CN115754044A - Application of taxus chinensis Qifen serving as metabolic marker for identifying male and female seedlings of taxus mairei - Google Patents
Application of taxus chinensis Qifen serving as metabolic marker for identifying male and female seedlings of taxus mairei Download PDFInfo
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Abstract
The invention provides application of taxus kirilowii in serving as a metabolic marker for identifying male and female seedlings of taxus cuspidata. And carrying out large-scale non-targeted high-throughput metabonomics analysis on the male and female taxus cuspidata seedlings by combining the chromatography with the UPLC-MS/MS technology. The metabolic profiles of female and male seedlings of the taxus cuspidata are obtained, and a small molecular metabolic marker taxus chinensis (Taxagifine) with obvious difference is obtained by screening and is obviously accumulated in the female seedlings of the taxus cuspidata. The invention provides a reliable and rapid identification method for distinguishing the male and female seedlings of taxus mairei, and provides a reliable molecular means for the protection, large-scale artificial cultivation and production of the endangered plant taxus mairei.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of taxus chinensis Qifen as a metabolic marker for identifying male and female seedlings of taxus cuspidata.
Background
Taxus chinensis is a tree plant of Taxus genus of Taxaceae family, and is native to Himalayan and southeast Asia. As a medicinal plant, the taxus chinensis is the most rare one of56 kinds of endangered species plants left over in the ancient fourth-class glacier period. At present, china has already listed the plants in the first-class protection scope of the state precious or endangered plants. Taxus plants have at least 15 varieties all over the world, and China has 4 varieties and one variety. The taxus chinensis can adapt to wide growing environment, including mountainous areas, temperate zones, warm temperate zones, tropical hills, mountainous areas and forests, such as deciduous forests, evergreens or mixed forests. In the global range, the altitude distribution of the taxus chinensis is approximately in the range of 900-3700 m.
Taxus chinensis contains paclitaxel. The statistical clinical experiment results show that the taxol also has remarkable curative effect on various cancers, the total effective rate reaches more than 75%, the taxol is mainly used for treating advanced breast cancer, lung cancer, ovarian cancer, head and neck cancer, soft tissue cancer and digestive tract cancer, is called as the last line of defense of advanced cancers, and is considered as a most promising anticancer medicament discovered in the last decade. Up to now, paclitaxel has been approved for clinical treatment in various countries, including English, french, japanese, italy, canada, sweden, germany, norway, switzerland, brazil, china. Recently, paclitaxel has been found to have good therapeutic effects on rheumatoid arthritis, senile dementia and the like. The Chinese yew tree is beautiful and elegant in shape, evergreen in four seasons, and drunken in the mature period of the fruit, can be widely applied to the aspects of water and soil conservation, gardening and appreciation and the like, is an excellent tree species for improving the ecological environment in the new century, building beautiful mountains and is commonly used for greening and appreciation of high-grade courtyards at present.
The dense-leaf taxus chinensis, also called Himalayan dense-leaf taxus chinensis, grows in a zone with the altitude of 2500-3400 m, is a male and female heterostrain, and is a specific taxus plant in Himalayan areas. The taxus densefruit has relatively strict requirements on the growth environment, belongs to the species of niche, has important ecological and economic values, and is a first-level key plant protection in China. Because the taxol contained in the plant has good curative effect on cancer and certain curative effect on diabetes, coronary heart disease and the like, the medicinal value of the taxol has attracted the wide attention of experts at home and abroad, and the taxol has considerable application prospect. However, the natural taxus cuspidata in China has narrow habitat and is endangered to be extinct, and effective protection and management measures are urgently needed. At present, the research on taxus mairei has made a preliminary progress, but the male and female seedlings thereof are highly similar in morphology and contain a certain amount of paclitaxel, which brings certain difficulties for the distinguishing and identifying work of the two. Therefore, the method can accurately and quickly identify and distinguish the male and female seedlings of the taxus mairei, and provides conditions for popularizing efficient main cultivars, producing effective components in a large scale and sustainably developing the resource protection of the wild taxus mairei.
Non-targeted metabonomics (unargeted metabonomics) refers to the technology such as LC-MS, GC-MS, NMR and the like, dynamic changes of all small molecule metabolites (mainly endogenous small molecule compounds with relative molecular weight within 1000 Da) before and after interference in cells, tissues, organs or organisms are detected in an unbiased manner, differential metabolites are screened out through biological information analysis, and relevant pathway analysis is carried out on the differential metabolites, so that the physiological mechanism for generating the series of changes is disclosed. Then collecting the nuclear-to-cytoplasmic ratio signal of the metabolite, analyzing the metabolic profile (metabolic profiling), thereby presuming a specific metabolic pathway to find the metabolite with characteristic indication effect. At present, the discovery of metabolic markers among different biological samples through metabolome technology has become an emerging research hotspot. However, in the field of botany and related fields, researches for screening metabolic markers and identifying plants by using the technology are rarely reported.
Disclosure of Invention
The invention aims to provide taxus chinensis-taxus chinensis marker for identifying female and male seedlings of taxus mairei aiming at the defects of the prior art, wherein the content of taxus chinensis-taxus mairei marker in female seedlings of taxus mairei is far higher than that of male seedlings of taxus mairei, so that a good technical means is provided for solving the problem of sex identification of rare and endangered plants in the breeding and forestation processes of taxus mairei.
The application of taxus kirilowii in identifying the metabolic markers of the male and female seedlings of taxus cuspidata has the following chemical characteristics: the molecular formula is C 37 H 44 O 13 Molecular weight is 696.74, and chemical structural formula is as follows:
preferably, the mass content of yew Qifen in female seedlings of the taxus densefolius is 10-30 times that of male seedlings of the taxus densefolius.
The invention also aims to provide the detection method for identifying the male and female yew plants, which comprises the following steps:
1) Grinding the branch sample of the male and female seedling of the taxus mairei, and extracting the metabolite by using a methanol solution with the volume content of 45-55%.
Weighing 120 mu L of methanol solution with volume content of 45-55%, precooling to-20 ℃, adding 25mg of ground taxus cuspidata sample, slightly shaking for 1min, incubating at room temperature for 10min, and standing at-20 ℃ overnight. Centrifuging at 4000 Xg for 20min, and storing the supernatant at-80 deg.C or drying;
2) Performing LC-MS analysis on the supernatant of the extracting solution in the step 1), judging according to the mass content of taxus kirilowii, and if the mass content of taxus kirilowii is higher than 2.1mg/g, determining the taxus cuspidati is a female taxus mairei strain, and if the mass content of taxus kirilowii is lower than 0.21mg/g, determining the taxus cuspidati is a male taxus mairei strain;
the conditions of the high performance gas chromatography were as follows:
the color phase system is: SCIEX UPLC system;
the chromatographic column temperature was: 35 ℃;
the chromatographic column comprises: agilent acid acrylamide columns (100mm x 2.1mm,1.7 μm, waters, UK);
mobile phase: mobile phase a was 25mM ammonium acetate +25mM ammonia, mobile phase B was 9 IPA of ACN +0.1% formic acid;
the mobile phase gradient was: 95% of mobile phase B in 0-0.5min, 95% to 65% of mobile phase B in 0.5-9.5min, 65% to 40% of mobile phase B in 9.5-10.5min, 40% to 95% of mobile phase B in 10.5-12min, and 95% of mobile phase B in 12.2-15 min;
mass spectrometry conditions were as follows:
high resolution mass spectrometer: tripleTOF5600plus type;
the shielding Gas is set to be 30PSI, the ion source Gas1 is 60PSI, and the ion source Gas2 is 60PSI;
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 60-1200Da.
Preferably, the samples of the male and female taxus densefolius strains are all seedlings.
The invention further aims to provide application of taxus chinensis qi in a kit for identifying female and male seedlings of taxus cuspidata.
The invention has the following beneficial effects:
1. the invention obtains a metabolite marker for identifying the male and female seedlings of taxus mairei by screening and provides a detection method thereof. According to the invention, the chemical components of the metabolites are identified by analyzing the metabolism spectra of the female and male seedlings of taxus mairei, and the metabolites which are obviously differentially accumulated are screened, so that the metabolite markers for identifying the female and male seedlings of taxus mairei are obtained.
2. The identification method has the advantages of high accuracy, high sensitivity, programmed operation and accurate and reliable result.
3. The invention can be used for samples collected in the field, does not need to be frozen for storage, and can be used for extracting and detecting the metabolic markers by simply drying and storing the collected samples at room temperature. The biomass of a detection sample required by the method is less and is only 25mg, and the method has little damage to endangered rare Chinese yew seedlings needing to be identified.
4. The invention utilizes a high performance chromatography and mass spectrometry combined method to obtain a non-target metabolic spectrum of the male and female taxus mairei seedlings, identifies a series of metabolites in tree species with different sexes, and obtains differential metabolites between the two by quantitative analysis. And screening to obtain the metabolites with obvious differences by a P value significance analysis method.
Drawings
FIG. 1 annotation results of metabolites of female and male plants of Taxus dense leaf;
FIG. 2 analysis of metabolite principal components of male and female young plants of Taxus dense leaf;
FIG. 3 liquid mass spectrum of Taxoqifen (Taxagifine) standard;
FIG. 4 is a verification of the reliability of the marked differential metabolites of Taxus dense leaf.
Detailed Description
In order to make the objects, techniques and features of the present invention more apparent, the present invention is further described in detail below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1: extraction of taxus mairei male and female seedling metabolite and control sample preparation
Treatment of experimental samples:
the small branches (twig) of taxus mairei are accurately weighed, and each 25mg is used for extracting metabolites.
Adding 120 mu L of precooled 45% -50% methanol solution into the ground taxus densefruit sample, and mixing in a centrifuge tube. Gently shaking for 1min, incubating at room temperature for 10min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20min, and storing the supernatant at-80 deg.C or drying;
treatment of control samples:
to better analyze the quantitative differences between the two samples of female and male Taxus wallichiana, a control sample was prepared. The control sample was prepared as follows: taking 10 independent female seedling test samples of taxus densefruit and 10 independent male seedling test samples of taxus densefruit, and mixing the 20 samples in equal volume. The mixture was divided into 10 equal portions to prepare control samples.
Example 2: annotation of metabolites of male and female seedlings of 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-proton ratio. The generated ion information was compared to its exact molecular weight and annotated by KEGG and HMDB online databases.
The present invention has a total of 1987 effective ion characteristics, and thus 685 successfully annotated metabolites were generated. Annotation of the results indicated that most of the identified metabolites could be assigned to at least one metabolic pathway. The annotated metabolites are mainly classified into metabolic pathways such as terpenoid metabolism, amino acid metabolism, fatty acid metabolism, nucleotide metabolism, energy metabolism, steroid metabolism, carbohydrate metabolism and the like, and specific results are shown in fig. 1.
Example 3: method for screening marked differential metabolites of male and female seedlings of taxus mairei
In order to screen the marked differential metabolites of male and female seedlings of taxus mairei, the invention detects two main quality control parameters including Coefficient of variation (Coefficient of variation) and Principal Component Analysis (PCA). The main component analysis results show that the variance values of PC1 and PC2 are 64.54% and 7.94%, 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 134 metabolites which are remarkably and differentially accumulated are obtained by co-screening, wherein 84 metabolites are remarkably accumulated in male taxus densefolius and 60 metabolites are remarkably accumulated in female taxus densefolius. The differential metabolites can be classified into a number of major metabolite classes, including alkaloids, amino acids, flavones, hormones, lipids, terpenes, phenylpropanoids, and carbohydrates. The main metabolites have great difference between male and female seedlings of taxus mairei. According to the invention, by analyzing the metabolites which are obviously differentially accumulated, the taxus chinensis Qifen is identified and obtained as the marked differential metabolite of the male and female seedlings of taxus cuspidata.
Example 4: taxifofin (Taxagifine) standard substance and liquid chromatography-mass spectrometry thereof
The standard product of taxus odd-shaped (Taxagifine) is purchased from a chemical source network and is characterized by comprising the following components: CAS number 81489-69-2; the molecular weight is 696.74; molecular formula C 35 H 47 NO 10 (ii) a The purity was 98%. The standard mass spectrum is shown in FIG. 3. And accurately measuring the content of the taxus chinensis Qifen in the taxus chinensis sample by using the standard substance.
Example 5: reliability verification example for distinguishing symbolic difference metabolites of male and female taxus cuspidata seedlings
The five-year-old female and male yew dense-leaf cultivation seedlings are planted in an experimental nursery garden of Hangzhou university. The collected samples are respectively numbered as follows: male1-6 (Male Taxus cuspidata strain) and Female1-6 (Female Taxus cuspidata strain).
Fully drying collected Taxus chinensis twigs in a 40-degree oven, filtering through a sieve with the aperture of 0.42mm, accurately weighing 0.5g of dry powder for each sample, adding 15mL of 100% methanol, and mixing uniformly. The mixture was treated with 150W,40kHz ultrasonic waves for 30min. After the lysate was centrifuged at 4000 Xg for 20min, 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 content of yew Qifen in female seedlings of the taxus mairei is obviously higher than that of male seedlings of the taxus mairei. The specific results are shown in FIG. 4. The above results demonstrate the reliability of taxus kirilowii as a marker metabolite for distinguishing female and male seedlings of taxus mairei. The invention obtains a marker metabolite for distinguishing the male and female seedlings of taxus mairei by screening based on a metabonomics technology, 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 (9)
1. TaxifofinThe application of the taxus chinensis in identifying the metabolic markers of the male and female seedlings of the taxus cuspidata is characterized in that the molecular formula of the taxus chinensis Qifen is C 37 H 44 O 13 Molecular weight is 696.74, and its chemical formula is shown as follows:
2. the use as claimed in claim 1, characterized in that the mass content of taxus cuspidata fischeri female seedlings is 10-30 times that of mandia androgens seedlings.
3. A method for identifying male and female strains of taxus mairei is characterized by comprising the following steps:
1) Grinding a sample of the male and female taxus densefolius plants, and extracting metabolites by using a methanol solution;
2) Performing LC-MS analysis on the supernatant of the extracting solution in the step 1), and judging whether the mass content of taxus chinensis Qifen is higher than 2.1mg/g, if so, determining that the taxus chinensis is a female taxus cuspidate plant, and if not, determining that the taxus cuspidate is a male taxus cuspidate plant;
the molecular formula of the taxus kirilowii is C 37 H 44 O 13 Molecular weight is 696.74, and its chemical formula is shown as follows:
4. the method of claim 3, wherein the step 1) of extracting the metabolites comprises: precooling 120 mu L of methanol solution with volume content of 50% to-20 ℃, adding the methanol solution into a 25mg ground taxus cuspidata sample, shaking for 1min, incubating at room temperature for 10min, standing at-20 ℃ overnight, centrifuging at 4000 Xg for 20min, and taking supernatant to stand at-80 ℃ for storage or drying for storage.
5. The method of claim 3, wherein the male and female Taxus cuspidata plants are seedlings.
6. The method of claim 3, wherein the samples of the male and female Taxus cuspidata are shoots.
7. The method of claim 3, wherein the weight content of taxus media in the female seedlings of taxus densefruit is 10-30 times that of the seedlings of mandilia male plants.
8. The method of claim 3, wherein said LC-MS analysis parameters comprise:
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 comprises: an Agilent acidic acrylamide column;
mobile phase: the mobile phase A is 25mM ammonium acetate and 25mM ammonia water, and the mobile phase B is IPA and CAN mixed solution with the volume ratio of 9;
the mobile phase gradient was: mobile phase B with volume content of 95% in 0-0.5min, mobile phase B with volume content of 95% -65% in 0.5-9.5min, mobile phase B with volume content of 65% -40% in 9.5-10.5min, mobile phase B with volume content of 40% -95% in 10.5-12min, and mobile phase B with volume content of 95% in 12-15 min;
the mass spectrometry conditions were as follows:
high resolution mass spectrometer: tripleTOF type 5600 plus;
the shielding Gas is set to be 30PSI, the ion source Gas1 is 60PSI, and the ion source Gas2 is 60PSI;
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 60-1200Da.
9.Application of taxus kirilowii in kit for identifying female and male seedlings of taxus cuspidata, which is characterized in that molecular formula of taxus kirilowii is C 37 H 44 O 13 Molecular weight is 696.74, and its chemical formula is shown as follows:
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