Screening And Functional Verification Of RcMYB Related To Stem Anthocyanin Synthesis In Ricinus Communis

Ricinus communis L.is an annual or perennial dicotyledonous herb of the Euphorbiaceae family.It has high application and economic value in the fields of agriculture,industry,medicine and textiles,and is a high-quality material for renewable bioenergy.Anthocyanins are natural pigments belonging to the flavonoid family of phenolic compounds,which play an important role in the formation of plant color and the ability of plants to withstand stress.The screening of castor stem color regulatory genes and the development of new castor germplasms with high resistance to castor are of great significance for stress-resistant breeding of castor,and also provide a new perspective for stress-resistant breeding of other crops.In this study,the transcriptome sequencing technology was used to sequence the leaves of the F₂generation extreme individuals after the hybrids of red-stemmed castor?3921?and green-stemmed castor?1101?were used for sequencing.From these,two screens related to the anthocyanin synthesis of castor R2R3-MYB transcription factor gene,and cloned,bioinformatics analysis,transient expression studies and function research of these two genes.The main research results are as follows:1. Transcriptome results showed that a total of 841 differentially expressed genes were identified,of which 343 differentially expressed genes were up-regulated and 498differentially expressed genes were down-regulated.Functional annotation,GO analysis,and KEGG enrichment analysis of these differentially expressed genes revealed that the most enriched genes were biological processes,followed by molecular functions,and at least the cell group classification;a total of 96 differential genes were enriched to metabolism Among the pathways,the main enriched metabolic pathways are phenylpropane biosynthesis and phenylalanine metabolism.84 genes related to the flavonoid biosynthetic pathway were further screened,and 11 genes were differentially expressed between red stem and green stem castor.Among them,Rc PAL,Rc CHS,Rc ANS and Rc DFR genes were up-regulated in red stem castor and green stem.The differential expression in castor was not significant.2. Two anthocyanin synthesis-related MYB transcription factor genes were cloned from the leaves of red-stemmed castor,named Rc MYB77 and Rc MYB114,respectively,and their complete CDS sequences were 738bp and 594bp in length,encoding 245 and198,respectively.Amino acids with predicted molecular weights of 27414.4k Da and47498.9k Da,isoelectric points of 6.4 and 5.2,belong to the R2R3-MYB transcription factor,and have a typical MYB family conserved domain and functional domain.3. The expression of Rc MYB77 in red-stemmed castor was significantly higher than that of green-stemmed castor,and the expression of Rc MYB114 in green-stemmed castor was significantly higher than that of red-stemmed castor.Rc MYB77 and Rc MYB114 are expressed in various tissues of Castor,and the expression of Rc MYB77 in leaves is higher than that in other tissues.Rc MYB77 was positively correlated with the expression of Rc CHS,Rc ANS and Rc DFR.Rc MYB114 is negatively correlated with anthocyanin content.4. Subcellular localization results showed that Rc MYB77 and Rc MYB114 proteins were expressed in the cell membrane.The genetic transformation test of Rc MYB77 and Rc MYB114 overexpression was carried out by using Agrobacterium-mediated genetic transformation method.The genetic transformation results showed that the over-expressed Rc MYB77 transgenic castor stalk did not show a significant change in color,but the leaves were red.Overexpression Rc MYB77 and Rc MYB114 had no phenotypic changes in T₂generation Arabidopsis.Other molecular biological identifications need further study.