The Cloning And Expression Of Chalcone Isomerase Gene And Chemical Composition Accumulative Study In Carthamus Tinctorius L.

Carthami Flos is a commonly used traditional Chinese medicine, and the original plant is Carthamus tinctorius L., which is from the compositae. Carthami Flos owe the effects of Invigorating the circulation of menstruation and scattering stasis pain. The efficacy related main effective components of flavonoids including chalcone, flavanone, flavanonol and flavonol etc. Chalcone isomerase gene is the first key gene exist in the branch of quinoid chalcone ketones and other types of flavonoids biosynthetic pathway. So CHI expression is closely related to the accumulation of various types of flavonoids.In this study, CHI was cloned, the expression profile of CHI in 1?7 d florescence was obtained, the prokaryotic expression of CHI study was finished, and the flavonoids content in 1?7 d florescence was detected, which laid the foundation for the relevance study between key gene expression and effective constituent, and the results are as follows:1. The cloning and analysising of CHIThe improved Trizol method was used to extract total RNA, PCR was used to clone CHI. the total sequence of CHI is 696 bp with a 633 bp complete open reading frame and can encode 210 amino acids, Blastp shows that the protein encodes by CHI belongs to Chalcone superfamily. The protein primary, secondary and tertiary structure was predicted by bioinformatics analysis.MEGA5.1 was used to the systematic tree construction, which shows to the safflower CHI is most similar to compositae chrysanthemum plants, the similarity up to 90%. Lay the foundation for study of CHI structure, function and the evolutionary relationships etc.2. Expression analysis of CHIThrough the standard curve, amplification curve, dissolve curve, established real-time PCR methods of safflower CHI and internal genes Ct60s, the expression profile in 1?7 d of safflower was obtained and the relative expression of CHI presents forur periods:1-3 d is the slow rise period.3-4 d is turning point period,4-6 d is rapid decline period, and 6-7 d is slow reducting period.Through enzyme primers digestion, PCR amplification (to insert enzyme loci in target gene), digestion and connection of target gene and carrier PET32 (to build a restructuring carrier), channel restructuring carrier to host Rosatta escherichia coli and IPTG was used to induce exogenous gene expression, the protein expression system of CHI was builded, realize the purpose of the exogenous gene CHI expressed in Rosatta escherichia coli, authenticated the relative molecular weight of CHI is about 24 KD. Lay the foundation for study of restructuring CHI properties, structure and function of the CHI etc.3. Analysis of safflower biomass-component content-gene expressionCM-5 spectral colour meter was used to detect each safflower sample color, ultraviolet spectrophotometer was used to detect flavonoids, HPLC was used to detect HSYA. The connection analysis of biomass-component content-gene expression shows that the biomass of 1-7 d safflower ample?favonoids and HSYA average content of each inflorescence as well as CHI relative expression present similar trends: gradually increased at first and peak in 3 d or 4 d, then gradually reduced; 3-4 d safflower powder samples change from yellow to red, which are bright-colored in reddish yellow. The color of 3-4 d samples changes from yellow to red, which is reddish yellow and bright-colored. DPS was used to analysis grey correlation degree of flavonoids/HSYA with color, which shows that the clarity and yellow degree is much more correlated to HSYA content, red degree is much more correlated to flavonoids content. AMOS was used to analysis the correlation of flavonoids and HSYA content with color, which shows that the yellow degree correlation of flavonoids and HSYA content is greater than the red degree and clarity. Composite medica records and this study, we put forward that:" Carthamus tinctorius L. should be picked in the summer in its full-blossom period, when the flowers changes from yellow to red, tubiform floret spread completely, the corolla top is golden yellow and the middle of tubiform floret is jacinth. " The Carthami Flos getted get the highest flavonoid constituent, the color is bright-colored in reddish yellow and get the best quality.Herbs active ingredients are always plant secondary metabolites, which is the material basis in determining the quality of medicinal materials; secondary metabolites are associated with individual development degree and tissue differentiation, it always affected by external factors stimulation by biosynthesis gene expression and regulation. Biosynthesis functional gene affect drug quality through regulation of effective composition accumulation. Genomics. genes over expression, or silencing could offer ideas and methods for functional genes excavation, gene expression and effective components accumulation study etc.