Microscopic Observation And Key Gene Mining Of Rose Prickle Development

Rose（Rosa rugosa Thunb.）is an important economic plant integrating ornamental,edible,medicinal and spice in the world.Its stem is covered with prickles,which is inconvenient in garden application,field management and flower picking.As a special form of trichome with developed secondary structure,the anatomical structure of rose has been widely concerned,but its development regulation is lack of in-depth study at the molecular functional level.Based on the previous research of the research group,this paper first investigated and classified the prickle types of different rose varieties,sequenced the transcriptome of different lateral branches of Rose Rugosa ’Zizhi’ with specific prickle traits,and analyzed the whole genome sequencing data of ’Purple Rose’,and screened out the candidate genes that may be closely related to the formation of rose prickles.Then we further constructed the overexpression vector of candidate genes,heterologously transformed Arabidopsis thaliana,verified its biological function,excavated the key genes that may regulate the formation of rose prickle,screened the transgenic plants with hairless or hairless phenotype,identified the downstream target genes directly regulated by the key genes of prickle development at the transcriptional level by transcriptome sequencing,and preliminarily discussed the molecular mechanism of its regulation,It lays a molecular theoretical foundation for the subsequent homologous transformation of key genes into roses and systematically revealing the gene regulatory network formed by prickles of rose.The main research results are as follows:（1）Using stereoscope to observe the prickle structure of 22 kinds of roses,it was found that 18 kinds of roses had both glandular prickles（GPs）and nonglandular prickles（NGPs）,and 3 kinds of roses had only NGPs.Among them,the NGPs of 9 kinds of roses and the GPS of 11 kinds of roses were naked,and the NGPs of 13 kinds of roses and the GPS of 8 kinds of roses were covered with trichome.R.Rugosa ’Zizhi’ has only NGPs and no trichome covered.The number of prickles decreases step by step on the primary collateral branch,secondary collateral branch and tertiary collateral branch,the characteristics of the prickle are single and regular.R.Rugosa ’Zizhi’ is an excellent plant material for studying the development of prickles.（2）Taking the primary collateral branch epidermis（FCB）,secondary collateral branch epidermis（SCB）,and tertiary collateral branch epidermis（TCB）of R.Rugosa ’Zizhi’ as plant materials,the transcriptome was sequenced using Illumina hiseq platform,and a total of 60.13 Gb data were obtained.The filtered high-quality clean reads were located in the purple rose genome for comparison,so as to obtain the expression profile information of corresponding genes.A total of 4588 differentially expressed genes（DEGs）were screened by differential expression analysis.The results of GO and KEGG enrichment analysis showed that during the development of rose prickles,the GO terms of response to stimulus and response to oxygencontaining compound were active,and plant hormone signal transduction pathways represented by auxin,cytokinin and gibberellin were significantly activated.From the DEGs,70 MYB and bHLH transcription factors highly related to epidermal hair were further screened and the expression profile was established.Based on the expression abundance,up-down regulation and related functional verification of homologous genes in the field of trichome,five candidate genes,RrbHLH130,RrCPC,RrMYB5,RrWER and RrCKX1,were selected as the follow-up research objects.（3）Based on the full-length sequences of RrCPC,RrMYB5 and RrWER genes cloned in the early stage of the laboratory,continued to clone the CDS region of RrbHLH130 and RrCKX1 genes to obtain the target fragment.The CDS of RrbHLH130 is 1293 bp long and can encode 431 amino acids;The CDS of RrCKX1 is 1644 bp long and can encode 547 amino acids.The protein structure and homology analysis of the five candidate genes showed that they were similar to the homologous proteins of Arabidopsis thaliana.Using the whole genome data of representative species such as Rosa rugosa,Rosa chinensis,Fragaria vesca,Arabidopsis thaliana and Oryza sativa,the identification and phylogenetic analysis of CKX gene family members were carried out.It was found that the evolution of CKX gene was relatively conservative among different species,and the CKX1 gene of Rosa rugosa,Rosa chinensis and Arabidopsis thaliana tended to be consistent in domain and gene structure,indicating that it may have the same function.（4）The pNC-Cam1304-35S overexpression vector of RrbHLH130,RrMYB5,RrWER and RrCKX1 genes and the pCAMBIA1304 overexpression vector of RrCPC gene were constructed by homologous recombination.The overexpression vector carrying five target gene fragments was heterologously transformed into Arabidopsis thaliana by Agrobacterium mediated inflorescence staining,and the corresponding transgenic Arabidopsis strains were screened by hygromycin.The results of RT-PCR showed that,five target genes were highly expressed in Arabidopsis.The transgenic Arabidopsis thaliana was continuously cultured to T3 generation and preliminary phenotypic identification was carried out.It was found that about 1/2 of the RrCPC transgenic Arabidopsis thaliana showed little or no surface fur on leaves,stems and inflorescences,while the other four transgenic lines had no obvious phenotypic changes compared with wild Arabidopsis thaliana.The transcriptome sequences of the strains with obvious phenotypic differences were further sequenced.The results of go enrichment analysis suggested that the DEGs of prickle development and epidermal hair development overlapped in function.In the case of RrCPC overexpression,the abundance of bHLH transcription factor GL3/EGL3 and WD40 repeat protein TTG1,which made up the MBW complex,remained unchanged or slightly increased.Meanwhile,MYB23（R2R3-MYB）,which promoted epidermal hair formation,was nearly completely inhibited,the expression decreased to 0.The activities of endogenous CPC and its homologous genes TRY and ETC1 were inhibited,while the expression of ETC2 was increased.The expression of GL2,a key initiation gene for trichome cell development and differentiation,was significantly inhibited,and the expression almost dropped to 0.These results suggest that RrCPC may be a key gene regulating the development of rose prickles.