CN115902002A - Application of Taxus cuspidata M as metabolic marker for identifying male and female seedlings of Taxus x media - Google Patents

Application of Taxus cuspidata M as metabolic marker for identifying male and female seedlings of Taxus x media Download PDF

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CN115902002A
CN115902002A CN202211386151.6A CN202211386151A CN115902002A CN 115902002 A CN115902002 A CN 115902002A CN 202211386151 A CN202211386151 A CN 202211386151A CN 115902002 A CN115902002 A CN 115902002A
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taxus
male
female
seedlings
media
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沈晨佳
侯凯琳
张洪山
梁雪霜
冯尚国
王慧中
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Hangzhou Normal University
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Abstract

The invention discloses application of Taxus cuspidata M as a metabolic marker for identifying male and female seedlings of Taxus x media. Through the combination of chromatography and UPLC-MS/MS technology, non-targeted high-flux metabonomics analysis is carried out on male and female Taxus media seedlings on a large scale. The metabolic spectrum of male and female seedlings of the taxus media is determined, and the screened taxol M is a small molecular metabolic marker with obvious difference and is obviously accumulated in male seedlings of the taxus media. The invention provides a reliable and rapid identification method for distinguishing the male and female seedlings of taxus media, and provides an effective molecular means for the protection and artificial cultivation of endangered rare plants.

Description

Application of Taxus M in identification of metabolic markers of male and female Taxus media seedlings
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of Taxus cuspidata M as a metabolic marker for identifying male and female seedlings of Taxus media.
Background
Taxus chinensis is a tree plant of Taxus genus of Taxaceae family. Taxus chinensis is a wiggle plant in the period of fourth glacier, and is a rare or endangered species in the world. Chinese yew is a special species in China, and is classified as a primary key protection plant in China. Taxus plants have at least 15 varieties all over the world, and China has 4 varieties and one variety. Taxus chinensis has a history of millions of years on the earth, belongs to prehistoric plants, is very precious, and is an activated stone in plant kingdom. Meanwhile, the taxus chinensis is a natural rare anti-cancer plant which is recognized to be endangered to be extinct in the world.
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, taxol has been approved for clinical treatment in English, faw, day, italy, canada, sweden, germany, norway, switzerland, brazil, china and many other countries. 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.
Taxus media (Taxus x media) is a natural hybrid variety of Taxus genus of Taxaceae family, its female parent is Taxus cuspidata, and its male parent is Taxus baccata, and evergreen conifer species are mostly shrub type, and its crown is ovate, and its bark is grey or tawny, has shallow crack, and its branch is flat or obliquely vertical and densely grows, and its 2-3 year-old branch is tawny or tawny. Taxus media is an evergreen medicinal plant of Taxus genus of Taxaceae family. The taxol content is as high as 0.03-0.04%, which is higher than the Chinese yew tree species, and is the current main cultivated species. Research shows that the taxol content of the male and female Taxus media plants is obviously different, and the male and female Taxus media plants are higher than the female and male Taxus media plants. Therefore, the commercial value of female seedlings of Taxus media is much higher than that of male seedlings. At present, the research on Taxus media has made a preliminary progress, but the male and female seedlings are highly similar in morphology, and the growing environments are highly overlapped, which brings certain difficulties for the differential identification 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 metabonomics (unordered metabonomics) refers to the unbiased detection of dynamic changes of all small molecule metabolites (mainly endogenous small molecule compounds with the relative molecular weight within 1000 Da) before and after stimulation or disturbance in cells, tissues, organs or organisms by adopting LC-MS, GC-MS and NMR technologies, and differential metabolites are screened by biological information analysis, and the pathway analysis is carried out on the differential metabolites to reveal the physiological mechanism of the changes. 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, the research of screening metabolic markers by utilizing metabonomics technology and identifying female and male woody plants is not much.
Disclosure of Invention
The invention aims to provide the application of Taxus M as a metabolic marker for identifying male and female Taxus media seedlings in view of the defects of the prior art, and the content of Taxus M in male Taxus media seedlings is far higher than that of female Taxus media seedlings, so that a good technical means is provided for solving the problem of tree species identification in the breeding and forestation processes of Taxus media.
The application of the taxol M as a metabolic marker for identifying the male and female taxus media seedlings is characterized in that the taxol M has the following chemical characteristics: the molecular formula is C 35 H 42 O 14 The molecular weight is 686.7, and the chemical structural formula is shown as follows:
Figure BDA0003929857630000021
preferably, the mass content of the taxol M in the male Taxus media seedlings is 30-55 times of that of the female Taxus media seedlings.
The invention also aims to provide a detection method of the male and female Taxus media, which specifically comprises the following steps:
1) And extracting metabolites from the ground branches of the male and female Taxus media plants by using a 45-55% methanol solution.
Adding 120 mu L of methanol solution with volume content of 45% -50% and precooled to minus 20 ℃ into a 25mg taxus chinensis sample, slightly shaking for 1min, incubating at room temperature for 10min, standing overnight at minus 20 ℃, centrifuging at 4000 Xg for 20min, and storing the supernatant at minus 80 ℃ or drying and storing;
2) Performing LC-MS analysis on the supernatant of the extracting solution, and judging according to the mass content of the taxol M, wherein if the mass content of the taxol M is more than or equal to 0.2mg/g, the seedlings are male Taxus media plants, and if the mass content of the taxol M is less than 0.006mg/g, the seedlings are female Taxus media plants;
the conditions of the high performance gas chromatography are as follows:
the hue system is: SCIEX UPLC system;
the chromatographic column temperature was: 35 ℃;
the chromatographic column is as follows: agilent acid acrylamide column (100 mm. Times.2.1mm, 1.7 μm, waters, UK);
mobile phase: the mobile phase A is 25mM ammonium acetate and 25mM ammonia water, and the mobile phase B is IPA and ACN mixed solution with the volume ratio of 9;
the mobile phase gradient was: the mobile phase B with the volume content of 95% is 0-0.5min, the mobile phase B with the volume content of 95-65% is 0.5-9.5min, the mobile phase B with the volume content of 65-40% is 9.5-10.5min, the mobile phase B with the volume content of 40-95% is 10.5-12min, and the mobile phase B with the volume content of 95% is 12.2-15 min;
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.
Still another object of the present invention is to provide the use of Taxus cuspidata M in a kit for identifying male and female seedlings of Taxus media.
The invention has the following beneficial effects:
1. the invention obtains a marker for distinguishing the male and female seedling metabolites of taxus media and provides a detection method thereof. The invention obtains the metabolite markers for distinguishing the male and female Taxus media seedlings by analyzing the metabolic spectrum of the male and female Taxus media seedlings, identifying the chemical components of the metabolites, and screening the metabolites which are obviously differentially accumulated.
2. The invention has the advantages of high precision, programmed operation, high sensitivity 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 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.
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 media seedlings, identifies a series of metabolites in different tree species, and obtains the differential metabolites between the two through quantitative analysis. And screening to obtain the metabolites with obvious difference by a P value significance analysis method.
Drawings
FIG. 1 is the annotation result of the metabolite of the male and female Taxus media;
FIG. 2 metabolite principal component analysis of male and female young Taxus media;
FIG. 3 is a liquid chromatogram of a standard Taxus M (taxine M);
FIG. 4 is a verification of the reliability of the marker differential metabolite of Taxus media.
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 media male and female plant seedling metabolite and control sample preparation
Treatment of experimental samples:
the small branches (twigs) of Taxus media were accurately weighed out at 25mg each for metabolite extraction.
120 mu L of precooled 45% -50% methanol solution is added into the ground Taxus media sample and mixed 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 sets of samples from the male and female Mandia plants, a set of control samples was prepared. The control sample was formulated as follows: taking 10 independent experimental samples of male taxus media strains and 10 independent experimental samples of female taxus media strains, and mixing the 20 samples in equal volume. The mixed solution was divided into 10 portions equally to prepare control samples.
Example 2: annotation of metabolites of male and female Taxus media
In the implementation process of the invention, a large amount of mass spectrum data including peak (peak), retention time (retention time), annotation information and the like 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 ion information generated was compared to its exact molecular weight and annotated by KEGG and HMDB online databases.
The present invention has obtained 2454 total effective ion characteristics and thus produced 1426 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 amino acid metabolites (321), sugar metabolites (143), basal metabolites (233), terpene metabolites (233), steroid metabolites (21), energy metabolites (54), nucleotide metabolites (54), fatty acid metabolites (123) and other metabolites (244), and specific results are shown in fig. 1.
Example 3: taxus media male and female young plant marker differential metabolite screening
In order to screen the marked differential metabolites of the taxus media and the taxus cuspidata, the main quality control parameter of Principal Component Analysis (PCA) is detected. The main component analysis result shows that the variance values of PC1 and PC2 are 53.53% and 9.32% respectively, which indicates that the difference of different genders of Taxus media is high, and the specific PCA analysis result is shown in FIG. 2. Statistical results show that 121 metabolites which are remarkably accumulated in a co-screening manner are obtained, wherein 80 metabolites are remarkably accumulated in male Taxus media, and the other 41 metabolites are remarkably accumulated in female Taxus media. 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 main metabolites are greatly different between the male and female Taxus media. The invention identifies and obtains the taxuschinensis M as the marked differential metabolite of the taxus media with different sexes by analyzing the remarkably differential accumulated metabolites.
Example 4: taxus M (taxine M) standard substance and liquid chromatography-mass spectrometry thereof
Buying Taxine M (Taxinine M) on chemical source network) The standard substance is characterized by comprising the following components: CAS number 135730-55-1; the molecular weight is 686.7; molecular formula C 35 H 42 O 14 . The standard mass spectrum is shown in FIG. 3. And accurately measuring the content of the taxol M in the taxus chinensis sample by using the standard substance.
Example 5: reliability verification example for distinguishing marker difference metabolites of taxus media with different sexes
The five-year-old male and female Taxus media plant cultivation seedlings are planted in the Hangzhou university experiment nursery. The collected samples are respectively numbered as follows: male1-6 (Male Taxus media) and Female1-6 (Female Taxus media).
Fully drying collected Taxus media twigs in a 40 ℃ 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 uniformly mixing. 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 average content of the taxol M in male taxus media seedlings is obviously higher than that of east female seedlings. The specific results are shown in FIG. 4. The above results demonstrate the reliability of taxol M as a marker metabolite for differentiating taxus media of different sexes. The invention screens and obtains a marked metabolite which can be used for distinguishing the female and male seedlings of taxus media based on the 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 (7)

1. Application of taxol M as a metabolic marker for identifying male and female seedlings of taxus media, which is characterized in that the molecular formula of taxol M is C 35 H 42 O 14 The molecular weight is 686.7, and the chemical structural formula is shown as follows:
Figure FDA0003929857620000011
2. a detection method for distinguishing male and female Mandia plants is characterized by comprising the following steps:
step (1): grinding a sample of the male and female Taxus media plant, and extracting metabolites from the sample with methanol solution;
step (2): performing LC-MS analysis on the supernatant of the extracting solution, and judging whether the mass content of taxol M is more than or equal to 0.2mg/g, if so, determining male seedlings of taxus media, and otherwise, determining female seedlings of taxus media;
the molecular formula of the taxol M is C 35 H 42 O 14 The molecular weight is 686.7, and the chemical structural formula is shown as follows:
Figure FDA0003929857620000012
3. the method of claim 2, wherein the weight content of Taxol M in male seedlings of Taxus media is 30-55 times that of female seedlings of Taxus media.
4. The method of claim 2, wherein the samples of male and female Taxus media are seedlings.
5. The method of claim 2, 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 is as follows: an Agilent acidic acrylamide column;
mobile phase: the mobile phase A is 25mM ammonium acetate +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: the volume content of the mobile phase B is 95% in 0-0.5min, the volume content of the mobile phase B is 95-65% in 0.5-9.5min, the volume content of the mobile phase B is 65-40% in 9.5-10.5min, the volume content of the mobile phase B is 40-95% in 10.5-12min, and the volume content of the mobile phase B is 95% in 12.2-15 min;
the 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.
6. The method according to claim 2, characterized in that step (1) is in particular: adding 120 μ L of methanol solution with volume content of 45% -50% pre-cooled to-20 deg.C into 25mg Taxus chinensis sample, shaking for 1min, incubating at room temperature for 10min, standing at-20 deg.C overnight, centrifuging at 4000 × g for 20min, and storing the supernatant at-80 deg.C or drying for storage.
7. Application of taxol M in a kit for identifying male and female seedlings of taxus media is characterized in that the molecular formula of taxol M is C 35 H 42 O 14 The molecular weight is 686.7, and the chemical structural formula is shown as follows:
Figure FDA0003929857620000021
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CN202211386151.6A 2022-11-07 2022-11-07 Application of Taxus cuspidata M as metabolic marker for identifying male and female seedlings of Taxus x media Pending CN115902002A (en)

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