Tissue Culture and Molecular Cloning of Quality Related Genes in Carthamus Tinctorius L.
|School||Second Military Medical University|
|Keywords||Safflower HSYA A (HSYA) Tissue culture Regeneration bud RACE Bioinformatic analysis|
Of safflower (Flos Carthami) for drying tubular flowers of the Compositae safflower (Carthmus tinctorius L.), expand coronary artery, anticoagulation, lowering blood pressure, anti-inflammatory, analgesic and other pharmacological effects, and a wide range of clinical applications. Which flavonoids chalcone ingredients - HSYA A (HSYA) is the main active ingredient of saffron, and the level of content HSYA A direct impact on the pros and cons of saffron quality. Therefore, from cDNA level with HSYA of closely related genes, will provide an effective way to regulation safflower varieties from the molecular level, level of nurturing from tissue culture vaccine for transgenic nursery prepare further verify the function of the gene has an important significance. Previous studies from our group hybrid F2 plants containing groups HSYA groups and without HSYA of screening by cDNA-AFLP technique the two safflower quality differences expressed fragments (TDF), were: exists only in Included in HSYA groups TDF-8, and exists only in free HSYA groups TDF-11. This study were determined by HPLC method of safflower in HSYA content, to get the groups containing HSYA without HSYA groups. According to the presence or absence were established in HSYA first strand cDNA library containing the HSYA A (HSYA) and without HSYA of A (HSYA) RACE (cDNA end rapid amplification) technology to provide a basis. TDF-11 fragment sequences are known by the TDF-8 were designed primers, RACE techniques to clone the TDF-8, the full-length sequence of the TDF-11, by sequencing splicing to give the exact length of the the TDF-8 full-length gene for 2315bp containing 1257bp length open reading frame encoding 419 amino acids, named as the WV-CPL GU380344 registry number. Bioinformatics analysis showed that the WV-cpl broad bean wilt virus has a high degree of homology (97%). Bioinformatics analysis and gene conserved regions speculated that The gene belongs to a member of the family of broad bean wilt virus, the virus could infect safflower, a long time ago and caused safflower protection mechanisms, such protection mechanisms mainly by generating secondary metabolites flavonoids against virus infection, the final of the virus to a symbiotic relationship with the saffron, evolution down from generation to generation. TDF-11, the exact length of the full-length gene obtained 1226bp, the largest open reading frame of not more than 141bp, named CT-WPR registry number GU227360. The bioinformatic analysis shows a CT-wpr trichocarpa glycoside predicted protein has 66% homology, gene sequence analysis revealed no significant open reading frame in the gene, suggesting that the gene might be non-encoding amino acid sequences, research shows that the role of non-coding the amino acid sequence of chain gene has a regulatory role, such as inhibition and activation. CT-wpr gene may adjust for and HSYA gene from modification and other effects, leading to inactivation of the gene or activation, ultimately HSYA A (HSYA) did not express in white flowers. In addition, preliminary cDNA library construction, we get a chalcone synthase of Contig 2566 EST homologous sequences, according to the EST primers were designed using the RACE technique to clone the full-length sequence of the gene for 1843bp containing 1164bp length of the open reading frame encoding 388 amino acids, of Contig 2566 speculated that the amino acids in the protein levels than multiple chalcone synthase homology of up to 70% or more, based on conservative sites and conserved sequence analysis, suggesting that the gene is one of the members of the family of chalcone synthase, The chalcone synthase chalcone synthesis key enzyme, HSYA the belonging to the chalcone constituents, therefore the synthesis of the gene and HSYA there are close links, HSYA The content plays a decisive role in determining the merits of safflower quality. To further validate the above three, and after the discovery of gene function, the establishment of safflower in vitro regeneration system is very necessary. In this study, based on access to domestic and international Compositae regeneration system established to establish safflower regeneration system, optimal hormone ratio of cotyledon and hypocotyl regeneration bud NAA0.5mg / L 6BA 2.0mg / L KT20 mg / L. The results of this study for the establishment of safflower regeneration system has laid the foundation, safflower quality full-length gene obtained homologous genes obtained, especially with the CHS to provide ideas for further study of the regulation of the safflower quality of functional genes, transgenic conditions and provide ideas for directional regulation of the quality of medicinal plants.