| Ginseng is recognized as a valuable Chinese herbal medicine in the world.Its seed germination rate is low,its growth period is long,it is susceptible to disease,and breeding work is progressing slowly.In the process of ginseng planting,there are often multiple stem morphological parameters.Compared with single stem ginseng,the photosynthetic area is increased and the yield is increased.In the ginseng cultivation and breeding work,the multi-stem morphology is more in line with the breeding target.According to previous studies,it is speculated that hormones have an important influence on the morphology of the ginseng.In this study,the important transcription factor BZR1 in the brassinosteroid signal transduction pathway was screened by single-and multi-stem ginseng transcriptome data analysis.The ginseng BZR1 gene was cloned by RT-PCR,and the ginseng callus and ginseng plants were used for gene transformation.In the experiment,qRT-PCR method was used to explore the expression of BZR1 gene under the salt stress condition of ginseng callus,and the expression characteristics of the gene were analyzed.The foreign gene transformation system based on ginseng callus was established to further study ginseng single and double.Provide a reference for stem morphogenesis.The main findings are as follows:1.Optimum screening of induced ginseng callus culture medium by orthogonal design method.The most suitable explants for ginseng callus induction are ginseng embryos.The most suitable induction medium is MS+6-BA(0.8 mg·L-1)+2,4-D(2.0mg·L-1)+IBA(0.1 mg·L-1)+KT(0.1 mg·L-1)+agar 6.5 g·L-1+sucrose 30 g·L-1,2,4-D and 6-BA is an essential hormone induced by ginseng callus.The most suitable medium for bud differentiation of ginseng callus is:MS+6-BA(1.0 mg·L-1)+2,4-D(0.2mg·L-1)+IAA(1.5 mg·L-1)+agar 6.5 g·L-1+sucrose 30 g L-1;the most suitable medium for adventitious root differentiation of ginseng callus is:MS+6-BA(0.5 mg·L-1)+NAA(1.0 mg·L-1)+agar 6.5 g·L-1+sucrose 25 g·L-1;and liquid culture of adventitious roots can accelerate the growth rate of adventitious roots.The liquid medium for adventitious root growth is:MS+6-BA(0.5 mg·L-1)+NAA(1.0 mg·L-1)+sucrose 25 g·L-1.2.The Brassinone signal transduction factor BZR1 gene was screened by single and multi-stem ginseng transcriptome data analysis.The differential expression of BZR1gene in single and double stem morphology was studied by real-time fluorescent quantitative PCR.Transcriptome data.3.Primers were designed based on the BZR1 gene sequence provided in the transcriptome data.A 966 bp ginseng BZR1 gene was cloned by RT-PCR and named as PgBZR1（GenBank accession number:）,and subcloned.Sequence analysis,bioinformatics analysis and prokaryotic expression analysis revealed that the gene encodes 321 amino acids and has the highest homology with the carrot BZR1 gene.The induced protein is consistent with the predicted molecular mass,and the size is34.5 kDa.4.Construction of PRI201-PgBZR1 plant expression vector,transformation of LBA4404 Agrobacterium by electroporation,identification of positive clones.5.Through the sensitivity screening test of antibiotics,the screening of 300mg·L-1Kan suitable for positive callus was obtained.The 50 mg·L-1 hygromycin was the most suitable antibacterial antibiotic for co-culture of ginseng callus.6.The ginseng plant transformation system was established by using the dip flower method,and the transformation system of ginseng callus was established by Agrobacterium tumefaciens.7.The expression of BZR1 gene in ginseng was detected under salt stress condition.The overall trend of BZR1 gene was down-regulated at 6h,and the expression level was increased after 12th.The expression of BZR1 gene was highest at 3h under 200mmol·L-1NaCl.It was preliminarily confirmed that PgBZR1 gene could enhance salt tolerance of ginseng callus under salt stress. |
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