Excavation And Application Research Of Genes Related To Prickly Formation In Rosa Rugosa

Rosa rugosa Thunb.is one of the traditional famous flowers in China,and is also a natural perfume plant widely used in the world.Its flowers can extract rose oil and make rose tea,which has high economic and health value.However,there are many inconveniences in the field cultivation management and flower picking process because of the multi-prickly stems of roses.Therefore,if we can cultivate less prickly or non-prickly varieties of roses,it will greatly improve the efficiency of field management and picking of roses,and also improve the economic benefits of roses.In this paper,the fZizhi Rose' with specific prickly traits was used as a test material.The differentially expressed genes in the first,second and third collateral branches of Rosa purpurea were identified by transcriptome sequencing.Screening for candidate genes that regulate the formation of rose prickles.The RrCPC gene associated with rose prick formation was cloned and homologously transformed into a rose to verify its function.The aim is to explore the key genes regulating the formation and development of rose prick,and lay the foundation for further understanding the molecular mechanism of the formation and regulation of rose thorns.lt will also lay the foundation for the reduction of the formation of rose prickles by genetic engineering means,and the cultivation of new germplasm with less thorns or thornless roses.The main findings are as follows:1.The transcriptome was sequenced using the skin tissue of the first,second and third side branches of'Zizhi Rose1.After removing low-quality reads,more than 98.22%of high-quality clean reads were obtained from the first,second,and third-stage side branch samples.The obtained sequencing data was assembled and spliced,and 87,071 Unigenes were obtained after redundancy.Differential protein detection and screening were performed using the DEseq2 method,and DEG detection was performed.It was found that the three-stage lateral branching showed more differential genes.Among them,the first and third collaterals have the most differential genes in the alignment,which is 3365.The difference in the second and third collaterals was the least,with 923 differences.The number of up-regulated genes in the first and second side branches was 324,and the down-regulated genes were 1287.In the first and third collaterals,the up-regulated genes were 1,660,and the down-regulated genes were 1,705.In the second and third collaterals,the up-regulated genes were 611 and the down-regulated genes were 312.Using the functional annotation,seven genes related to the function of rose prick formation were screened from the 30 genes with the largest difference.Furthermore,the RrCPC gene associated with prick formation was cloned homologously from 'Zizhi Rose'.2.The temporal and spatial expression patterms of the eight genes mined were studied by real-time PCR.The results showed that the expression of RrCPC gene in the first,second and third lateral branches of rose increased gradually during the development of the lateral branches of rose.The expression level of MYB5 gene showed a decreasing trend from grade to grade.The CKX1 gene has the lowest expression level in the primary side branch and the expression level in the seeondary and tertiary side branches is equivalent.The CKX6 gene and bHLH130 gene showed a gradual decline.The expression levels of PHYB gene and WER gene are comparable in primary and secondary collaterals,and lowest in tertiary collaterals.The expression of UF3GT gene expression was not obvious,and the expression level in the first to third collaterals increased first and then decreased.3.Expression vectors of RrCPC,RrWER and RrMYB5 genes which may be associated with rose prick formation were constructed and genetically transformed into roses.The results showed that the pCAMBIA1304-RrCPC overexpression vector,the Crispr/Cas9 vector of pCaS9-RrWER and pCas9-RrMYB5,and the pCAMBIA1304 empty vector were successfully constructed.Then,using Agrobacterium-mediated somatic embryo transformation,three expression vectors were transferred to 'BaoBai Rose'.The pCAMBIA1304,RrCPC,RrWER and RrMYB5 transgenic somatic embryos were initially obtained by GUS staining and DNA verification.