screened and for the further phenotypic analysis. Constructed two genes into the vector p EGAD-3XHA-LUC, transferring soybean roots by Agrobacterium rhizogenes K599. But examined by LUC, have not got the over-expression root. The conditions should be optimized for further transformation.