| Hairy root induction system of Rehmannia glutinosa was established in this paper.To screen the elicitors that can promote content of acteoside in R.glutinosa hairy roots,different elicitors were added into the culture medium and acteoside content was determined.Key genes involved in the biosynthesis of acteoside were identificated by RNA-seq.The MYB of R.glutinosa were identified.Expression of MYB transcription factor was studied in different development period,under shading and the condition of continuous cropping,and main effect of MYB was analysed.All these studies can provide the basis for further research of the molecular mechanism of the biosynthetic pathway of acteoside.The primary results of research are as follows:1.R.glutinosa hairy roots were induced by different Agrobacterium strains and different cultivars.It is confirmed by PCR analysis that the Ri plasmid of Agrobacterium containing the rol B and rol C sequences of DNA had been integrated into hairy roots of R.glutinosa.A4 is proved to be the best inducing strain,and it has different effects on different varieties of R.glutinosa.As a result,the induction efficiency of BJ-3 and 85-5 is the highest,the QH-1 and SDZ induction efficiency is in middle,BZY induction efficiency is the lowest.2.Culture period of R.glutinosa hairy roots is determined 30 days according to the law of biomass of 85-5-A4-1 BJ-3-A4-1 in liquid medium.And WP medium is the most suitable for the growth of BJ-3-A4-1.The effects of different concentration SA,Me JA,Ag +,Put on 85-5-A4-1 growth of and acteoside content were compared.It is showed that as the best choice SA of 25 μ M not only can promote the growth of hairy roots,but also can significantly improve acteoside content.Acteoside content of5 clutivars of R.glutinosa hairy roots induced by A4 were compared,the results show that BZY content is lowest and SDZ content is the highest.QH-1 and 85-5 content are approximate,slightly lower than BZY,slightly higher than that of BJ-3.3.Using RNA-seq to R.glutinosa hairy roots were induce by 25 μM SA and the transcription group was analyzed transcriptome analysis on 0h,12 h and 24 h.Compared to SA0,SA12 samples had 2140 genes up-regulated and 1576 genes down regulated expression.Compared to SA24,SA0 has 2401 genes up-regulated and1617 genes down regulated.Compared to SA24,SA12 has 1472 genes up-regulated and 1246 genes down regulated.There were 272 genes that were differentially expressed in the 3 kinds of comparison method.Differentially expressed genes were annotated to three major functional items by GO function significant enrichment analysis: biological process,cell composition and molecular function.A large number of them were involved in the process of metabolism,binding and catalytic activity.Among the 15 most significant pathways enriched by KEGG database,differentially expressed genes all accounted for 2.24% of the total in these 3 kinds of contrast.The enzyme genes,PAL,C4 H,C3H,Ty DC/DODC,CUAO,Ty HO,ALDH were identified.They are the catalytic enzyme fragment of c DNA genes that may be involved in the biosynthesis of acteoside.The selected genes were verified by q RT-PCR,and the results showed that there were significant differences in the expression patterns of SA and other 3 kinds of genes.4.Forty unique MYB genes with complete ORF were isolated.With Batch CD(CDD)search software and SMART software for analysis,that 40 with a complete sequence of ORF are confirmed as MYB gene c DNA.The data were submitted to the Gen Bank NCBI database and registration number is KR780077-KR780116.The homology comparison was carried out on NCBI.Thirty-six MYB genes were highly consistent with the sesame MYB.Three MYB genes were highly consistent with sequence of homologous protein of Salvia miltiorrhiza.One MYB genes were highly consistent with sequence of homologous protein of Baikal Skullcap.Expression quantity of forty MYB genes were detected with RNA-seq under different conditions.As a result,seventeen Rg MYB was higher in the leaves than roots,and the rest twenty-three Rg MYB was higher in roots.After shading,twenty-eight MYB genes were up-regulated and twelve genes were down regulated in different degrees in the leaves;but twenty-one MYB genes were up-regulated and ninteen genes were down regulated in roots.Twenty-seven MYB genes were down regulated in seedling root of replanted R.glutinosa.While twenty-eight genes were up-regulated in pull period root.More than 80% genes were down regulated in early stage.5.Expression of Rg MYB7,Rg MYB15 and Rg MYB22 in four elicitors treatment were measured by q RT-PCR.Results show that,after SA treatment 3 h,9 h,12 h and24 h Rg MYB7 were up-regulated,while in the other three kinds of elicitor treatment were not up-regulated.Therefore,Rg MYB7 is a specific response to SA treatment gene. |
