1. A method for rapidly breeding Armillaria mellea of Gastrodia elata is characterized by comprising the following steps:

(1) constructing a mathematical model of an external evaluation index of gastrodin synthesis of armillaria mellea accompanied with rhizoma gastrodiae;

(2) determining external evaluation index of gastrodin synthesis of armillaria mellea to be detected, and predicting gastrodin content in the gastrodia elata when the armillaria mellea strain accompanies with the gastrodia elata;

(3) and (3) selecting the armillaria mellea target strain according to the gastrodin content predicted in the step (2).

2. The method for rapidly breeding Armillaria mellea of Gastrodia elata according to claim 1, wherein the mathematical model for constructing the extrinsic evaluation index of gastrodin synthesis of Armillaria mellea accompanied by Gastrodia elata is specifically as follows: separating Armillaria mellea from the same original strain and with different passage times, measuring the amount of converting p-hydroxybenzyl alcohol into gastrodin by the Armillaria mellea strain and the gastrodin content of the co-cultivated gastrodia elata of the same Armillaria mellea, and then constructing a mathematical model of the amount of synthesizing the gastrodin by bioconversion of the Armillaria mellea and the gastrodin content of the co-cultivated gastrodia elata by taking the amount of synthesizing the gastrodin by bioconversion as an abscissa and the gastrodin content of the co-cultivated gastrodia elata as an ordinate.

3. The method for rapid breeding of Armillaria mellea of Gastrodia elata according to claim 1, wherein the operation of isolating Armillaria mellea is as follows: the isolation was performed from the top 0.5cm of the halimasch rhizomes.

4. The method for rapid breeding of Armillaria mellea of Gastrodia elata according to claim 3, wherein more than 5 strains of Armillaria mellea are selected from the same Armillaria mellea.

5. The method for rapid breeding of Armillaria mellea of Gastrodia elata according to claim 1, wherein the in vitro bioconversion conditions of Armillaria mellea are as follows: initial pH4.5, transformation liquid 30mL, resting cell amount of Armillaria mellea 6.5g, p-hydroxybenzyl alcohol 3mg/mL, 1.5% Tween 80, 3% glucose, 28 deg.C, culture for 6 d.

6. The method for rapidly breeding Armillaria mellea of Gastrodia elata according to claim 1, wherein the resting cell culture conditions of Armillaria mellea are as follows: culturing and activating for 4-6 days at 23 ℃ on a PDA culture medium, adding 1mL of bacterial suspension into 30mL of PDA liquid culture medium, culturing for 4 days at 23 ℃ and 120rmp, centrifuging for 15min at 4 ℃ and 10000rmp, removing supernatant, washing for 2 times by using sterile water, and collecting bacterial resting cells.

7. The method for rapidly breeding Armillaria mellea of Gastrodia elata according to claim 1, wherein quantitative analysis of gastrodin is performed by RP-HPLC; c18 reversed phase column, mobile phase: 0.02% phosphoric acid solution pH =2.6, methanol volume ratio 5:95, detection wavelength 220nm, flow rate 0.5ml/min, column temperature 35 ℃.

Background

The gastrodia elata is an unrooted and green leaf-free orchidaceae plant, is one of traditional rare Chinese medicinal materials, has quite long edible history and can be used as a raw material of health-care food; the planting of the traditional Chinese medicinal materials can fully utilize resources under the forest or resources such as barren mountains and wild mountains, so that land competition with grains, fertilizer competition and the like are avoided, and products with high added values can be produced.

Taking the wild-imitating planting of gastrodia elata in a Chongqing Shizhu forest as an example, the planting mode is gradually popularized, and the quality of fresh gastrodia elata is superior and is deeply favored by the market; the artificial cultivation of the gastrodia elata relies on the assistance of germination bacteria and armillaria mellea to promote the germination of gastrodia elata seeds and the growth of the gastrodia elata, particularly, the armillaria mellea is an indispensable accompanying bacterium of the gastrodia elata and mainly provides nutrient substances for the gastrodia elata and promotes the synthesis of gastrodin, so that the quality of the armillaria mellea strain is closely related to the yield and the quality of the gastrodia elata. The reasons for the decrease in yield and quality of gastrodia elata during continuous asexual cultivation mainly include the degeneration of gastrodia elata seeds, germinal bacteria and armillaria mellea, wherein the degeneration influence of the armillaria mellea is more obvious.

Because the reproduction of the armillaria mellea is mainly asexual reproduction at present, the armillaria mellea which is degenerated by multiple asexual reproduction is mainly characterized in that the adaptability and the stress resistance are reduced, and the growth of rhizomorph is slow; then the funiculus is inelastic and fragile, and even becomes a hollow shell. As the number of transfer generations increases, the Armillaria mellea forms a brownish black fungus cable net which is thick, long, numerous and branched on the fungus material. The Armillaria mellea strain is obtained mainly by separating field dominant strains and breeding indoors, but the breeding of the excellent Armillaria mellea strain at present mainly adopts appearance growth indexes such as growth rate, branch number, robustness, yield and quality of the accompanying cultivated Gastrodia elata and the like as references, and the breeding before the accompanying cultivation does not adopt the index of Gastrodin synthesis as a reference index. For example, in the prior art (study on genetic diversity of symbiotic armillaria mellea of gastrodia elata in Qinba mountain area and breeding of excellent strains [ D ]. Shaanxi institute of technology, 2016.), 18 strains of armillaria mellea and gastrodia elata are adopted to carry out companion planting tests, and the results show that in the asexual propagation stage of gastrodia elata, the yield of the gastrodia elata combining ZY-17 with red and black gastrodia elata is highest, and then ZY-9, the yield of the gastrodia elata combining ZY-9 with red gastrodia elata and the yield of the gastrodia elata combining ZY-17 with black gastrodia elata are highest in the sexual propagation stage of gastrodia elata, and the yield of the gastrodia elata is used for evaluating the armillaria mellea.

At present, excellent Armillaria mellea strains can be screened out only through a large number of comparison cultivation experiments, so that the workload is high, the period is long, and strains with high gastrodin synthesis are probably ignored due to lack of external index reference of gastrodin synthesis evaluation.

Disclosure of Invention

In order to solve the problems that the traditional breeding method of the Armillaria mellea is low in efficiency, and especially the external evaluation index for gastrodin synthesis of the Armillaria mellea accompanying cultivated gastrodia elata is lacked, the invention establishes a mathematical model of the external evaluation index for the Armillaria mellea gastrodin synthesis, which is used for quickly breeding Armillaria mellea, directionally screening strains with high gastrodin synthesis amount, improving breeding efficiency, reducing comparison cultivation screening workload, shortening breeding period and having good practicability.

The technical scheme of the invention is as follows:

a method for rapidly breeding Armillaria mellea of Gastrodia elata comprises the following steps:

(1) constructing a mathematical model of an external evaluation index of gastrodin synthesis of armillaria mellea accompanied with rhizoma gastrodiae;

(2) determining external evaluation index of gastrodin synthesis of armillaria mellea to be detected, and predicting gastrodin content in the gastrodia elata when the armillaria mellea strain accompanies with the gastrodia elata;

(3) and (3) selecting the armillaria mellea target strain according to the gastrodin content predicted in the step (2). Specifically, under the condition that the appearance indexes meet the requirements, the condition that the predicted value of the gastrodin content is low is eliminated, or the condition that the appearance indexes meet the requirements and the predicted value of the gastrodin content is high is selected.

The method fills the blank that no external evaluation index for synthesizing gastrodin by the armillaria mellea strain exists at present, can improve the oriented screening efficiency of the armillaria mellea strain on the basis of the existing breeding, and provides a method for breeding the armillaria mellea strain and judging the quality of the strain.

In a further scheme, the mathematical model for constructing the external evaluation index of gastrodin synthesis of armillaria mellea accompanied and planted gastrodia elata specifically comprises the following steps: separating Armillaria mellea from the same original strain and with different passage times, measuring the amount of converting p-hydroxybenzyl alcohol into gastrodin by the Armillaria mellea strain and the gastrodin content of the co-cultivated gastrodia elata of the same Armillaria mellea, and then constructing mathematical models of the amount of synthesizing the gastrodin by bioconversion of the Armillaria mellea and the gastrodin content of the co-cultivated gastrodia elata by taking the amount of synthesizing the gastrodin by bioconversion as an abscissa and the gastrodin content of the co-cultivated gastrodia elata as an ordinate. The amount of gastrodin synthesized by the armillaria mellea bioconversion is the amount of gastrodin converted from p-hydroxybenzyl alcohol by the armillaria mellea strain.

In a further embodiment, the isolation of Armillaria mellea is performed as follows: the isolation was performed from the top 0.5cm of the halimasch rhizomes. More than 5 isolated strains of the same armillaria mellea are selected.

In a further embodiment, the conditions for the in vitro bioconversion of Armillaria mellea are: initial pH4.5, transformation liquid 30mL, resting cell amount of Armillaria mellea 6.5g, p-hydroxybenzyl alcohol 3mg/mL, 1.5% Tween 80, 3% glucose, 28 deg.C, culture for 6 d.

The conversion liquid is in particular water.

In a further embodiment, the culture conditions of the resting cells of Armillaria mellea are as follows: culturing and activating for 4-6 days at 23 ℃ on a PDA culture medium, adding 1mL of bacterial suspension into 30mL of PDA liquid culture medium, culturing for 4 days at 23 ℃ and 120rmp, centrifuging for 15min at 4 ℃ and 10000rmp, removing supernatant, washing for 2 times by using sterile water, and collecting bacterial resting cells.

In a further scheme, the quantitative analysis of gastrodin adopts an RP-HPLC method; waterssymetry C18 reverse phase column (4.6 mm. times.150 mm, 5um), mobile phase: 0.02% phosphoric acid solution pH 2.6, methanol volume ratio of 5:95, detection wavelength of 220nm, flow rate of 0.5ml/min, column temperature 35 ℃.

The invention has the beneficial effects that:

the invention provides a method for directionally and rapidly screening gastrodia elata associated bacteria armillaria mellea strains, which is characterized in that only a mathematical correlation model of an external evaluation index for gastrodin synthesis needs to be established for strains, then the content of the external bioconversion gastrodin of the strains is measured, the gastrodin content of the concomitantly planted gastrodia elata can be predicted according to the mathematical model, the problem of long directional screening period of excellent armillaria mellea is solved, the operation is convenient, the screening period is short, the practicability is high, and the method is suitable for popularization and application.

The invention establishes a mathematical model of external evaluation of gastrodin synthesis for the first time, improves the strain breeding efficiency, fills the blank of directionally screening the high-yield gastrodin strains of the armillaria mellea, can effectively improve the strain breeding efficiency and shorten the breeding time, and can be used for detecting the strain quality of the armillaria mellea and providing high-quality strains for the production of the gastrodin.

The method predicts the content of gastrodin by detecting the external biotransformation synthesis of armillaria mellea, can effectively eliminate degenerated and aged strains, does not need a long cultivation contrast experiment, can effectively shorten the breeding time, and has good value in actual production.

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

Example 1 Rapid Breeding of Armillaria mellea

Materials and equipment:

the original strain of Armillaria mellea is collected from Xinxiang wash in Shizhu county, Chongqing, is the main strain of Armillaria mellea cultured by Gastrodia elata Blume at present and is an excellent strain cultured by local Gastrodia elata Blume, but the strain is degenerated during use.

The incubator is a 250L artificial climate box with constant Shanghai, the shaking table is Saimei Feilansis solaris2000, and HPLC is adopted to determine the content of gastrodin.

Inoculating an Armillaria mellea strain from the same original strain after passage for 1-5 times to a PDA (potato dextrose agar) culture medium for culture, preparing the Armillaria mellea on the PDA culture medium into a bacterial suspension by using the PDA liquid culture medium, inoculating the bacterial suspension into the PDA liquid culture medium, culturing for 4d, collecting hyphae, culturing for 6d under the conditions of an initial pH value of 4.5, a conversion liquid of 30mL, an Armillaria mellea resting cell amount of 6.5g, p-hydroxybenzyl alcohol of 3mg/mL, tween 80 of 1.5%, glucose of 3% and 28 ℃, and determining the content of gastrodin by using an HPLC (high performance liquid chromatography) method, wherein the content of the gastrodin is the content of the biosynthetic gastrodin of the Armillaria mellea. Each strain was replicated 3 times and the average was recorded.

5 strains with different biosynthesis gastrodin contents are selected from the Armillaria mellea strains, and wild-imitating tending is carried out under the forest to accompany the cultivation of the Gastrodia elata. And (4) after the gastrodia elata is collected, determining the quality and gastrodin content of the gastrodia elata.

TABLE 1 content of gastrodin biosynthesized in Armillaria mellea, and yield and gastrodin content of concomitantly cultivated Gastrodia elata

Data processing and mathematical model construction: taking the amount of gastrodin biosynthesized by the armillaria mellea as an independent variable and the gastrodin content in the gastrodia elata cultivated with the armillaria mellea as a dependent variable, performing linear regression analysis by adopting excel software, and establishing a regression equation of the gastrodin content and the externally biosynthesized gastrodin: 0.0857X-0.0271, R²＝0.988。

Prediction of gastrodin accompanied with gastrodia elata: and (3) selecting one strain from the Armillaria mellea strains after passage for 1-5 times according to the established mathematical model, determining that the biosynthesis amount of gastrodin by the Armillaria mellea is 4.87mg/L, and obtaining Y which is 0.3904 according to the model. The content of gastrodin in the cultured rhizoma Gastrodiae is 0.3944%. Therefore, the amount of gastrodin in the gastrodia elata predicted by the regression equation is consistent with the actually measured gastrodin content.

For the strain which is obviously degenerated after more than 5 passages, 0.5cm of the tip of the halimasch strongyllum is taken, the halimasch is cut into small sections with the diameter of 2mm, the pith layer is removed, the pith layer is left, the halimasch is mutagenized for 2 minutes by using 1% ammonia water, the mutagenized rejuvenation strain is obtained, 5 strains are selected to measure the biosynthesis gastrodin amount of the halimasch, the gastrodin content of the gastrodia elata cultivated concomitantly is predicted, the gastrodin content of the cultivated gastrodia elata is measured, and the results are shown in table 2.

TABLE 2 content of biologically synthesized gastrodin and content of concomitant gastrodin by Armillaria mellea rejuvenation strains

The content of gastrodin in the concomitantly planted gastrodia elata cannot be predicted by traditional halimasch breeding, and the influence of the strain on gastrodin synthesis can be evaluated only by taking the gastrodia elata and measuring the gastrodin content after a gastrodia elata cultivation contrast experiment. The results show that the gastrodin content of the accompanied gastrodia elata is calculated to be consistent with the actual content of the accompanied gastrodin according to the amount of the gastrodin biosynthesized outside the armillaria mellea, and the method is proved to be accurate and reliable. The traditional comparison cultivation experiment of breeding consumes nearly 3 years, the period is long, and the screened strain probably has lower gastrodin synthetic amount. The directional screening of the invention can effectively improve the screening efficiency, and proves that the method can be quickly used for directional breeding of strains, has strong practical application and can be popularized.

According to the result of the embodiment 1, the method can be used for the oriented screening of the breeding of the armillaria mellea strain, the external evaluation index of the biosynthesis of the gastrodin is increased on the basis of the existing screening of the armillaria mellea strain, the method is used for the oriented screening and the content prediction of the accompanied gastrodia elata, the screening efficiency can be effectively improved, the resource waste is reduced, and the screening period is shortened.

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.