CN116195443B - Method for improving patchouli alcohol content - Google Patents
Method for improving patchouli alcohol content Download PDFInfo
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- CN116195443B CN116195443B CN202310263000.XA CN202310263000A CN116195443B CN 116195443 B CN116195443 B CN 116195443B CN 202310263000 A CN202310263000 A CN 202310263000A CN 116195443 B CN116195443 B CN 116195443B
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- GGHMUJBZYLPWFD-UHFFFAOYSA-N patchoulialcohol Chemical compound C1CC2(C)C3(O)CCC(C)C2CC1C3(C)C GGHMUJBZYLPWFD-UHFFFAOYSA-N 0.000 title claims abstract description 84
- GGHMUJBZYLPWFD-MYYUVRNCSA-N Patchouli alcohol Natural products O[C@@]12C(C)(C)[C@H]3C[C@H]([C@H](C)CC1)[C@]2(C)CC3 GGHMUJBZYLPWFD-MYYUVRNCSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 41
- 235000011751 Pogostemon cablin Nutrition 0.000 claims abstract description 46
- 238000005286 illumination Methods 0.000 claims abstract description 23
- 241001529821 Agastache Species 0.000 claims abstract description 12
- 241000222666 Boerhavia diffusa Species 0.000 claims abstract 4
- 230000001965 increasing effect Effects 0.000 claims description 12
- 241000196324 Embryophyta Species 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 230000014509 gene expression Effects 0.000 abstract description 6
- 240000002505 Pogostemon cablin Species 0.000 description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 4
- 239000000306 component Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000000341 volatile oil Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001072961 Pogostemon Species 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 1
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- -1 sesquiterpene compound Chemical class 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
- A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Cultivation Of Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for improving patchouli alcohol content, which comprises the following steps: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m 2 ·s ‑1 ～24μmol·m 2 ·s ‑1 Red light 12 mu mol.m 2 ·s ‑1 ～16μmol·m 2 ·s ‑1 Infrared light 0.8 mu mol.m 2 ·s ‑1 ～1.2μmol·m 2 ·s ‑1 . In the invention, when the patchouli tissue culture seedling is subjected to illumination treatment, blue light in the light quality is removed, and other light quality is reserved, so that the expression level of patchouli alcohol synthase gene can be obviously improved, and the patchouli alcohol content is improved.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for improving patchouli alcohol content in patchouli and a planting method of patchouli.
Background
Pogostemon cabin (Blanco) Benth is a plant of genus Pogostemon of Labiatae, and has effects of removing turbid pathogen with aromatics, stimulating appetite, relieving vomiting, relieving exterior syndrome, and relieving summer-heat. The patchouli volatile oil obtained by distilling patchouli is widely applied to the fields of food, medicines, daily chemicals and the like.
Patchouli alcohol is a sesquiterpene compound and is the component with the highest content in patchouli volatile oil. The content of patchouli alcohol in the patchouli dry medicinal material is not less than 0.10% specified in Chinese pharmacopoeia. At present, the annual yield of the global patchouli alcohol and the essential oil mixture thereof exceeds 1000 tons, but the annual demand is up to 2000 tons, and the 90 percent yield of the global patchouli alcohol is from Indonesia, and the supply of the local patchouli alcohol in China is seriously insufficient.
Research shows that exogenous application of the plant hormone methyl jasmonate can increase the content of patchouli alcohol in patchouli, but the method not only greatly increases the production cost, but also can influence the quality and safety of patchouli due to excessive application of the plant hormone.
Therefore, the method for economically and safely improving the content of the patchouli alcohol is found, and has important significance for relieving the requirements of medical treatment and production life on the patchouli alcohol.
Disclosure of Invention
Based on the above, the invention aims to provide a method for improving the patchouli alcohol content and a planting method of patchouli.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the present invention, there is provided a method for increasing patchouli alcohol content, said method comprising the steps of: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m 2 ·s -1 ～24μmol·m 2 ·s -1 Red light 12 mu mol.m 2 ·s -1 ～16μmol·m 2 ·s -1 Infrared light 0.8 mu mol.m 2 ·s -1 ～1.2μmol·m 2 ·s -1 。
In a second aspect of the present invention, there is provided a planting method of pogostemon cablin, the method comprising the steps of: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m 2 ·s -1 ～24μmol·m 2 ·s -1 Red light 12 mu mol.m 2 ·s -1 ～16μmol·m 2 ·s -1 Infrared light 0.8 mu mol.m 2 ·s -1 ～1.2μmol·m 2 ·s -1 。
In the invention, when the patchouli tissue culture seedling is subjected to illumination treatment, blue light in the light quality is removed, and other light quality is reserved, so that the expression level of patchouli alcohol synthase gene can be obviously improved, and the patchouli alcohol content is improved.
Drawings
FIG. 1 is a graph showing the peak of the mass spectrum measurement of patchouli alcohol content in patchouli leaves under different illumination conditions in test example 1 of the present invention.
FIG. 2 is a graph showing the peak area results of the mass spectrometry peak diagram in FIG. 1.
FIG. 3 shows the results of detecting the expression level of patchouli alcohol synthase gene in patchouli leaves under different illumination conditions in test example 2 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In some embodiments of the present invention, a method of increasing patchouli alcohol content is disclosed, the method comprising the steps of: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m 2 ·s -1 ～24μmol·m 2 ·s -1 Red light 12 mu mol.m 2 ·s -1 ～16μmol·m 2 ·s -1 Infrared light 0.8 mu mol.m 2 ·s -1 ～1.2μmol·m 2 ·s -1 。
The inventor of the invention unexpectedly found in experiments that when the patchouli tissue culture seedlings are subjected to illumination treatment, blue light in the light is removed, other light (green light, red light and infrared light are kept unchanged), and under the operation, the expression level of patchouli alcohol synthase genes can be obviously improved, so that the patchouli alcohol content is improved.
In some embodiments, the number of days of light treatment is 50 to 70 days.
In some embodiments, the number of days of light treatment is from 55 days to 65 days.
In some embodiments, the time of the daily light treatment is 10 to 14 hours.
In some embodiments, the time of the daily light treatment is 11-13 hours.
In some of these embodiments, the intensity of the light treatment is 2500lux to 3500lux.
In some embodiments, the plant height of the patchouli tissue culture seedling is 5 cm-6 cm.
In some of these embodiments, the light treatment temperature is from 23 ℃ to 27 ℃.
In other embodiments of the present invention, a method of planting pogostemon cablin is disclosed, the method comprising the steps of: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m 2 ·s -1 ～24μmol·m 2 ·s -1 Red light 12 mu mol.m 2 ·s -1 ～16μmol·m 2 ·s -1 Infrared light 0.8 mu mol.m 2 ·s -1 ～1.2μmol·m 2 ·s -1 。
In some embodiments, the number of days of light treatment is 50 to 70 days.
In some embodiments, the time of the daily light treatment is 10 to 14 hours.
In some of these embodiments, the intensity of the light treatment is 2500lux to 3500lux.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1A method for increasing patchouli alcohol content
The method comprises the following steps:
1. pogostemon cablin leaf surface alcohol disinfection
And (3) cleaning the surfaces of the picked blades by adopting a third pair of blades of patchouli, and then sucking residual water drops on the surfaces of the blades. After placing the leaves in 75% ethanol, rinsing back and forth for 10 seconds, transferring the leaves to a first bottle of sterile water, shaking for 30 seconds, and rotating the leaves along with the water. And after the shaking is finished, the blades are sequentially placed into the second bottle and the third bottle of sterile water, and the shaking time is 60 seconds.
2. Pogostemon cablin leaf surface mercury lift disinfection
Adding 0.1g of mercuric powder into 100ml of pure water to prepare 0.1% mercuric solution, performing ultrasonic treatment until the mercuric is completely dissolved, and dripping one drop of Tween-80 to prepare the mercuric disinfectant.
The leaf sterilized by alcohol is transferred into mercuric chloride sterilizing liquid and is shaken for about 8 minutes. The shaking force is not too large, the blades can shake along with water, and damage to the blades is increased due to too large force. Care was taken to observe the state of the blade during shaking. After shaking, the leaves are sequentially placed into the fourth to seventh bottles of sterile water, and the shaking time is 30 seconds, 60 seconds and 60 seconds in sequence, namely the sterilization is completed.
3. Induction of patchouli cluster buds
Cutting the sterilized leaves into small pieces of about 0.5cm multiplied by 0.5cm on an ultra-clean workbench, placing the small pieces on a patchouli cluster bud induction medium (the culture medium components are shown in table 1), placing the small pieces in a culture room at the temperature of (25+/-2), culturing the small pieces in the culture room, illuminating the small pieces by using a fluorescent lamp, wherein the illumination time is 12h/d (8:00-20:00), and the illumination intensity is 2500lux. After 2-3 months, the patchouli cluster buds are induced.
TABLE 1
4. Inducing rooting of patchouli cluster buds into tissue culture seedlings
Transferring the strong patchouli cluster buds to rooting culture medium (the components of the culture medium are shown in Table 1), placing the patchouli cluster buds in a culture room at the temperature of (25+/-2), culturing the patchouli cluster buds in the culture room, illuminating the patchouli cluster buds by using a fluorescent lamp for 12h/d (8:00-20:00), culturing the patchouli cluster buds for about 2 months at the illumination intensity of 2500lux, and inducing rooting.
5. Treatment of tissue culture seedlings
When the plant height of the rooted patchouli tissue culture seedling reaches 5-6 cm, the treatment can be carried out.
Placing the tissue culture seedlings into a climatic incubator, and receiving illumination under the following conditions: blue light: 0; green light: 22 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Red light: 14 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the And (3) infrared light: 1 mu mol m 2 ·s -1 . The temperature of the incubator is adjusted to 25 ℃, the incubator is cultivated for two months, and the patchouli leaves are collected.
Example 2A method for increasing patchouli alcohol content
The method comprises the following steps:
steps 1 to 4 are the same as in example 1.
5. Treatment of tissue culture seedlings
When the plant height of the rooted patchouli tissue culture seedling reaches 5-6 cm, the treatment can be carried out.
Placing the tissue culture seedlings into a climatic incubator, and receiving illumination under the following conditions: blue light: 0; green light: 20 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Red light: 15 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the And (3) infrared light: 1.2. Mu. Mol.m 2 ·s -1 . The temperature of the incubator is regulated to 26 ℃, the incubator is cultivated for two months, and the patchouli leaves are collected.
Example 3A method for increasing patchouli alcohol content
The method comprises the following steps:
steps 1 to 4 are the same as in example 1.
5. Treatment of tissue culture seedlings
When the plant height of the rooted patchouli tissue culture seedling reaches 5-6 cm, the treatment can be carried out.
Placing the tissue culture seedlings into a climatic incubator, and receiving illumination under the following conditions: blue light: 0; green light: 24 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Red light: 12 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the And (3) infrared light: 0.8 mu mol m 2 ·s -1 . The temperature of the incubator is regulated to 24 ℃, the incubator is cultivated for two months, and the patchouli leaves are collected.
Comparative example
The comparative example is a control experiment, the patchouli tissue culture seedlings with the same growth period as in the example 1 and the same growth vigor are taken and put into a climatic incubator to receive illumination, and the illumination condition is normal illumination, namely: blue light: 14 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Green light: 22 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Red light: 14 mu mol m 2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the And (3) infrared light: 1 mu mol m 2 ·s -1 . The temperature of the incubator is adjusted to 25 ℃, the incubator is cultivated for two months, and the patchouli leaves are collected.
Test example 1 Effect of different illumination conditions on patchouli alcohol content in patchouli leaves
The leaves of example 1 and comparative example were cut. Samples of the same area were obtained on the same blade age of example 1 and comparative example using a 6mm diameter punch. And (3) measuring the content of patchouli alcohol in the volatile substances by adopting an Agilent 8890-5977B gas chromatograph-mass spectrometer and a solid phase microextraction-headspace sampling-gas chromatograph-mass spectrometer (SPME-HS-GC-MS) detection method.
The specific method comprises the following steps: the patchouli leaves were added to 15mL headspace sample bottles, incubated at 50deg.C for 10min, extracted with 50/30 μm CAR/PDMS/DVB extraction head at 50deg.C for 5min, and desorbed at 250deg.C for 1min. The column was an Agilent HP-5MS capillary column (0.25 mm. Times.30 m,0.25 μm). The carrier gas is high-purity helium, the flow rate of the carrier gas is 1.2mL/min, the temperature of the sample inlet is 250 ℃, and the split ratio is 20:1. Programming temperature: the initial temperature was 45℃for 0.5min, and the temperature was raised to 280℃at 10℃per min for 0.5min. The ion source EI source has electron energy of 70eV, and the scanning mode is full scanning, and the mass range m/z is 50-450. Compound identification and data processing: the relative content of the compounds was calculated by chromatographic peak area normalization and compound identification was performed by Agilent MassHunter chemical workstation in combination with NIST2017 standard mass spectrum library.
The mass spectrum measurement peak diagram of volatile matters in patchouli leaves is shown in figure 1, and the peak area (namely patchouli alcohol content) of the peak diagram corresponding to figure 1 is shown in figure 2. As is clear from the results of fig. 2, the content of patchouli alcohol in example 1 (treatment group in the figure) was significantly increased compared to the comparative example (control group in the figure), which means that the increase of patchouli alcohol content was significantly promoted by adjusting the blue light in the light irradiation to 0.
Test example 2 Effect of different light conditions on the transcriptional level of patchouli leaf patchouli alcohol synthase
The patchouli leaves of the example 1 and the comparative example are cut, and the expression level of patchouli alcohol synthase genes in the leaves is detected by adopting fluorescence quantitative PCR, and the specific method comprises the following steps:
1. extraction of total RNA from plant tissue
Placing the sample into a 1.5ml centrifuge tube, grinding the sample by liquid nitrogen, and adding 1ml Trizol; mixing, and standing on ice for 5 min; adding 0.2ml chloroform, shaking or mixing upside down, and standing on ice for 5 min; 12000g,4 ℃, centrifugation for 12 minutes; taking the supernatant into a new centrifuge tube, adding 0.5ml of isopropanol, uniformly mixing, and standing at-20 ℃ for 10-30 minutes; 12000g,4 ℃, centrifugation for 12 minutes; discarding the supernatant, rinsing the precipitate with 75% ethanol for 2-3 times, airing, dissolving with a proper amount of DEPC water, and preserving at-20 ℃ for standby.
2. Synthesis of first strand of reverse transcribed cDNA
Using TaKaRaRT reagent Kit With gDNA Eraser (Takara, RR 047A), the first step of the reverse transcription was carried out using 10. Mu.L of the reaction system, and the system was prepared as shown in Table 2.
TABLE 2
| Totalvolume | 10μl |
| TotalRNA | 1μg |
| 5×gDNAEraserBuffer | 2μl |
| gDNAEraser | 1μl |
| RNAaseFreedH 2 O | upto10μl |
The reaction mixture was stirred by flicking the EP tube and centrifuged instantaneously, and incubated at 42℃for 2 minutes (on a PCR apparatus). The second step of reverse transcription was then carried out and the system was prepared as shown in Table 3 using 20. Mu.L of the reaction system.
TABLE 3 Table 3
The reagents were mixed with a flick EP tube, centrifuged briefly, and incubated at 37℃for 15 minutes, 85℃for 5 seconds, and stored at-20 ℃.
3. Fluorescent real-time quantitative PCR
20. Mu.L of cDNA product obtained by reverse transcription was subjected to ddH 2 O was diluted 10-fold for use. The reaction system was prepared by 10. Mu.L of the reaction system, and the system was prepared as shown in Table 4.
TABLE 4 Table 4
Amplification was performed using a Roche fluorescent quantitative PCR apparatus, the reaction procedure was:
4. the reaction data were processed by LightCycler480 Software1.5 and then byThe analysis method calculates the gene expression level.
In this test example, transcript levels of patchouli alcohol synthase (PatPTS) in patchouli leaves were detected by fluorescence quantitative PCR, and the results are shown in FIG. 3. As can be seen from FIG. 3, the transcript level of the patchouli alcohol synthase gene in example 1 (the treatment group in the figure) was significantly increased relative to the transcript level of the patchouli alcohol synthase gene in the comparative example (the control group in the figure), indicating that the patchouli alcohol content was increased due to the increase of the transcript level of the patchouli alcohol synthase gene.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (4)
1. A method for increasing patchouli alcohol content, comprising the steps of: collecting herba Agastaches leaves after illumination treatment of herba Agastaches tissue culture seedling; the light treatment includes: green light 20 mu mol.m.s -1 ~24 μmol·m²·s -1 Red light 12 mu mol.m.s -1 ~16 μmol·m²·s -1 Infrared light 0.8 mu mol.m.s -1 ~1.2 μmol·m²·s -1 The method comprises the steps of carrying out a first treatment on the surface of the The number of days of the illumination treatment is 50-70 days; the time of the illumination treatment is 10-14 hours per day; the intensity of the light treatment is 2500 lux~3500 lux; the plant height of the patchouli tissue culture seedling is 5 cm-6 cm; the temperature of the light treatment is 23-27 ℃.
2. The method for increasing patchouli alcohol content according to claim 1, wherein the number of days of the light treatment is 55 days to 65 days.
3. The method for increasing patchouli alcohol content according to claim 1, wherein the time of the light treatment per day is 11-13 h.
4. A method for planting patchouli, which is characterized in that the method for improving patchouli alcohol content in patchouli is applied according to any one of claims 1 to 3.
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