阅读说明：本技术 超敏蛋白在提高茶树叶片儿茶素和氨基酸含量上的应用 (Application of hypersensitive protein in improving content of theanine and amino acid in tea leaves ) 是由 李荣林 杨亦扬 万青 *** 李欢 艾则宜 于 2020-06-23 设计创作，主要内容包括：本发明公开了超敏蛋白在提高茶树叶片儿茶素和氨基酸含量上的应用,特别地,超敏蛋白可以使酯型儿茶素EGCG比例增加,且同时提高茶树叶片儿茶素总量和氨基酸含量。本发明对于茶树鲜叶品质调控具有重要的意义。(The invention discloses application of a hypersensitivity protein in improving the content of tea leaf catechin and amino acid, in particular, the hypersensitivity protein can increase the proportion of ester catechin EGCG and simultaneously improve the total amount of the tea leaf catechin and the content of the amino acid. The invention has important significance for regulating and controlling the quality of the fresh tea leaves.)

1. The application of the hypersensitive protein in simultaneously improving the content of catechin and amino acid in tea leaves.

2. Use according to claim 1, characterized in that hypersensitivity proteins are used on tea to increase the proportion of ester catechins EGCG.

3. The use according to claim 1, wherein the hypersensitive protein is derived from erwinia amylovora bacteria (f: (a))Erwinia amylovora) Protein encoded by gene hrp N.

4. The use according to claim 1, wherein the hypersensitive protein solution is applied to the tea tree by irrigation, smearing or spraying.

5. The use according to claim 4, wherein the concentration of the hypersensitive protein solution is 50mg/L to 1000 mg/L.

6. The use according to claim 4, wherein the amount of the harpin protein powder is 5-100g per mu.

Technical Field

The invention belongs to the field of tea production, and particularly relates to application of a hypersensitive protein in improving the content of theanine and amino acid in tea leaves.

Background

Catechin is the most important substance component in tea, which is the main substance basis of tea as a delicious beverage, and is the main substance basis of tea with slight bitterness and refreshing feeling. The fine control of enzymatic or non-enzymatic oxidation of catechins is a critical reference condition for the tea processing technology. Catechin also plays an important role in the process of tea tree resisting diseases and pests. Catechin is a key substance of tea with health care function, and a large number of researches prove that the tea has the capacities of resisting oxidation, cancer and mutation and preventing cardiovascular and cerebrovascular diseases and is closely related to catechins.

The amino acid is the main material basis of fresh taste of tea, the amino acid and the aroma of the tea form great relevance, and the amino acid is the essential material basis for the growth of tea trees. The phenol-ammonia ratio is the main reference condition for the adaptability of fresh tea leaves. Amino acid is one of main nutrients required by human beings, so that the amino acid has close relation with the drinking value and the health care value of tea leaves. Theanine is an amino acid with the most important function in the tea, is the basis of nitrogen metabolism of tea trees, and is a main contributor to fresh taste of tea. Theanine also has important effects of regulating nerve function and promoting lipid metabolism.

Based on the above, in the process of breeding the tea tree variety, the screening has important significance in simultaneously having high catechin content and amino acid content. The catechin content in tea tree leaf is 6-8%, the amino acid content is 0.5-0.8% (fresh weight), and the specific content level is related to variety, season, etc. The C metabolism mainly synthesized by catechin in tea plant and the N metabolism synthesized by protein amino acid have competition effect, and fresh leaves with high catechin content and high amino acid content are difficult to obtain under the conventional condition.

Methods for regulating plant growth and secondary metabolism by utilizing phytostimulants are widely concerned, for example, salicylic acid or jasmonic acid can regulate the synthesis of terpenoids and increase the content of terpenoids, and brassinolide can improve the content of leaf amino acid and improve the growth capacity of crops. Also, Benzothiadiazole (BTH) and beta-aminobutyric acid (BABA) can increase the activities of tea polyphenol oxidase and peroxidase.

The hypersensitive protein used in the patent is a protein elicitor extracted from bacterial protein of the erwinia amylovora, HarpinEa is in contact with a signal receptor (receptor) on the surface of a plant after being sprayed on the surface of the plant, a simulated pathogen attack signal is given to the plant, and then the expression of various genes is caused through information transmission, so that various defense systems in the plant can be activated within a few minutes to obtain resistance, after 30 minutes, the plant shows a resistance reaction to the pathogen, and meanwhile, the physiological and biochemical activities of the plant are promoted through the transmission of jasmonic acid and ethylene signals, but at present, the influence of the hypersensitive protein on the content of catechin and amino acid in leaves is not reported in public researches of tea trees.

Disclosure of Invention

The purpose of the invention is as follows: the research of the invention finds that when hypersensitivity protein (Harpin) is properly used on tea trees, the physiological and biochemical characteristics of the tea trees are promoted to generate obvious favorable reaction, the total content of catechin and the content of amino acid in tea tree leaves can be simultaneously improved, and therefore, the application of the hypersensitivity protein in simultaneously improving the content of tea element and the content of amino acid in the tea tree leaves is provided.

The invention discovers that the hypersensitive protein is applied to tea trees to increase the proportion of ester catechin EGCG.

Preferably, the hypersensitive protein is a protein encoded by the Erwinia amylovora (Erwinia amylovora) gene hrp N.

Specifically, the hypersensitivity protein is prepared into a solution and then acts on the tea trees in a root irrigation, smearing and spraying mode.

Preferably, the concentration of the hypersensitive protein solution is 50mg/L-1000 mg/L.

The dosage of the hypersensitivity protein powder is 5-100g per mu.

In a preferred embodiment, the catechin and amino acid content of tea tree leaves is increased by:

1) dissolving hypersensitive protein in water to prepare a stock solution of 1000mg/L, diluting with water to a required concentration, and performing root irrigation, smearing and spraying treatment on the tea trees in the growing season of the tea trees (3 months to 10 months per year in Yangtze river basin);

2) after the hypersensitivity protein (harpin) is used for the 1 st time, 1000mg/L hypersensitivity protein stock solution is used again at an interval of 48h, the hypersensitivity protein stock solution is diluted to the required concentration by water, and the tea tree is subjected to root irrigation, smearing or spraying treatment for the 2 nd time;

3) sample analysis 24h after using hypersensitivity protein (harpin) at 2 nd time. Whether the processing needs to be continued or not can be determined according to the result of the sampling analysis.

Has the advantages that:

through experimental research, the tea tree is proved to have obvious favorable reaction for promoting the physiological and biochemical characteristics of the tea tree when hypersensitive protein (Harpin) is properly used on the tea tree, and after the hypersensitive protein of 50mg/L-1000mg/L is tested for being used on the tea tree, the PPO and POD activities in fresh tea leaf tissues can be obviously increased, and the trend that amino acid and catechin are obviously increased simultaneously is proved.

The specific implementation mode is as follows:

measuring catechin and gallic acid by HPLC method, collecting ground sample 1g, adding 10mL methanol solution (containing 0.1% formic acid), leaching for 2h, centrifuging 4000g, collecting supernatant, filtering with microporous membrane, quantifying by HPLC, separating with C18 column (4.6mm × 250mm, 5 μm), mobile phase A comprising acetonitrile, acetic acid and water at volume ratio of 6: 1: 193, mobile phase B comprising acetonitrile, acetic acid and water at volume ratio of 60: 1: 139, eluting with gradient of phase B initial concentration of 20%, linearly increasing to 65% within 35min, immediately decreasing to 20%, maintaining for 5min, and ending at 40min, and measuring flow rate of 1 mL/min^-1The column temperature is 25 ℃, the detection wavelength is 280nm, and the sample injection amount is 10 mu L.

The total amount of free amino acid is measured by ninhydrin colorimetry. 1g of ground sample is taken, 10mL of methanol solution (containing 0.1% formic acid) is added, leaching is carried out for 2h,4000g of the mixture is centrifuged, and the supernatant is reserved. Taking 1mL of supernatant, adding 0.5mL of phosphate buffer with pH8.0 and 0.5mL of 2% ninhydrin solution, heating in boiling water bath for 15min, cooling, adding water to constant volume to 25mL, standing for 10min, and performing color comparison at 570 nm. The glutamic acid was used as a standard substance for quantification.

The present invention will be described in further detail with reference to specific examples. The examples will help to understand the present invention given the detailed embodiments and the specific operation procedures, but the scope of the present invention is not limited to the examples described below. In the following examples, the hypersensitive protein (Harpin) was purchased from science and technology development Co., Ltd, Nongdago, Hunan, with a product approval number of HNP32153-E0594 and a product standard number of Q/3201SKSH007-2007.

In the following examples, tea trees were treated by irrigating, smearing, and spraying with a hypersensitive protein solution during 3-10 months of the tea tree growing season.

(1)4, 15 days in the morning, 75mg/L of hypersensitivity protein solution is sprayed on the tea trees, and the liquid consumption per mu is 45 liters. Spraying clear water to the control area. And 4, spraying 75mg/L hypersensitive protein solution on the tea trees again in 16-day morning of 4 months, and using 45 liters of liquid for each mu. Spraying clear water to the control area. The sampling test on 17 days at 4 months showed that the total amount of catechins (fresh weight) was 11.87% as compared with the control, and the amount of amino acids was 1.09% (fresh weight) as compared with the control, and the amount was 29.76% as compared with the control, as shown in Table 1.

TABLE 1

(2) In the evening of 6 months and 10 days, 200mg/L of hypersensitivity protein solution is sprayed on tea trees, and the liquid consumption per mu is 45 liters. Spraying clear water to the control area. In the evening of 6 months and 11 days, 200mg/L of hypersensitivity protein solution is sprayed on the tea trees again, and the liquid consumption per mu is 100 liters. Spraying clear water to the control area. After 24h, sampling was performed, and the results are shown in Table 2, in which the total amount of catechins (fresh weight) was increased by 12.95%, 134.34%, and the total amount of amino acids (fresh weight) was increased by 1.10%, 65.66%.

TABLE 2

(3) After 20 days after 6 months, 50mg/L of the hypersensitive protein solution is sprayed on the tea trees, and the liquid consumption per mu is 45 liters. Spraying clear water to the control area. And after 24 hours, spraying the tea trees again with 100mg/L of the hypersensitive protein solution, and using liquid quantity of 45 liters per mu. Spraying clear water to the control area. After another 24-hour sampling test, the results are shown in Table 3, in which the total amount of catechins (fresh weight) was increased by 132.54%, and the total amount of amino acids (fresh weight) was increased by 52.25%.

TABLE 3

(4)7, spraying the tea trees by 300mg/L of the hypersensitivity protein solution for 1 day in 7 months, wherein the liquid consumption per mu is 45 liters, spraying the tea trees by 100mg/L of the hypersensitivity protein solution again after 24 hours, and the liquid consumption per mu is 45 liters. Spraying clear water to the control area. After 24h sampling analysis, the total catechin content was increased by 139.93% (fresh weight) compared with the control as shown in Table 4. The total amino acid amount of 1.18% (fresh weight) is increased by 69.59% compared with the control.

TABLE 4

(5) After 8 months and 5 days, 300mg/L of the hypersensitivity protein solution is sprayed on the tea trees, the liquid consumption per mu is 45 liters, after 24 hours, the hypersensitivity protein solution of 100mg/L is sprayed on the tea trees again, and the liquid consumption per mu is 45 liters. Spraying clear water to the control area. After 24h sampling analysis, the total content of catechin increased by 96.57% in 14.69% and the total content of amino acid increased by 41.18% in 0.96% as shown in Table 5.

TABLE 5

In conclusion, when hypersensitive protein (Harpin) is properly used on tea trees, the physiological and biochemical characteristics of the tea trees are promoted to generate obvious favorable reactions, and after the hypersensitive protein of 50mg/L-1000mg/L is tested on the tea trees, the PPO activity and the POD activity in fresh tea leaf tissues can be obviously increased, and the trend that amino acid and catechin are simultaneously increased is found. The invention has important significance for regulating and controlling the quality of the fresh tea leaves.