| Soybean genetic transformation is an important means of studying gene function and breeding new soybean varieties.Agrobacterium-mediated cotyledon node transformation is the common method to transform exogenous genes into soybean.This method has several advantages,such as low copy-number,high genetic stability,low cost,and the gene silencing phenomenon is rare.But there are also many disadvantages including strong genotype dependence,low elongation rate,difficulty in screening,serious browning phenomenon and the long process.BBM(BABY BOOM)gene,a embryo genesis related gene,promotes cell proliferation and embryogenesis,and can induces the shoot regeneration during tissue culture.Researches show ectopic expression of BBM can promote the transformation efficiency in tobacco(Nicotiana tabacum L.),Chinese white poplar(Populus tomentosa Carr.)and sweet pepper(Capsicum L).Therefore,we would like to transfer the BnBBM gene to soybean to improve the explant regeneration and elongation rate in this study.In addition,several factors affecting the regeneration and effeciency of agrobacterium-mediated cotyledon node soybean transformation were optimized,including the soybean genotype,the direction of explants incision surface,the selection concentration of glufosinate ammonium,and the concentration of Vc and AgNO3 for preventing browning.The results of optimization of agrobacterium mediated cotyledonary node transformation are as follows:① We obtained one T0 soybean plant overexpressing 35 S:BnBBM in Tian Long 1 background.The one T0 plant and two T1 generation plants are all positive by PCR confirmation.Now 666 T2 seeds were harvested.Whether ectopic expression of BnBBM can improve the regeneration and transformation efficiency of soybean remains to be further tested.② We identified soybean varieties with high transient transformation effeciency by GUS staining,including Jack,Tian Long No.1 and Da Li-huang,whereas the GUS transient expression rate are all above 94%in these varieties.③ The GUS transient expression rate was higher when the explants incision surface was put up.④The results showed that 7 mg·L-1 glufosinate ammonium is the best selection concentration overall.⑤ Adding different concentrations of Vc or AgNO3 to the medium failed to reduce the browning of explants,but improved the elongation and transformation efficiency significantly.Adding Vc can improve the elongation rate from 7.9%to 30%,and adding AgNO3 can improve the transformation efficiency from 3.2%to 5.50%.WRKY transcription factors play an important role in regulating plant growth and development,and participating in plant response to abiotic and biotic stresses.Arabidopsis WRKY28 transcription factor is required for resistance to necrotroph pathogen and abiotic stress.To explore the function of a gene similar to AtWRKY28 in soybean,we cloned GmWRKY28-like(Glyma14g03280.1)and employed bioinformatics analysis,sub-cellular localization and tissue expression pattern of it.We also analyzed the expression levels of GmWRKY28-like under ABA,PEG and NaCl stress treatments.Finally,we studied the phenotypes of the Arabidopsis transformed with Gm WRKY28-like under NaCl and mannitol stress treatments.Our results showed:① The coding DNA sequence(CDS)of Gm WRKY28-like gene is 1008 bp in length,encoding 335 amino acids.② GmWRKY28 protein contains a conserved WRKY domain,and has 22 serine,1 threonine and 2 tyrosine,without any transmembrane domain or signal peptide.③ Phylogenetic analysis showed that the WRKY28 from Phaseolus vulgaris is most similiar with GmWRKY28-like when comparing with WRKY28 from other plant species.④ GmWRKY28-like protein was located in the nucleus.⑤ GmWRKY28-like expressed at low levels in roots and seeds while high levels in true leaves,flowers and shoot apical meristem.⑥ The promoter of GmWRKY28-like contains a variety of cis-elements,such as MBS,W box,ABRE,and box-W1 which are involved in biotic and abiotic stresses.⑦ The expression of GmWRKY28-like is induced by ABA,PEG and NaCl,and the GmWRKY28-like overexpressing transgenic Arabidopsis showed significantly enhanced tolerance to high concentrations of NaCl or mannitol.Therefore,we hypothesized that GmWRKY28-like may be involved in soybean response to these abiotic factors. |
