Cloning And Characterization Of MYB Transcription Factor Gene In Rheum Palmatum L.

As one of the commonly used traditional Chinese medicine in China,Rhei Radix et Rhizoma is widely used in clinic,and its wild resources are scarce,at present,the application in the market is mainly its cultivated products,and the cultivation process is affected by many factors,such as temperature,light,altitude and so on.Most of the active components of traditional Chinese medicine come from the secondary metabolites of medicinal plants.The biosynthesis of active components is closely related to the quality of medicinal materials.Cell signal transduction or stress affects the synthesis of active components and quality formation by acting on the biosynthesis pathway of secondary metabolites in medicinal plants.As a large family of plant-specific transcription factors,MYB plays an important role in plant growth and development,secondary metabolism or stress adaptation.This study based on Rheum palmatum L was used as the object,and six MYB transcription factors were screened from transcription data of one year old R.palmatum L.,and bioinformatics analysis such as protein physicochemical properties,structural domains or molecular evolution was carried out.Then,using q RT-PCR method detected the expression patterns of those genes in response to hormone and abiotic stress.Finally,ascertaining whether the encoded protein has transcriptional activity in yeast,is helpful for the further study of its molecular role in the synthesis and regulation of R.palmatum L.anthraquinone active ingredients.Furthermore,it is that for the scientific connotation of quality formation and genetic improvement in Rhei Radix et Rhizoma providing theoretical guidance.The specific research contents are as follows:1.Based on the differential gene analysis of transcriptional data from different tissues of R.palmatum L.,six MYB transcription factors with complete open reading frames were screened.The sequence lengths of Rp MYB1,Rp MYB2,Rp MYB3,Rp MYB4,Rp MYB5 and Rp MYB6,were 1128 bp,1002 bp,828 bp,738 bp,882 bp,and 876 bp,respectively.The number of amino acids is 375 aa,333 aa,275 aa,245 aa,293 aa,and 291 aa,respectively.The target gene sequences were cloned and verified by RT-PCR technique,and the registration number of Gen Bank was submitted as MW269443～MW269448.Protein structure analysis showed that Rp MYB1,Rp MYB2,and Rp MYB5 belonged to the R1-MYB subfamily,in which Rp MYB1、Rp MYB2 was a transmembrane protein,and Rp MYB3,Rp MYB3,and Rp MYB6 were typical members of the R2R3-MYB subfamily.The six MYB transcription factors were all non-secretory proteins,and the prediction of subcellular localization was consistent in the nucleus.Phylogenetic analysis showed that Rp MYB1,Rp MYB2,and Rp MYB5 were clustered in the R1-MYB subfamily,Rp MYB3,Rp MYB4 was clustered in the S4 subfamily,and Rp MYB6 was classified as the S20 subfamily.2.Real-time fluorescence quantitative RT-PCR analysis showed that the expression of Rp MYB1,Rp MYB2,and Rp MYB6 was the highest in roots,while the expression of Rp MYB3 and Rp MYB4 in leaves was 4.39,14.84 times higher than that in roots,and the expression of Rp MYB5 in petioles was 2.24 times higher than that in roots.Compared with the Mock treatment group,ABA can induce the expression of Rp MYB1 and Rp MYB5 at 1 h and 24 h,which are 2.63 and 2.66 times that of CK treatment,respectively.Me JA stimulation can significantly induce the expression of Rp MYB6 at 24 h,up to 10.30 times,and at 12 h,and inhibited Rp MYB3 expression at 24 h,low to 0.08 times that of ck treatment,meanwhile,SA induced the most significant Rp MYB6 expression at 3 h,reaching 21.84 times.Compared with the abiotic stress treatment and Mock treatment groups,the response of Rp MYB1 and Rp MYB2 to high temperature stress was promoted most significantly at 24 h,which was16.88 and 5.19 times that of CK treatment,respectively.The response of Rp MYB3 to salt stress at 24 h increased slightly.1.77 times;stimulated by wound the expression of Rp MYB4 was significantly induced at 3 h to 3.91 times that of ck treatment,while the expression of Rp MYB5 was significantly inhibited to 0.14 times at 24 h;the expression of Rp MYB6 was significantly inhibited by salt and injury at 24 h low to 0.09 times,but high temperature stress significantly promotes its expression at 24 h up to 4.68 times.3.The recombinant expression vectors Rp MYB1-p GBKT7,Rp MYB2-p GBKT7,Rp MYB3-p GBKT7,Rp MYB4-p GBKT7,Rp MYB5-p GBKT7 and Rp MYB6-p GBKT7 were constructed and transformed into yeast AH109 competent cells respectively to verify the transcriptional activity of MYB transcription factors.The results showed that Rp MYB1,Rp MYB4,and Rp MYB5 had obvious transcriptional activity,while Rp MYB2,Rp MYB3,and Rp MYB6 had no or weak transcriptional activity alone,and might need to be combined with other proteins to play a transcriptional regulatory role.