Tissue Culture And Molecular Cloning Of Quality Related Genes In Carthamus Tinctorius L.

Flos Carthami, dried flower petals of safflower Carthamus tinctorius L., is an important crude drug in traditional Chinese medicine for promoting blood circulation and removing obstruction in the channels. The main chemical composition hydroxysafflor yellow A (HSYA) is the main active component. The quality of Flos Carthami was influenced by the content of the HSYA directely. It is important to find out the functional genes to regular pattern of heredity about the safflower quality. As a result, identification the genes linked to HSYA of Carthamus tinctorius L. at the cDNA molecular level will lay a foundation for breeding spineless cultivars at the molecular level. Meanwhile, the purpose of this study was to develop a plantlet regeneration system for a Safflower breeding program with the final objective of applying transgenic techniques to prepare for further validation of gene function.Our group found that two transcript-deprived fragments (TDFs) related to quality of the safflower by cDNA-AFLP from the F2 generation of hybrid: TDF-8 and TDF-11. The TDF-8 is only present in the population with HSYA, while the TDF-11 is only present in the population without HSYA. In this study, the HSYA content was analysised by high performance liquid chromatography (HPLC), discovering the population with HSYA and population without HSYA. According to the content of the HSYA, we constructed two different first-strand cDNA libraries. We designed primers according to the given sequences of the TDF-8 and TDF-11. We obtained the full-length of the TDF-8 and TDF-11 by RACE. The full length of TDF-8 obtained 2315 bp cDNA and was designated as WV-prl (GenBank accession No. GU380344). It contains a 1257bp open reading frame (ORF), encoding a protein of 419 amino acids. Homologous analysis at the protein level showed that WV-prl carried a highly homology (97%) to Broad bean wilt virus. Carthamus tinctorius L. is one of the hosts of Fabavirus virus. Flavonoids are a diverse group of compounds with a wide range of biological effects, especially anti-viral activity against a range of plant viruses. so that, it may affect the biosynthesis and accumulation of secondary metabolites to some extent. The full-length cDNA sequence of TDF-11 gene obtained 1226 bp cDNA, designated as CT-wpr (GenBank Accession No. GU227360) was cloned. Nucleotide sequence alignment revealed that CT-wpr shared 66% identity with Populus trichocarpa in GenBank. But it has no open reading frame (ORF) larger than 141bp. It is likely a non-coding RNA (ncRNA). Biologists have discovered that ncRNAs exercise many key roles in the cell, including gene silencing, gene regulation, chromatin remodeling, gene localization, gene modification and DNA imprinting. It suggested that CT-wpr, the non-coding RNA gene may have the function of gene regulation. The presence of CT-wpr may affect the expression of genes related to HSya to some exstent, activating or repressing some of them. As a result, some of the genes related to HSYA can't express anymore. In additional, we obtained one Contig 2566 EST sequence with the homologous sequence of Chalcone synthase(CHS) by initial cDNA library construction. Primers were designed by the sequence of the Contig 2566 EST. The full length of Contig 2566 obtained is 1843 bp which contains a 1164bp open reading frame (ORF), encoding a protein of 388 amino acids. Homologous analysis at the protein level showed that the full length of Contig 2566 carrying a homology (70%) to CHS. We conclude that protein is a member of the CHS family which is the key enzyme of the CHS biosynthesis by analysis of the conservative sites and consequences. HSYA belongs to chalcone, so the gene is closely related to biosynthesis of the HSYA, playing a main role in determining the content of the HSYA.To vertificate the function of these three genes and more genes which may be found in the future, it is necessary to establish in vitro regeneration system of safflower. In current study, we will established regeneration system of safflower by referring to the papers about establishing in vitro regeneration system of Compositae home and abroad. MS basal medium supplying with 0.5mg/L NAA + 2.0mg/L 6BA +20 mg/L KT was found to be the best for shoot regeneration.Our finding is the significant foundation of in vitro regeneration system of the safflower, and is also provide idea for target for medical plant functional studies.