| Rosa rugosa Thunb has a long history of cultivation in China,and is a famous natural fragrance plant.Its flowers can extract rose essential oil,make rose tea,flower cakes,etc.,and it is widely used in fragrance,cosmetics,food and other industries.Nowadays,the rose industry has formed a complete industrial chain with considerable economic benefits:from cultivation to intensive processing and product development.As an economic plant mainly picking flowers,the branching habit of rose determines the growth and quantity of its lateral branches,and is also an important factor affecting the biological yield of flowers.Therefore,to explore the key genes that regulate the occurrence of lateral branches of rose and elucidate its internal regulation mechanism can not only enrich the related theories of the development of lateral branches of plants,but also lay a solid theoretical foundation for the future development of new multi-branch and high-yield rose varieties by means of genetic engineering.In this paper,Rosa rugosa 'Zizhi' was used as the test material,transcriptome sequencing and gene expression profiling were used to extract and screen out transcription factor genes related to the development of lateral branches of rose,and the key transcription factor RrRAX3 was cloned and its expression characteristics were analyzed.The contents of plant hormones in different branches were determined,and the relationship between the expression of RrRAX3 and the development of branches and the content of hormones was analyzed.The CRISPR/Cas9 vector and overexpression vector of RrRAX3 gene were further constructed to homologous transform the rose and verify the function of RrRAX3 in regulating the development of lateral branches of the rose.The main research results are as follows:1.The primary,secondary and tertiary branches of Rosa rugosa 'Zizhi' were used as experimental materials,59,052 expression profiles of the lateral branches of rose were established by transcriptome sequencing,and the high abundance genes indicated that lateral branch development involves active structural construction and functional regulation.1789 differentially expressed genes were screened through the differential expression analysis,enrichment analysis found that the carbohydrate metabolism and chloroplast(chlorophyll)synthesis are active in lateral branches,in particular,the plant hormone signal transduction pathways represented by auxin,gibberellin and brassinolide were significantly activated,synthesized the complex interactions between proteins,suggested that the form and development of lateral branches of rose involve complex metabolic regulation and control process.2.18 candidates transcription factors associated with roses collateral development were choosed,including 7 WRKY,5 ERF,5 bHLH,what's more RAX3,TCP18 and WIN1 each one member were needed,high-low of homology,expression abundance,up-down multiple and corresponding functional mechanism of homologous genes were comprehensive considered,RAX3 gene(UniGene 25215)was selected as the target gene for further study.3.The RrRAX3 gene of Rosa rugosa was cloned and its expression characteristics were analyzed.Meanwhile,the contents of plant hormones in the primary,secondary and tertiary branches of 'Rosa glaucca pourr' were determined.The results showed that the rose RrRAX3 gene contained the typical R2R3-myb protein domain and nuclear localization signal,and was highly homologous to the RAX3 of Rosa chinensis.The gene expression level of RrRAX3 in the primary,secondary and tertiary branches of Rosa rugosa 'Zizhi' showed a decreasing trend step by step,and there was a significant difference in the expression level of each lateral branch.The expression quantity of was positively correlated with auxin(IAA)and gibberellin(GA3),negatively correlated with cytokinin c-Zeatin and abscisic acid(ABA),but not significantly correlated with GA1.4.After obtaining the target gene,the CRISPR/Cas9 vector pCas9-RrRAX3 and the over-expression vector pCANBIA1304-RrRAX3 were successfully constructed.Then,the empty vector pCAMBIA1304 and the overexpressed vectors pCAMBIA1304-RrRAX3 and gene knockout vector pCas9-RrRAX3 were homologous transformed into Rosa rugosa'Baobai' by agrobacterium-mediated somatic embryo transformation.The transgenic somatic embryo was obtained and germinated after GUS staining verification. |
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