Cloning And Expression Analysis Of Anthocyanin Biosynthesis Related Genes In Rosa Rugosa

Rosa rugosa, a kind of important ornamental plants, belongs to the family Rosaceae,applied to landscape greening, food industry and pharmaceutical industry, which has great landscape and ecnomic value. Flower color is one of the most important ornamental characters of Rosa rugosa. Rosa rugosa includes a wide diversity of species, but the flower color is simplex. The barriers of interspecific hybridization incompatibility and self-incompatibility in Rosa rugosa, which limit the innovation of the flower color and the development of Rosa rugosa at abroad. Molecular breeding is a new method of cultivating new varieties. The studies about the molecular basis of the flower color of Rosa rugosa are lacking, which limits the molecular breeding of flower color modification. Anthocyanin is the principal pigment in petals, its biosynthesis pathway has been studied extensively in model organism. Most structural genes and regulatory genes in the pathway have been isolated.However, there are no reports on this subject in Rosa rugosa. Therefore, the cloning and expression analysis of structral genes in Rosa rugosa will promote further studies on new cultivars breeding.A full cDNA of(CHS?CHI?F3H?DFR)gene and partial cDNA of(ANS)gene involved in anthocyanins synthesis were cloned from the flower petals of Rosa rugosa ‘Zi zhi' by RT-PCR. The expression of CHS?CHI?F3H?DFR and ANS genes in different developing stages of flower and different organs were carried on analysis by Quantitative RT-PCR.The main results were as follows:1. Five cDNAs of CHS?CHI?F3H?DFR and ANS were isolated by RT-PCR technology from the flower petals of Rosa rugosa ‘Zi zhi', named RrCHS, RrCHI, RrF3 H, RrDFR, RrANS,respectively. A full cDNA length of(RrCHS?RrCHI?RrF3H?RrDFR) gene was 1167 bp,711bp, 1092 bp, 1047 bp, encoding 389, 237, 364 and 349 amino acids respectively. A partial cDNA length of(RrANS) gene was 562 bp. Sequence comparative analysis by NCBI blastp tools showed that those five genes shared a high homology with relevant genes of other plants.The accession number in GenBank: ALR74720.1, ALR74721.1, ALR74723.1, ALR74719.1,ALR74722.1, respectively.2. The molecular weight, theoretical pI, signal peptide, transmembrane domain,phosphorylation sites, O-glycosylation sites, second structure and conserved structuraldomain of those five genes were analysed by bioinformatics software. The phylogenetic tree showed that the system evolution of those genes were consistent with plant taxonomy and had obvious features of genus and species.3. The amount of anthocyanins of Rosa rugosa with different flower colors in five developmental stages was determined. The results showed that the amount of anthocyanins in the Rosa rugosa ‘Zi zhi' was significantly higher than that in the Rosa rugosa ‘Fen zizhi'.Rosa rugosa ‘Bai zizhi' had the lowest amount of anthocyanin content. The amount of anthocyanins was gradually decreased in five development stages of Rosa rugosa with different flower colors, conforming to the change of colours.4. The expression patterns of RrCHS, RrCHI, RrF3 H, RrDFR and RrANS, in five developmental stages of Rosa rugosa with different flower colors were determined for the first time by RT-PCR. The results indicated that the transcripts of all five genes were detected in five development stages of Rosa rugosa with different flower colors. The gene differential expression patterns in different flower colors exist significant difference at different growth stages.The expression of RrCHS and RrDFR presented a gradually decreasing trend in five development stages of Rosa rugosa with different flower colors, and reached the minimum expression in the end of flowering. The expression level of RrCHI was raised rapidly and then decreased with the developing of flower. The transcripts of RrF3 H and RrANS showed no significant rules in three cultivars. The formation and change of anthocyanins has a close relationship with RrCHS, RrCHI and RrDFR. The relative transcription level of RrF3 H and RrANS have no obvious corresponding relationships with the formation of anthocyanins,indicating involved in other metabolic processes besides anthocyanin biosynthesis.5. The expression patterns of RrCHS, RrCHI, RrF3 H, RrDFR and RrANS genes in different organs(stems, leaves, stamens, pistil, petals) of Rosa rugosa ‘Zi zhi' were carried on analysis by quantitative RT-PCR. The results showed that the transcripts of all five genes were detected in five different organs of Rosa rugosa‘Zi zhi'. The expression of five genes showed tissue-specific expression profiles. The results of expression analysis in different organs showed that RrCHS, RrCHI and RrDFR had the similarity expression feature that the expression level in petals all higher than stems, leaves, stamens and pistil. In the meanwhile,the expression level of RrF3 H was high in stems and leaves but lower in stamens, pistil and petals. The expression level of RrANS was higher in stems than other organs, indicating involved in other metabolic processes besides anthocyanin biosynthesis.