CN110531001B - Metabolism marker for distinguishing taxus yunnanensis and taxus mairei and detection method thereof - Google Patents
Metabolism marker for distinguishing taxus yunnanensis and taxus mairei and detection method thereof Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a metabolic marker for distinguishing taxus yunnanensis and taxus mairei and a detection method thereof. The application of the taxane cyanogenic glycoside as a metabolic marker for identifying the taxus yunnanensis and the taxus mairei, wherein the mass content of the taxane cyanogenic glycoside in the taxus yunnanensis is 5-10 times that of the taxus mairei. The invention can be used for samples collected in the field, does not need to be frozen for storage, and the collected samples can be used for extraction and detection of metabolic markers by simple drying and storage at room temperature. The biomass of a detection sample required by the invention is less and is only 25mg, and the damage to rare taxus chinensis seedlings to be identified is less.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a metabolic marker for distinguishing taxus yunnanensis and taxus mairei and a detection method thereof.
Background
Taxus chinensis belongs to shallow root plants, has unobvious main roots and developed lateral roots, is a natural rare anti-cancer plant which is known to be endangered to extinct in the world, is an ancient wiggle tree species which is left over by the fourth glacier, and has a history of 250 ten thousand years on the earth. Because the growth rate of the taxus chinensis is slow and the regeneration capability is poor under natural conditions, a large-scale taxus chinensis raw material forest base has not been formed in the world for a long time.
Taxus chinensis has great economic value, and taxol and derivatives thereof extracted from the plant body are one of the most widely applied antitumor drugs in the world at present. Paclitaxel was the first antitumor active ingredient isolated from the bark of Taxus brevifolia, and is a specific drug for treating ovarian cancer and breast cancer. With the progress of research, paclitaxel has also been found to be effective against other cancers, such as: lung cancer, esophagus cancer, etc. also have obvious curative effect. The whole plant of Taxus chinensis can be used as medicine for treating diabetes, menoxenia, hypertension and other urinary system diseases. In addition, the yew has wide development prospect in garden appreciation. By utilizing the special red fruit of the taxus chinensis, the taxus chinensis forest becomes a landscape tree in high-grade districts, parks and roads at present.
Taxus yunnanensis (A) and (B)Taxus yunnanensi) Is evergreen arbor of Taxus of Taxaceae, with height of 20 m and diameter at breast height of 1 m; big branch developing, golden and yellow winter bud, narrow bud scale, tapered tip, longitudinal ridge on the back, thin and soft leaf texture, needle-like or needle-like strip shape, arranged in two rows, tapered or slightly sharp tip at the tip, dark green or green top, and luster. Taxus yunnanensis is a precious tree species in Yunnan province, is also a key protection tree species in Yunnan province, and is a main tree species for producing taxol medicaments in China. The red pigment is distributed in the northwest and the west of Yunnan China, in the southwest of Sichuan China and in the southeast of Tibet, and also distributed in the northern areas of Dane and Burma, and grows in high mountain areas with the altitude of 2000-3500 m. Taxus chinensis var mairei (Taxus mairei) Evergreen arbor, whose bark is light gray, is longitudinally split into long slices; the top end of the bud scale is blunt or sharp, and falls off or partially stays at the base of the twig. 2 rows of leaves, which are in a shape of a sickle and are 1.5-4.5 cm long, and the midrib belt on the back has no papilla cutin protrusions or is distributed sporadically sometimes, or 1 to a plurality of papilla cutin protrusions are arranged on two sides of the midrib adjacent to the stomatal belt, the color of the papilla cutin protrusions is different from that of the stomatal belt, the papilla cutin protrusions are light green, and the border width is obvious. The water-soluble fertilizer is distributed in provinces in the south of Yangtze river basin, Henan and Shaanxi. The national level emphasizes the protection of wild plants. At present, the research on taxus yunnanensis and taxus mairei has been carried out primarily, but the seedlings are highly similar in morphology, contain a certain amount of paclitaxel, and the growing environments are partially overlapped,this brings some difficulty to the discrimination work of the two. Accurate and rapid identification and differentiation are the precondition for popularizing high-efficiency main cultivars and developing the resource protection of wild yew.
Non-targeted metabolomics is a recently developed technology for studying small molecule metabolites in organisms. The technology can comprehensively analyze all metabolites of a specific organism, a specific cell or a specific physiological condition for quantitative and qualitative analysis, and find out the metabolites with characteristic difference. As an emerging discipline for the study of small molecule metabolites, metabolomics technology is a discipline that has been developed following transcriptome and proteomics. Through multiple means such as chromatogram, mass spectrum and even nuclear magnetic resonance, the non-targeted metabonomics technology accurately detects the content difference of metabolites in the material, and is widely applied to the research fields such as disease diagnosis, medicine research and development, environmental detection and basic theories of botany. Collecting the nuclear-to-cytoplasmic ratio signal of the metabolite, analyzing the metabolic profile (metabolic profiling), and presuming a specific metabolic pathway to find the metabolite with characteristic indication function. With modern metabolome technology, the discovery of metabolic markers between different biological samples is currently a focus of research. In the field of botany, there are not many studies for screening metabolic markers using metabolomics technology and for identification between plant species.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a metabolome marker-Taxus cyanamide for identifying Taxus yunnanensis and Taxus chinensis var mairei, wherein the content of the metabolome marker-Taxus cyanamide in Taxus yunnanensis is far higher than that in Taxus chinensis var mairei, so that a good technical means is provided for solving the problem of tree species identification in the breeding and forestation processes of Taxus chinensis.
The application of the taxane cyanogen glycoside as the metabolic marker for identifying the taxus yunnanensis and the taxus mairei has the following chemical characteristics: the molecular formula is C14H17NO7The molecular weight is 311.3, and the chemical structural formula is shown as follows:
the weight content of the taxane cyanoglycoside in the taxus yunnanensis is 5 to 10 times of that of the taxus mairei.
Preferably, the method is used for efficiently distinguishing the taxus yunnanensis seedlings from the taxus mairei seedlings.
The invention also aims to provide a detection method of the taxus yunnanensis and the taxus mairei, and particularly relates to a detection method of the taxus yunnanensis and the taxus mairei
1) And extracting the metabolites of the grinded taxus yunnanensis or taxus mairei branch samples by using 45-55% methanol buffer solution.
Adding 120 μ L of 50% methanol precooled to-20 deg.C by volume into 25mg Taxus chinensis sample, shaking gently for 1 min, incubating at room temperature for 10 min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20 min, and storing the supernatant at-80 deg.C or drying;
2) performing LC-MS analysis on the supernatant of the extracting solution, and finally judging according to the mass content of the taxane cyanoglycemia, wherein if the mass content of the taxane cyanoglycemia is higher than 0.12 mg/g, the taxus yunnanensis is obtained, and if the mass content of the taxane cyanoglycemia is lower than 0.02 mg/g, the taxus mairei is obtained;
the conditions of the high performance gas chromatography were as follows:
the hue system is: SCIEX UPLC system;
the chromatographic column temperature was: 35 ℃;
the chromatographic column is as follows: an acidic acrylamide column (100 mm × 2.1mm, 1.7 μm, Waters, UK);
mobile phase: mobile phase A is 25 mM ammonium acetate +25 mM ammonia water, and mobile phase B is 9:1 IPA of ACN +0.1% formic acid;
the mobile phase gradient was: 95% of mobile phase B in 0-0.5 min, 95% to 65% of mobile phase B in 0.5-9.5 min, 65% to 40% of mobile phase B in 9.5-10.5 min, 40% to 95% of mobile phase B in 10.5-12 min, and 95% of mobile phase B in 12.2-15 min;
the mass spectrometry conditions were as follows:
high resolution mass spectrometer: TripleTOF type 5600 plus;
the shielding Gas is set to be 30 PSI, the ion source Gas1 is 60 PSI, and the ion source Gas2 is 60 PSI;
the heating temperature is 650 ℃;
for the positive ion mode, the ion spray voltage is 5000V respectively, and for the negative ion mode, the ion spray voltage floating is-4500V respectively;
the mass spectrum data acquisition adopts an IDA mode, and the TOF mass range is 60-1200 Da.
The invention also aims to provide application of the metabolic marker in a kit for identifying taxus yunnanensis and taxus mairei. The kit comprises a standard substance of the taxane cyanoglycane and/or a detection reagent for detecting the taxane cyanoglycane.
The invention has the following beneficial effects:
1. the invention has the advantages of high precision, programmed operation, high sensitivity and accurate and reliable result.
2. The invention can be used for samples collected in the field, does not need to be frozen for storage, and the collected samples can be used for extraction and detection of metabolic markers by simple drying and storage at room temperature. The biomass of a detection sample required by the invention is less and is only 25mg, and the damage to rare taxus chinensis seedlings to be identified is less.
3. The invention utilizes a method of combining high performance chromatography and mass spectrometry to obtain non-target metabolic spectra of the taxus yunnanensis and the taxus mairei, identifies a series of metabolites in different tree species, and obtains differential metabolites between the two by quantitative analysis. By passingPAnd (4) screening to obtain the metabolites with obvious differences by a value significance analysis method.
Drawings
FIG. 1 is an annotated map of Taxus yunnanensis and Taxus mairei metabolites.
FIG. 2 shows the results of PCA analysis of metabolites of Taxus yunnanensis and Taxus mairei.
FIG. 3 is a chromatogram of taxane glycoside standard.
FIG. 4 shows the result of the content of cyanogen glycoside in YN1-10 (Taxus yunnanensis) and NF1-10 (Taxus mairei).
Detailed Description
In order to make the objects, techniques and features of the present patent more apparent, the following detailed description of the present patent will proceed with reference being made to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not limiting upon the present patent.
Example 1: extraction of taxus yunnanensis and taxus mairei metabolites and control sample preparation
Treatment of experimental samples:
the small branches (twig) of Taxus chinensis are accurately weighed, and each 25mg is used for extracting metabolites.
And adding 120 mu L of precooled 45% -50% methanol extract into the ground taxus chinensis sample, and mixing in a centrifuge tube. Shaking gently for 1 min, incubating at room temperature for 10 min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20 min, and storing the supernatant at-80 deg.C or drying;
treatment of control samples:
to better analyze the quantitative differences between the two groups of samples from Yunnan and southern Yew, a group of control samples was prepared. The control sample was prepared as follows: taking 10 independent taxus yunnanensis experimental samples and 10 independent taxus yunnanensis experimental samples, and mixing the 20 samples in equal volume. The mixture was divided into 10 equal portions to prepare control samples.
Example 2: notes on metabolites of Taxus yunnanensis and Taxus mairei
In the implementation process of the invention, a large amount of mass spectrum data including peak (peak), retention time (retention time) and annotation information are detected. The obtained mass spectrum original data is converted into an XML format through software such as XCMS, CAMERA and metaX. The obtained data of different ions are identified by retention time and nuclear-to-proton ratio. The generated ion information was compared to its exact molecular weight and annotated by KEGG and HMDB online databases.
The present invention yielded a total of 7234 valid ion signatures, and thus yielded 5345 successfully annotated metabolites. Annotation of the results indicates that most of the identified metabolites can be assigned to at least one metabolic pathway. The annotated metabolites are mainly classified into metabolic pathways such as amino acid metabolism, carbohydrate metabolism, terpenoid metabolism, lipidation metabolism, nucleotide metabolism, energy metabolism, and the like, and the specific results are shown in fig. 1.
Example 3: screening of marker differential metabolites of taxus yunnanensis and taxus mairei
In order to screen the marked differential metabolites of taxus yunnanensis and taxus mairei, the invention detects two main quality control parameters including Coefficient of Variation (Coefficient of Variation) and Principal Component Analysis (PCA). Among them, the coefficient of variation value was less than 28%, showing higher reproducibility. The main component analysis results show that the variance values of PC1 and PC2 are 4.20% and 69.41%, respectively, which indicates that the discrimination of the two taxus species is high, and the specific PCA analysis results are shown in FIG. 2. Statistical results show that a total screening results in 311 metabolites with significant differential accumulation, 150 of which are significantly accumulated in taxus yunnanensis, and 161 of which are significantly accumulated in taxus mairei. The above differential metabolites can be classified into a plurality of main metabolite classes including alkaloids, amino acids, flavones, hormones, lipids, terpenes, phenylpropanoids, saccharides, and the like. The above major metabolites are largely different between Taxus yunnanensis and Taxus mairei. According to the invention, by analyzing the metabolites which are obviously differentially accumulated, the taxus chinensis cyanogen glycoside is identified and obtained as the marked differential metabolite of the taxus yunnanensis and the taxus mairei.
Example 4: taxus cyanamide (Taxiphyllin) standard substance and liquid chromatography-mass spectrometry thereof
A standard product of taxane cyanoglycine (Taxiphyllin) purchased from a chemoattractant network is characterized by comprising the following components in percentage by weight: CAS number 21401-21-8; the molecular weight is 311.3; molecular formula C14H17NO7(ii) a The density is 1.6 +/-0.1 g/cm3
Boiling point 586.7 + -50.0 deg.C (760 mmHg). The standard mass spectrum is shown in FIG. 3. The content of the cyanogen glucoside of the taxus chinensis in the taxus chinensis sample is accurately determined by utilizing the standard substance.
Example 5: reliability verification example for distinguishing marker differential metabolites of taxus yunnanensis and taxus mairei
The five-year-old taxus yunnanensis cultivation seedlings and the five-year-old taxus yunnanensis cultivation seedlings are planted in the Hangzhou university experiment nursery. The collected samples are respectively numbered as follows: YN1-10 (Taxus yunnanensis) and NF1-10 (Taxus mairei).
Fully drying collected Taxus chinensis twigs in a 40-degree oven, filtering through a sieve with the aperture of 0.42 mm, accurately weighing 0.5g of dry powder for each sample, adding 15 mL of 100% methanol, and mixing uniformly. The mixture was treated with 150W, 40 kHz, ultrasonic waves for 30 min. After the lysate was centrifuged at 4000 Xg for 20 min, the supernatant was transferred to a new centrifuge tube. After vacuum drying, the mixture was dissolved in 50% methanol again for use. And diluting the obtained sample by 10 times by volume, and carrying out UPLC-MS/MS quantitative detection. The detection result shows that the average content of the taxane glycoside in the taxus yunnanensis is obviously higher than that in the taxus mairei, and the specific result is shown in fig. 4. The above results demonstrate the reliability of taxane glycoside as a marker metabolite for distinguishing taxus yunnanensis from taxus mairei. The invention obtains a marker metabolite for distinguishing taxus yunnanensis and taxus mairei by screening based on a metabonomics technology, wherein the required sample is only 25mg, and the accuracy rate is 100%.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (6)
1. The application of the taxane cyanogen glycoside as the metabolic marker for identifying the taxus yunnanensis and the taxus mairei is characterized in that the molecular formula is C14H17NO7The molecular weight is 311.3, and the chemical structural formula is as follows:
2. the use of claim 1, wherein the weight content of the taxane glycoside in the taxus yunnanensis is 5-10 times that of the taxus mairei.
3. The use of claim 1, wherein the taxus yunnanensis and taxus mairei are young plants.
4. A detection method for distinguishing taxus yunnanensis from taxus mairei is characterized by comprising the following steps:
1) after grinding, two taxus branch samples are extracted by methanol solution to obtain metabolites:
adding 120 μ L of 50% methanol precooled to-20 deg.C by volume into 25mg Taxus chinensis sample, shaking gently for 1 min, incubating at room temperature for 10 min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20 min, and storing the supernatant at-80 deg.C or drying;
2) performing LC-MS analysis on the supernatant of the extracting solution, and finally judging according to the mass content of the taxane cyanoglycemia, wherein if the mass content of the taxane cyanoglycemia is higher than 0.12 mg/g, the taxus yunnanensis is obtained, and if the mass content of the taxane cyanoglycemia is lower than 0.02 mg/g, the taxus mairei is obtained;
the molecular formula of taxane cyanoglycoside is C14H17NO7The molecular weight is 311.3, and the chemical structural formula is as follows:
5. application of taxane cyanoglycane in a kit for identifying taxus yunnanensis and taxus mairei is characterized in that the molecular formula of the taxane cyanoglycane is C14H17NO7The molecular weight is 311.3, and the chemical structural formula is as follows:
6. the use of claim 5, wherein the kit comprises a standard of taxane cyanamide and/or a detection reagent for taxane cyanamide.
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| CN109486982A (en) * | 2017-09-11 | 2019-03-19 | 杭州百迈生物股份有限公司 | For identifying the SNP marker and application of taxus chinensis in northeast |
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| CN109486982A (en) * | 2017-09-11 | 2019-03-19 | 杭州百迈生物股份有限公司 | For identifying the SNP marker and application of taxus chinensis in northeast |
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| Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei);Chunna Yu等;《Tree Physiology》;20170717;第37卷;第1659-1671页 * |
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