Molecular Mechanism Of MYB Transcription Factor Regulating Anthocyanin Biosynthesis In Three Rehmannia Species

Anthocyanins have good antioxidant,anti-tumor and vision-protective activities,and play an important role in maintaining human health.Rehmannia glutinosa is a traditional bulk medicinal material and one of the famous "Four major huaiqing medicines".R.chingii and R.henryi are the related species of R.glutinosa,which also have a long history of folk medicine.The flowers of Rehmannia are rich in catalpol,acteoside,polysaccharide and other medicinal components,which have important development and utilization value.R.glutinosa,R.chingii and R.henryi have significant differences in flower color,which was presumed to be caused by the different types and contents of anthocyanins.To study the material basis of the anthocyanins difference in Rehmannia,as well as the key genes and molecular mechanisms regulating the anthocyanins biosynthesis.This will lay the foundation for the full development and utilization of the flower resources in Rehmannia,provide genetic resources for germplasm resources innovation and molecular breeding of R.glutinosa and also lay a theoretical foundation for studying the molecular regulation of anthocyanin accumulation.In order to investigate the role of MYB transcription factors in regulating anthocyanin biosynthesis in Rehmannia glutinosa,R.chingii and R.henryi,the total anthocyanin content in the corolla at different developmental stages of the three species was determined in this study.The qualitative and quantitative analysis of anthocyanin composition in the corolla of the three species was carried out by anthocyanin-targeted metabolomics,and the material basis of anthocyanin accumulation differences in the corolla of the three species was initially analyzed.The enzyme genes that may be involved in anthocyanin biosynthesis were mined by transcriptome sequencing techniques from R.glutinosa,R.chingii and R.henryi,respectively,and MYB transcription factors that may regulate anthocyanin biosynthesis were identified.Six MYB genes were cloned and their sequence characteristics were analyzed.Overexpression and CRISPR/Cas9 gene editing vectors were constructed to investigate the molecular function of MYB genes regulation of anthocyanin biosynthesis using Agrobacterium-mediated genetic transformation.The promoters of Nt DFR and Nt ANS genes were further cloned from tobacco,and the promoters of endogenous ANS genes were cloned from R.glutinosa,R.chingii and R.henryi,respectively.And a dual luciferase reporter vector was constructed to analyze the effect of six MYB transcription factors on the promoter expression activity.The main studies are as follows:1.The reference genes for real-time fluorescence quantitative PCR analysis of R.chingii and R.henryi have not been reported.In this study,based on the transcriptome data of corolla at five different developmental stages and leaves of R.chingii and R.henryi,six housekeeping genes with stable expression were screened as candidates in the two species.The expression abundance of candidate genes in different samples was analyzed by q RT-PCR,and the stability of the candidate reference genes was evaluated using three different algorithms,Ge Norm,Norm Finder and Best Keeper.The results showed that the candidate reference genes with higher stability in R.chingii were Rc TIP41 and Rc18 S,and in R.henryi were Rh Actin and Rh18 S.Moreover,four functional genes were screened from R.chingii and R.henryi,respectively,and the stability and accuracy of the reference genes were verified.Correlation analysis of quantitative results and transcriptome data confirmed that Rc TIP41 and Rc18 S,Rh Actin and Rh18 S were the most suitable reference genes for R.chingii and R.henry q RTPCR analysis.2.The total anthocyanin content in corolla at different developmental stages and leaves of R.glutinosa,R.chingii and R.henryi was determined and revealed that the content varied dynamically.And the total anthocyanin content in corolla at the same developmental stage was significantly defferent,with the highest total anthocyanin content in the corolla of R.glutinosa,followed by R.chingii,and the least in R.henryi.Using anthocyanin-targeted metabolomics,25,29 and 26 anthocyanin components were detected in the corollas of R.glutinosa,R.chingii and R.henryi,includeding cyanidin,pelargonidin,peonidin,delphindin and petunidin.In addition,cyanidin 3-O-rutinoside was the most abundant anthocyanin component in the corollas of the three species,while its content was highest in R.chingii(958.14 μg·g-1),followed by R.glutinosa(812.28 μg·g-1)and lowest in R.henryi(431.06 μg·g-1).Transcriptome sequencing(RNA-Seq)analysis was further performed on the corollas at five different developmental periods and leaves of R.glutinosa,R.chingii and R.henryi,and100,891,91,010 and 80,035 Unigenes were obtained by assembly,respectively.The differential genes were screened by comparing the transcriptome data of flowers and leaves,and six key enzyme genes of the anthocyanin biosynthesis pathway were identified from the transcriptomes of R.glutinosa,R.chingii and R.henryi.In addition,341,335 and315 MYB transcription factor genes were identified in the transcriptomes of R.glutinosa,R.chingii and R.henryi.Combining the accumulation pattern of anthocyanins in different developmental stages of corollas and the expression pattern of anthocyanin biosynthesis enzyme genes,six candidate MYB genes that may regulate anthocyanin biosynthesis were finally identified in R.glutinosa,R.chingii and R.henryi.3.The Rg MYB41,Rg MYB42 and Rg MYB43 of R.glutinosa,Rc MYB1 and Rc MYB3 of R.chingii,and Rh MYB1 gene of R.henryi were cloned by polymerase chain reaction(PCR),and the expression patterns of the six MYB genes were analyzed by q RT-PCR.Amino acid sequence analysis revealed that the six MYB transcription factors contained two SANT superfamily MYB-binding structural domains and belonged to the R2R3-MYB subfamily.Six overexpression vectors of MYB genes were constructed and genetic transformation into leaves of tobacco and three species of Rehmannia using Agrobacterium-mediated method.In tobacco,heterologous expression of six MYB genes was able to redden the color and significantly increase the anthocyanin content of different tissues such as callus,seedlings,leaves and floral organs,with the Rc MYB3 gene being the most functional.The q RT-PCR analysis revealed that heterologous expression of six MYB genes in tobacco could also significantly increase the expression level of anthocyanin biosynthesis-related enzyme genes.Similar to the tobacco results,overexpression of MYB genes significantly increased the total anthocyanin content and the expression levels of anthocyanin synthase genes in leaves,veins and tuberous roots(roots)of R.glutinosa,R.chingii and R.henryi.This result demonstrated that the six MYB genes are positive regulators of anthocyanin biosynthesis.4.The promoter sequences of Nt DFR、Nt ANS、Rg ANS、Rc ANS and Rh ANS genes were obtained by conventional PCR techniques or genome walking method,respectively.Predictions using promoter analysis software revealed that the five different gene promoter sequences contained MYB-binding cis-acting elements.The dual luciferase reporter vectors containing the target promoter sequences were constructed and transfected with six MYB genes in tobacco leaves,and the regulatory effects of the six MYB transcription factors on promoter activity were analyzed by fluorescence value measurements.The results showed that Rg MYB41,Rg MYB42,Rg MYB43,Rc MYB1,Rc MYB3 and Rh MYB1 were able to significantly enhance the activity of p Nt DFR and p Nt ANS promoters,and the intensity of their regulatory activities was proportional to the content of anthocyanins in different tissues of tobacco.In addition,the R.glutinosa,R.chingii and R.henryi MYB transcription factors were also able to enhance the activity of p Rg ANS,p Rc ANS and p Rh ANS promoters,and the difference in their regulatory intensities may be related to the differential accumulation of anthocyanin content in the three Rehmannia species.5.To establish a CRISPR/Cas9 gene editing system for R.chingii,the Rc PDS1 gene with a length of 8,041 bp was cloned using PCR,and its coding region was 1,743 bp in length,containing 14 introns and 15 exons.The target site was designed on the 4th exon of Rc PDS1 gene and the gene editing vector for p KSE401:35S:sg Rc PDS1 was constructed.Fifty-seven kanamycin-resistant regenerated lines were obtained by Agrobacterium-mediated genetic transformation of R.chingii leaves,of which 34(59.6%)lines had an albino phenotype.Sequencing revealed that plants with albino phenotypes had different types of mutations such as base deletions,substitutions and insertions in the gene target sites.It is shown that CRISPR/Cas9-based gene editing can be used to target the genome of R.chingii.The gene editing vector of Rc MYB3 was further constructed to study its function in regulating anthocyanin biosynthesis.Recently,an Rc MYB3 gene edited plant with significantly whiter flower and lower anthocyanin content was obtained.The results further demonstrate that the Rc MYB3 gene plays an important role in positively regulating the anthocyanin biosynthesis in R.chingii.