Cloning And Characterization Of Early Nodulation Genes In Soybean

Soybean (Glycine max), originated from China, is one of the most important food crops. It has been widely accepted by people since ancient times. One of the most important characteristics of soybean is its biological nitrogen fixation ability. The root nodules and rhizobium of soybean have symbiotic relationship, which can be used to provide nitrogen for the growth and development of soybean through the absorption of N by rhizobium. In this study, the functional verification based on expression of high content in the root nodule of thee transcription factors GmTGA4, GmbHLH93, GmWRI1,and to explore the signal transduction mechanism of symbiotic nitrogen fixation in legume rhizobia, which provide a theoretical basis for know legume nodule. The results of this paper are as follows:In order to identify the signal transduction mechanism of symbiotic nitrogen fixation GmTGA4, GmbHLH93, GmWRI1 in legume rhizobia, we use transgenic soybean rooting technique was verified on overexpression and gene silencing under the influence of the number of soybean nodules, The results showed that: in the over expression of GmTGA4, GmbHLH93, GmWRI1 gene, with the control group contrast, found a significant increase in the number of root nodules; gene silencing in GmTGA4,GmbHLH93, GmWRI1, though the comparison with the control group, significantly reduced the number of root nodules found. The above results show that GmTGA4,GmbHLH93 and GmWRI1 play an important role in the process of soybean nodulation.The effect of GmSymRK, GmNSPl and GmNODULIN27 on GmTGA4, GmbHLH93 and GmWRI1 in the process of nodulation signal transduction was verified by quantitative PCR. The results showed that overexpression of GmTGA4, GmbHLH93 and GmWRI1,respectively, led to the increase of GmSymRK expression, decrease and increase. The expression of GmNSP1 increased, decreased and increased. The expression of GmNODULIN2 7 increased,decreased and increased. Gene silencing GmTGA4,GmbHLH93, GmWRI1, respectively, led to decreased expression of GmSymRK, GmNSP1 expression decreased, GmNODULIN27 expression increased. Gene silencing GmTGA4,GmbHLH93, GmWRI1, respectively, led to decreased expression of GmSymRK, GmNSPl expression decreased, increased, increased, GmNODULIN27 expression increased. The experimental results show that: GmTGA4, GmbHLH93, GmWRI1 may be located upstream of GmSymRK, located in GmNSPl, GmNODULIN27 downstream.In order to examine promoters in transgenic soybean hairy roots, we cloned GmTGA4, GmbHLH93, GmWRI1 gene 5 'upstream regulatory region, construction of the success of the GmTGA4, GmbHLH93, GmWRI1 promoter GUS fusion gene, and through agrobacterium K599 induced transgenic hairy root, then GUS stained.The expression of GUS reporter gene driven success of the downstream gene specific expression in the nodule, which shows more than 3 transcription factors in the location within the nodule and play a important role.For the study of GmTGA4, GmbHLH93, GmWRIl ,we successfully constructed bait vector, screening of soybean nodule cDNA library by yeast two hybrid techniques were successfully screened with GmTGA4 interacting protein GmTTGl, shows that GmTGA4 may play a regulatory role in the metabolism of flavonoids within the nodule. The laboratory experiments have been carried out, and the GmbHLH93 gene was successfully screened by using the soybean saponin gene. The results showed that GmbHLH93 might play an important role in the regulation of saponin synthesis.