Molecular Cloning, Expression Of Vector Construction And Genetic Transformation Of GmZFP4Gene Of Soybean

In recent years, the environment has been changing dramatically and the extreme weather occurs frequently. The arid climate, caused by global woarming, has emerged regionally to be a negative factor which leads to the reduction of crop yield. Fortunately, plants obtained some drought resistence during the long evolutionary time. For instance, massive studies have revealed that the transcription factors paly a pivotal role in the drought response of plants. C2H2zinc finger protein is considered as a typical example of this factors. However, comapred to other plants, soybean has a higher water demand, which requires more studies to analysis its molecular mechanism evolved in drought response. In addition, the function of C2H2zinc finger protein in soybean remians elusive.We obtained the cDNA of GmZFP4, the gene of the Shanxi soybean C2H2zinc protein transcription factor, from RT-PCR. The amino acid sequence coded by GmZFP4contains a highly conserved domain QALGGH which exists in C2H2zinc finger protein. The sequence analysis shows GmZFP4transcription factor is highly conserved and has no difference in JinDa53, JinDa74and JinDa78. Furtehrmore, the sequence of cDNA of GmZFP4exact matches to the genome DNA. Thus, we assume that the drought resistance of the drought-resistant variety JinDa74is likely to related to the downstream genes regulated by GmZFP4, rather than the GmZFP4per se.The GmZEP4was cloned by T-A cloning and the clones were sequenced and analysed which was proven to be identical with the results of the previous study. Based on the T-A cloning, specific primers were designed to introduce Bam H I and Sac I restrict emzyme sites to the ends of target gene separately. Subsequently, Bam H I and Sac I Double enzyme digestion system was established to digest the target gene and expression vector pBI121. Digested target gene and vector were ligated and the35S:GmZFP4expression vector was built. Finally, the35S:GmZFP4vector was transformed into E. coli JM109and the positive strain was selected and transformed into Agrobacterium EHA105by freeze-thaw method.Arabidopsis thaliana was transformed by floral dip method and the seed of T1was collected. The seeds was disinfected with8%NaCIO and incubated in the screening culture medium added25mg/ml kanamycin and25mg/ml for2weeks. Thirty-nine resistant shoots with giant and green leaves were selected, while the others show signs of yellowing. The emergence rate of GmZFP4transgenic Arabidopsis was0.19%. After transplanting,18shoots were survived.electrophoresis showed that12samples have target bands. The true positive conversion rate GmZFP4transgenic Arabidopsis is about0.06%.JinDa53, JinDa74, JinDa75, JinDa78were planted in the greenhouse, and there the immature cotyledonary nodecotyledonary nodes as explant. We optimized principally two key factor in the immature scutellum section induction system of soybean:2,4-D and KT. The results suggested that the embryonic callus induction rate which was induced by2,4-D of20mg/L was higher than the2,4-D of40mg/L, but it had not significant difference. The two2,4-D concentration Levels could significantly affect the embryonic callus induction rate. The consentration of KT in the culture medium could significantly affect the germination of somatic embryos. A0.2mg/L was the best KT concentration for the germination of all the five genetypes in the culture medium.