Tissue difference analysis of flavonoid components and their key enzyme gene expression in BeiChaihu and narrow-leaf Chaihu.

North Chaihu Bupleurum chinense DC, narrow-leaf chaihu B.scorzonerifolium Willd. is an umbrella-shaped Chahu genus perennial herb.
the 2015 edition of the Chinese Pharmacopoeia, which stipulates that the dry roots of Bei Chaihu and Narrow Leaf Chaihu are medicinal sage, and have a history of more than 2,000 years for the treatment of inflammation, deheating, malaria, etc.
Chaihudo to root into the drug, however, a number of reports show that part of the plant is rich in flavonoids, flavonoids in the growth and development of plants play an important role in the protection of plants from ultraviolet rays, bacteria, fungi and other injuries, but also make plants show different colors. At the same time, the
, flavonoids play an important role in the treatment and prevention of cancer, cardiovascular and other diseases.
but in the process of drug-taking, part of the wood-hu ground is often discarded, the ground part is not fully utilized, no doubt caused by the waste of medicinal plant resources.
the unique biological activity of flavonoids has attracted the attention of researchers, the research on the metabolic synthesis pathways and functional genes of medicinal plant flavonoids has made great progress, but the research on the biosynthesis pathways of flavonoid sestake in the northern chaihu and narrow-leaf chaihu is still lagging behind, and the rare reports of jaundice biosynthesis pathwayfunctional genes in the plants of chaihu.
Sui and other similation group sequencing (NCBI login number: GSE51187) on the root tissues of BeiChaihu and Narrow Leaf Chaihu, from which a number of genes involved in the synthesis of secondary metabolites were found.
the aromatic group of isoflavone synthase (ISOflavone synthase, IFS) catalytic dihydroflonoid compounds from C-2 to C-3 to produce isoflavoneses; F3H) can catalyze dihydroflonic compound C-3 hydroxyl to produce dihydroflonol compounds, dihydroflonol reductase (dihydroflflol 4-reductase), is an important regulatory branch of plant anthocyanin symtonia synthesis.
analysis of the expression of the key enzyme genes of flavonoid synthesis and the association with the content of flavonoid ses, can provide a basis for studying the accumulation and regulation mechanism of flavonoid components in Chaihu.
the study of north Chaihu and narrow leaf Chaihu root, stem, leaf, fruit as an experimental material, using high-efficiency liquid chromatography method to determine the content of 4 kinds of flavonoids in different tissues (reed, quercetin, shanbabin, isobin), ULTRA-violet photophotometric method to determine the total wheyone content, using real-time real-time The fluorescent quantitative PCR method (qRT-PCR) instrument measured the expression of three homologous compound synthesis key enzyme genes (IFS, F3H, DFR) in different tissues of Bei Chaihu and narrow leaf Chaihu, and studied the relationship between the synthetic key enzyme genes of three flavonoids and the content of flavonoids.
with a view to promoting the development of chaihu secondary metabolic engineering through the study of the biosynthesis of the monitaths of beechaihu and narrow-leaf Chaihu, and providing technical support for the full utilization of Chaihu resources, improving the quality of medicinal herbs, quality control, etc.
1, Materials and Equipment 1.1 Materials North Chaihu was collected on September 3, 2017 in Jilin Agricultural University Pharmaceutical Park, and wild narrow-leaf Chaihu was collected on September 5, 2017 from the northwest of the platform town of Baicheng, Jilin Province, about 2.71 km (N45 s 74', E Meadow grasslands, respectively, randomly collected 10 plants of Bei Chaihu, narrow leaf Chaihu each, identified by Professor Yang Limin of the School of Traditional Chinese Medicine of Jilin Agricultural University as the authentic of The North Chaihu B. chinense DC, the narrow-leaf Chaihu B. scorzonefolriwillwill.
will collect fresh samples in the ice box to keep fresh, bring back after the experiment carefully washed the sediment with clean water, with filter paper to suck water, then the north Chaihu and narrow leaf Chaihu decomposed into roots, stems, leaves, fruit 4 parts, the north Chaihu, narrow leaf Chaihu different tissues Cut into small pieces with a small knife after sterilization, north Chaihu, narrow leaf Chaihu different tissue parts of the small pieces mixed evenly in the frozen storage tube, immediately placed in liquid nitrogen in the frozen, stored in the ultra-low temperature cold storage tank for follow-up experiments.
the remaining clean cleaning of the north Chaihu, narrow leaf chaihu tissue (root, stem, leaf, fruit) in the oven low temperature drying to a constant quality, with a shredder mill into fine powder, used in 4 flavonoid ingredients and total flavonoid content determination.
1.2 reagent control products reeddine (quality score 91.9%, batch number 100080-201610) and quercetin (quality score 99.1%, batch number 100081-201610) purchased at the China Food and Drug Inspection Research Institute; K1624010) was purchased by Shanghai Aladdin Biochemical Technology Co., Ltd.; methanol, acetylene (chromatography, FisherScience), methanol (extractor) and other reagents are domestically analyzed pure; polysaccharide plant total RNA extraction kit purchased at Changchun Baijin Co., Ltd.; RT rt reagent with gDNA dna reincarnations redo kit, SYBR®
1.3 Instrument Agilent 1260 High Efficiency Liquid Chromatography (Agilent Corporation, U.S.); SMART-N Pure Water Machine (Shanghai Kanglei Analysis Instrument Co., Ltd.); SB-800 Ultrasonic Cleaner (Ningbo Xinzhi Biotech Co., Ltd.); AUY220 Electronic Balance (Japan's Shimizu Company); UV-1700 UV-based spectrophotometer (Japan Shimadzu) Company); Mx3000P fluorescent quantitative PCR (Aligent, U.S.); MDF-382E ultra-low temperature refrigerator (Japan SANYO Company); Heraeus Fresco 21 microcentrifuge (Thermo U.S.); NanoDrop2000nucleic acid/protein quantameter (U.S.A.); SIM-F140 Ice Machine (Japan S. S.
2, method 2.1 HPLC method to determine the content of flavonoids 2.1.1 control solution preparation took reed, quercetin, oxycodone control 10 mg, precision weighing, carefully placed in 10 mL bottle.
due to the low solubility of isopherin, another precision isido leus control 10 mg, carefully placed in a 100 mL bottle.
with methanol to the scale, shake evenly, that is, to obtain each control of the mother liquor (reed, quercetin, mountain slug quality concentration of 1 mg/mL, the concentration of the quality of the hematin weight of 0.1 mg/mL).
are precise lying, quercetin, sage control mother liquid each 1 mL, ishes leus control mother liquid 5 mL placed in 10 mL bottle, methanol fixed to the scale, shake, get mixed control solution (reed, quertin, mountain slug quality concentration of 0.1 mg/mL, ishes l concentration of 0.05 mgL).
2.1.2 Preparation of the test solution precision called North Chaihu or narrow leaf Chaihu root, stem, leaf, fruit powder 2.0 g, set plug cone-shaped bottle, precision addition volume fraction 70% methanol 40 mL, ultrasonic (power 800 W, frequency 40 kHz) Treatment of 30 min, filtered, repeated extraction 3 times after the combined filter placed in the evaporation dish, 50 degrees C water bath swayed, residue with 70% methanol dissolved and fixed to 10 mL bottle, shaken well, after 0.22 m membrane is supplied with the test solution.
due to the high content of some reeds on the ground of Bei Chaihu and Narrow Leaf Chaihu, it is necessary to further dilute 10 to 25 times the supply of the test solution.
2.1.3 Chromatography conditions refer to Wei Yingjie and et al.
Elitt Hypersil ODS2 (250 mm x 4.6 mm, 5 m) column, flow phase 0.1% phosphoric acid (A) - acetylene (B), gradient elutation, conditions: 0 to 15 min, 5 % to 20%B; 15 to 30 min, 20% to 25% B; 30 to 60 min, 25% to 45% B; 60 to 70 min, 45% to 50% B.
volume flow of 1.0 mL/min, detection wavelength 360 nm, column temperature 35 degrees C.
2.1.4 The standard curve of the drawing of flavonoids reed, quercetin, grouse, isobin mixed control solution, in turn diluted the concentration is the initial concentration of 1/50, 1/20, 1/5, 2/5, 3/5, 4/5, 1/1 flavonoid mixed control solution.
in accordance with the "2.1.3" item chromatography conditions, in turn, the sample 20 sL, with the peak area of the vertical coordinates (Y), the solution mass concentration is horizontal coordinates (X), the standard curve is drawn, the result of a good linear relationship, the regression equation see table 1.
2.1.5 4 flavonoids content determination was taken from 3 samples of north Chaihu or narrow-leaf chaihugen, stem, leaf and fruit, and the test solution was prepared according to the "2.1.2" method, and the content of 4 of the flavonoid sr.co.uk was calculated by regression equation.
2.2 UV photophotoofometer method to determine the total flavonoid content of the sample 2.2.1 control solution prepared with reed seditin control 10 mg, precision weighing, carefully placed in a 10mL bottle, with methanol fixed to the scale, shaking, that is, reed-in-control solution (reed-in-quality concentration of 1 mg/mL).
2.2.2 preparation of the test solution is prepared according to the method of "2.1.2".
2.2.3 The method of drawing the graph reference Zhou Yafu, etc.
precision-taking reed-in control solution 0, 0.02, 0.10, 0.20, 0.40, 0.80, 1.60, 2.40 mL in 10 mL bottles, plus 60% ethanol fixed to 4 mL, each plus 5% sodium nitrite solution 0.4 mL, shake 6 min, add 10% aluminum nitrate solution 0.4 mL, shake well, place 6 min, each add 4% sodium hydroxide solution 4 mL, with 60% ethanol solution set to 10 mL, shake well, place 15 min, with distilled water for blank control, at 510 nm to detect absorbance (A) value.
with a value of A in ordinate (Y), the total flavonoid mass is horizontal coordinate (X), the standard curve is plotted, the result is 0.02 to 2.40 mg, the total flavonoid mass and A value linear relationship is good, the regression equation is Y-0.772 X-0.008 2, r2-0.999 5.
2.2.4 sample total flavonoid content determination is taken from the north Chaihu or narrow leaf Chaihu root, stem, leaf, fruit sample 3 copies, in accordance with the "2.2.2" method to prepare the test sample solution, take 1 mL for the test solution according to the "2.2.3" method operation, through the regression equation to calculate the total flavonoid content.
2.3 qRT-PCR method to determine the expression of each gene 2.3.1 total RNA extraction and cDNA synthesis took the north Chaihu or narrow leaf Chaihuroot, stem, leaf, fruit tissue sample 50 to 200 mg in liquid nitrogen grinding powder, according to Changchun Baijin's total RNA extraction kit operation procedures to extract the total RNA, using nanoDrop 2000 detector to determine the total concentration of different tissues.
use PrimeScript RT reagent Kit withgDNA Eraser reverse transcript ionatum to synthesize total RNA reverse transcription cDNA and save it at a spare degree of 20 degrees C.
2.3.2 qRT-PCR method uses qRT-PCR to detect tissue expression patterns of the key enzyme genes synthesized by flavonoids in Bei Chaihu and Narrow-leaf Chaihu.
beiChaihu with extension factor EF1 alpha-based because of incarnosis, narrow leaf chaihu with microtube protein beta-tubulin base because of incarinas.
qRT-PCR method detects IFS, F3H, DFR gene expression in different tissues (root, stem, leaf, fruit) respectively (the ifs of the Northern Chaihu and narrow-leaf hutong source IFS in the transcription database are HHH_rep_c6754, ZZZ_rep_c363; the homologous F3H gene number is ZZZ_rep_c8447, HHH_c9371, and the same source DFR gene number is HHH_rep_c2965, ZZZ_rep_c2235, respectively).
each reaction was repeated 3 times, each gene with the expression in the root as a control, using the analysis results of the 2-s.
the reaction system: sterilized water 7.5 sL, SYBR® Premix Ex TaqTM 10?L, primers each 0.5?L, 50 x ROX Dye II 0.5?L, cDNA template 1 sL, total 20 ?L.
reaction procedure: 94 degrees C pre-degeneration 30 s; 45 cycles (94 degrees C degeneration 5 s, 55 degrees C annealing 30 s, 72 degrees C extension 20s). The sequence of primers used in
can be shown in Table 2.
3, results and analysis of 4 flavonoids content in different tissues HPLC method to determine the content of 4 flavonoids in beichai hu and narrow leaf chaihuroot, stem, leaf, fruit as shown in Table 3, the overall north chaihugen, leaves, fruit 4 kinds of flavonoid content is higher than Narrow leaf Chaihu, reed in 4 kinds of flavonoid stakes in the proportion of very high, accounting for more than 95% of the total 4 ingredients, quercetin, sage, istronosis content is very low, in the north Chaihu, narrow leaf chaihu root and stem difficult to detect the cresify and istros.
the distribution pattern sedofs in different tissues in North Chaihu were leaves and stems, and reeds had the highest content of leaves in different tissues in North Chaihu, reaching 106.961 mg/g;
3.2 total flavonoid content in different tissues uv-light light.