Identify The Function Of The NTHK1 Gene And Transformation With Agrobacterium-mediated In Soybean

NTHK1 is cloned from tobacco as an ethylene receptor and could be induced by damage, NaCl and PEG. It seems to participate in the signal transfer process of salt and drought stresses. Thus, NTHK1 might relate to many adverse conditions. And the function of the gene is not affirmed in the soybean. In order to facilitate its application in transgenic breeding, we must make clear the function of the gene in soybean.The results showed:1. We constructed a new Agrobacterium rhizogenes and a new Agrobacterium tumefaciens expression vectors and tested their correction with restricted endonuclease, BamHâ… and Notâ… .2. We discovered that lower bacterium concentrations(0.101,0.213)were desirable for inducing hairy roots and assuring the hairy roots numbers or higher transgenic efficiency.3. NTHK1 was proved to be integrated into the plant tissues and showed higher activity by using PCR, RT-PCR, GUS staining and paraffin section methods.4. We screened the critical hygromycin concentration for hairy roots was 10mg/L.5. After hygromycin filtration, the hairy roots were treated with different NaCl concentrations. We found that both of them decreased with the NaCl concentration increased. But the transgenic hairy roots grew better than that of control at the same NaCl concentration. Comparison of the water content showed that water content of transgenic hairy roots was higher than that of control at the same salt concentration stress, which meant that transgenic hairy roots had higher resistance or tolerance to salt. So NTHK1 gene had the salt-tolerant function. We also did the salt-resistance experiment outdoors. It showed that the transgenic hairy roots were more salt-resistant than control at low NaCl concentration, and there were no much differences between them at high NaCl concentration. We also analyzed the hairy roots activity, the content of proline, the content of MDA. It showed that hairy roots activity decreased gradually with the NaCl concentration increased, while the content of proline and the content of the MDA did the reverse. We also find the activity of the peroxidase isozyme increased with the NaCl concentration increased.6. Phenotype of hairy roots did not change with PEG filtration. But water contents of different PEG concentrations showed that water contents of transgenic hairy roots were higher than that of control in every different PEG concentration stress, which indicated that transgenic hairy roots could uptake more water than control at the same PEG concentration so as to decrease the damage of drought stress. From this we concluded that NTHK1 had the drought resistant function. According to the analysis, we concluded that the NTHK1 gene had the salt-tolerance (salt-resistance)and drought-tolerance (drought-resistance)function in soybean.In addition, we obtained fifteen transgenic soybean plants in the experiment by using the cotyledonary as the explant and Agrobacterium-mediated co-transformation system. Three of those transgenic plants were proved to be positive by glufosinate-resistant experiment and PCR, which indicated that the NTHK1 gene was possibly integrated into the genome of soybean and was successfully expressed. Thereby it established the base for obtaining the stress-resistance varieties.