Preparation method of artemisia apiacea diene-containing distant hybridization tobacco material
Through the distant hybridization of China Tobacco 300 and Yinsheng tobacco Nicotiana umbratica, a new variety F1158 containing artemisinin was successfully obtained, which solved the problem of low success rate of distant hybridization and a shortage of artemisinin, and provided plant-derived natural artemisinin diene, which promoted the in vitro synthesis of artemisinin.
Patent Information
- Application Number
- CN202510075210.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art is difficult to successfully obtain new varieties containing artemisinin through distant hybridization, and the natural source of artemisinin is limited, resulting in a problem of supply and demand.
By using Zhongchi 300 as the parent and Yinsheng tobacco Nicotiana umbratica as the parent, the distal hybrid F1158 containing Artemisia diene was successfully obtained. The method includes collecting pollen from the parent, de-pollinating the parent, and obtaining distant hybrid seeds through planting and management.
It has achieved the successful acquisition of new varieties containing artemisinin in distant hybridization, provided natural artemisinin diene from plant origin, promoted the in vitro synthesis and development of artemisinin, and solved the problem of insufficient supply of artemisinin.
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Figure CN119563544A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant breeding, and in particular to a method for preparing a distant hybridization tobacco material containing artemisinadiene. Background Art
[0002] Distant hybridization is a special hybridization method, which involves different species, genera, and even species with more distant relationships. This hybridization method can break through the limitations of reproductive isolation, bring together the excellent traits of different species, greatly expand the scope of genetic variation, and is of vital importance for variety improvement, the creation of new varieties, and the enrichment of germplasm resources. However, due to the reproductive differences between different species, distant hybridization often faces many difficulties. For example, hybrid incompatibility, difficulty in successful fruiting, and early death or infertility of hybrid offspring often occur, which makes the probability of success of distant hybridization small and the technical difficulty is quite high. At present, people often use a variety of means to overcome these difficulties, such as extensive test crosses to screen out effective female parents, adopt repeated pollination, early or delayed pollination, mixed pollination, etc., as well as media method, chromosome doubling method, etc. Although distant hybridization technology is difficult and has a low success rate, as long as the hybridization is successful, new varieties with excellent specific traits that cannot be produced by close hybridization can be obtained, thereby providing diversified and strong support for germplasm innovation and enriching breeding materials.
[0003] Amorphadiene, also known as amorphadiene, belongs to the sesquiterpenoids and is a natural metabolite with a molecular formula of C 15 H 24 , with a molecular weight of 204.35. Terpenoids are widely used in many fields such as industry, medicine, flavors and fragrances due to their special properties. Artemisinin is a new antimalarial drug extracted from the traditional Chinese medicinal material Artemisia annua, and artemisinin is an important precursor for the synthesis of artemisinin. After ring-opening peroxidation, artemisinin diene can generate the peroxylactone compound artemisinin. At present, artemisinin is the first choice for the treatment of malignant malaria worldwide. It has good therapeutic effects and high demand, but its natural sources are limited, the actual production volume is low, and the supply is insufficient. Artemisinin diene, as a precursor for the synthesis of artemisinin, is particularly important in the development of antimalarial drugs. In addition, studies have found that artemisinin diene may have certain activities in anti-inflammatory and anti-tumor aspects. In terms of anti-inflammatory, artemisinin diene may participate in related cell signaling pathways to regulate inflammatory responses; in anti-tumor studies, artemisinin diene may have growth inhibitory effects on certain tumor cells. Therefore, studying more natural artemisinadiene from plant sources is of great significance for the development of in vitro synthesis of artemisinin. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a distant hybrid tobacco material containing artemisinin to solve the problems existing in the above-mentioned prior art. The present invention uses Zhongyan 300 as a female parent and shade-grown tobacco Nicotiana umbratica as a male parent for distant hybridization to obtain a distant hybrid F1158 containing artemisinin (neither of the parents contains artemisinin), which is of great significance for the development of in vitro synthesis of artemisinin.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The invention provides a method for preparing a distant hybridization tobacco material containing artemisinin, comprising the steps of performing distant hybridization with Zhongyan 300 as a female parent and shade-grown tobacco Nicotiana umbratica as a male parent.
[0007] Preferably, pollen from the male wild shade-growing tobacco Nicotiana umbratica is collected, and the female Zhongyan 300 is emasculated; the pollen is applied to the pistil of the female parent for pollination; and distant hybrid seeds are collected after maturity;
[0008] The distant hybrid seeds are planted.
[0009] Optionally, the anthers of the male parent are collected when the anthers have reached a mature state but have not yet cracked; the collected anthers are placed in an environment with a temperature of 25° C. until the anthers mature, crack and release pollen.
[0010] Optionally, before the flowers of the female parent open, well-developed flower buds are selected for emasculation.
[0011] Optionally, the pollination is repeated pollination to increase the success rate.
[0012] The present invention also provides application of the distant hybridization tobacco material containing artemisinin obtained according to the preparation method in producing artemisinin.
[0013] The present invention also provides the use of the distant hybridization tobacco material containing artemisinin obtained according to the preparation method in the production of artemisinin.
[0014] The present invention discloses the following technical effects:
[0015] The present invention uses Zhongyan 300 as a female parent and shade-growing tobacco Nicotiana umbratica as a male parent for distant hybridization, and obtains a distant hybrid F1158 containing artemisinin (neither of the parents contains artemisinin). After testing, the leaves and flowers of the hybrid F1158 contain artemisinin. With the artemisinin standard as a reference, the artemisinin content is quantitatively analyzed and found to be 182.5768 mg / kg in the flowers and 876.3185 mg / kg in the leaves. The present invention can provide natural artemisinin from plant sources, which is of great significance for the development of in vitro synthesis of artemisinin. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0017] Figure 1 This is the phenotype diagram of the hybrid F1158;
[0018] Figure 2 This is the result of SPME-GCMS method to determine the volatile aroma components of hybrid F1158 flower buds;
[0019] Figure 3 Chromatogram for the determination of aroma components in the leaves of hybrid F1158;
[0020] Figure 4 Chromatogram showing the determination of aroma components in the flowers of hybrid F1158. DETAILED DESCRIPTION
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0026] The flue-cured tobacco variety Zhongyan 300 and the wild shade-grown tobacco Nicotiana umbratica used in the present invention are provided by the National Tobacco Mid-Term Bank.
[0027] Example 1
[0028] 1. Material creation method
[0029] 1.1 Parent selection
[0030] Distant hybridization was carried out with the flue-cured tobacco variety Zhongyan 300 as the female parent and the wild shade-growing tobacco Nicotiana umbratica as the male parent.
[0031] 1.2 Pollen collection and processing
[0032] When the anthers of the male parent shade-growing tobacco Nicotiana umbratica have reached a mature state but have not yet cracked, the anthers are collected. The collected anthers are placed in a clean and uncontaminated culture dish, the lid is opened, and it is placed in a clean and tidy environment at a temperature of 25°C until the anthers mature, crack and release pollen. After the anthers release pollen, cover them in time to prevent pollen from being contaminated.
[0033] 1.3 Zhongyan 300 emasculation
[0034] Before the flowers of the mother plant Zhongyan 300 bloom, select well-developed buds and carefully remove all anthers using tweezers to avoid damaging the pistil.
[0035] 1.4 Distant hybridization pollination
[0036] In clear weather, select fresh pollen with high vitality, that is, pollen when the anthers have just cracked. Use a cotton swab to apply it to the pistil of the mother plant, ensuring that the entire pistil is evenly coated. After pollination is completed, mark and bag the plants to prevent pollen cross-pollination.
[0037] 1.5 Post-pollination management
[0038] Observe the flowering of the female plant every day and remove the male plants in time to prevent pollination. Due to the incompatibility of distant hybridization, regularly re-pollinate the flowers that have been pollinated to increase the success rate of distant hybridization fertilization.
[0039] After pollination, follow-up surveys are conducted regularly to understand the development of the fruit. After hybridization, field management of the plants is strengthened, fertilizer and water, soil moisture, pest and disease control are regularly done, and regular pruning is performed to ensure that the hybrid fruit is adequately nutritious and grows normally. When the distant hybrid seeds mature, all distant hybrid seeds are collected.
[0040] 2. Screening and trait characteristics of distant hybrid offspring
[0041] According to the conventional method, the distant hybrid seeds harvested were planted and one hybrid germinated normally. After germination, at the two-leaf stage, most of the hybrid roots and stems turned brown and then died. They could not grow normally, with a survival rate of about 0.05%. The hybrid offspring F1 was infertile. It was named distant hybrid F1158.
[0042] The agronomic characteristics of hybrid F1158 at full flowering stage are shown in Table 1 and Figure 1 As shown: the average plant height is 160cm, leaf length is 44.30cm, leaf width is 33.70cm, number of leaves is 22, petiole is 10.50cm, and there are leaf ears.
[0043] The survey data of agronomic traits of the female parent Zhongyan 300 in the flowering period are shown in Table 1: the average plant height is 166.0 cm, the leaf length is 65.20 cm, the leaf width is 29.375 cm, the number of leaves is 28, there are no petioles, and there are leaf ears.
[0044] Table 1 Agronomic traits of hybrid F1158 and its female parent Zhongyan 300
[0045]
[0046] 3. Detection and analysis of aroma components of hybrids
[0047] 3.1 Determination of aroma components of hybrid F1158 fireworks by solid phase microextraction (SPME)
[0048] Take the hybrid F1158 fireworks sample into a 15mL headspace bottle. Note that the sample volume does not exceed 1 / 2 of the bottle capacity, leaving space above the headspace bottle. Insert the SPME needle through the sample bottle injection septum and into the headspace bottle. Gently push the handle bar to extend the extraction fiber head so that it is exposed to the headspace of the sample. Adsorption equilibrium for 20 minutes. Carefully push back the fiber head, pull out the needle, quickly insert it into the gas chromatography (GC) injection port, push out the fiber head, desorb the sample analytes adsorbed on the fiber head, and enter the chromatographic column for separation and analysis.
[0049] The present invention uses solid phase microextraction SPME-GCMS technology to detect the volatile components in the flower buds of the parents and distant hybrids. The analysis shows that the flower bud sample of the distant hybrid F1158 has a unique product at the peak time of 21.8745min, with a peak area of 259264337.471082 and a molecular formula of C 15 H 24 The mass spectrum was compared with the NIST library and the results showed that this compound was artemisinic acid diene (cas: 92692-39-2) (see Figure 2 ), but neither parent contains artemisinin. Therefore, the present invention successfully obtains distant hybridization breeding materials containing artemisinin through distant hybridization.
[0050] 3.2 Determination of aroma components in hybrid F1158 leaves and fireworks using gas chromatography-mass spectrometry (GC-MS)
[0051] According to the material-liquid ratio of 1:8 (g / mL), 5g of leaves and flowers of hybrid F1158 were taken respectively, ground with liquid nitrogen, quickly placed in 40mL of ether, mixed and sealed. Extract and soak for 2h at room temperature and 100rpm, filter coarsely to remove larger impurities. Blow ether with nitrogen to 5mL, remove water through anhydrous sodium sulfate, pass through 0.22μM organic phase filter membrane, and prepare GCMS samples. Take 300μL for gas chromatography-mass spectrometry (GC-MS) analysis. Injection volume: 1μL. Chromatographic conditions: keep warm at 60℃ for 3min, heat to 280℃ at 10℃ / min, keep for 5min, helium flow rate 1ml / min. Mass spectrometry conditions: ion source temperature 300℃, interface temperature 250℃, scan mode acquisition m / z50-500. GC-MS detection results show that a total of 1 product was detected at 14.997min. Its mass spectrum was compared with the NIST library, and the results showed that this compound was artemisinic acid diene.
[0052] The leaves and flowers of the hybrid F1158 of the present invention were tested by SPME-GCMS and GCMS technology and found that both the leaves and the fireworks contained artemisinin. The leaf sample had a unique product at the peak time of 14.997 min, with a peak area of 3641812 and a molecular formula of C 15 H 24 (See Figure 3); The flower sample has a unique product at the peak time of 14.997min, with a peak area of 4067657 and a molecular formula of C 15 H 24 (See Figure 4 ), the mass spectra of the leaves and flowers were compared with the NIST library, and the results showed that the compounds were all artemisinadiene.
[0053] Using artemisinin standard as reference, a quantitative analysis of the content of artemisinin in the flowers and leaves of F1158 material revealed that the content in the flowers was 182.5768 mg / kg and the content in the leaves was 876.3185 mg / kg.
[0054] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for preparing a distant hybridization tobacco material containing artemisinadiene, characterized in that: The method comprises the steps of performing distant hybridization with Zhongyan 300 as the female parent and shade-growing tobacco Nicotiana umbratica as the male parent.
2. The preparation method according to claim 1, characterized in that The following steps are involved: Collect pollen from the male wild shade-growing tobacco Nicotiana umbratica, emasculate the female Zhongyan 300, apply the pollen to the pistil of the female to pollinate, and collect distant hybrid seeds after maturity; The distant hybrid seeds are planted.
3. The preparation method according to claim 2, characterized in that: When the anthers of the male parent reach a mature state but have not yet cracked, the anthers are collected; the collected anthers are placed in an environment with a temperature of 25° C. until the anthers mature, crack and release pollen.
4. The preparation method according to claim 2, characterized in that: Before the flowers of the female parent open, well-developed flower buds are selected for emasculation.
5. The preparation method according to claim 2, characterized in that: The pollination is repeated pollination to increase the success rate.
6. Use of the distant hybridization tobacco material containing artemisinin obtained by the preparation method according to any one of claims 1 to 5 in the production of artemisinin.
7. Use of the distant hybridization tobacco material containing artemisinadiene obtained by the preparation method according to any one of claims 1 to 5 in the production of artemisinin.