Tea signal peptide drought-resistant inducer and application thereof

Abstract

The invention relates to the technical field of plant drought resistance, in particular to a tea signal peptide drought resistance inducer and application thereof. Active substances of the tea signal peptide drought-resistant inducer comprise one or more of CLE25 signal peptide, jasmonic acid methyl ester and cis-3-hexene acetate; the amino acid sequence of the CLE25 signal peptide is as follows: RKVPNGPDPIHN. The tea signal peptide drought-resistant inducer improves the drought resistance of tea trees and improves the content of chlorophyll, carotenoid and other substances of the tea trees.

Description

Technical Field

[0001] The invention relates to the technical field of plant drought resistance, in particular to a tea signal peptide drought resistance inducer and application thereof. Background Art

[0002] Tea trees are temperature-sensitive plants, and drought damage to tea trees can occur all year round. Summer drought and autumn drought (September-November) are more common, especially the continuous high temperature and drought in summer will lead to a decline in tea yield and quality. In the summer of 2022, a severe drought occurred in southern my country, and the continuous high temperature and drought had a huge impact on the growth of tea trees. Among them, Hangzhou City, Zhejiang Province is the provincial capital city with the most cumulative high temperature days in the country, with 29 days of high temperature weather. The high temperature and drought in summer have the greatest impact on the "Longjing 43" variety, mainly because it is an asexual cutting planting. In 2022, tea was affected by drought, including 30,000 acres of Pu'er tea gardens in Yunnan and 110,000 acres of tea gardens in Songyang County, Zhejiang. The severe drought caused serious losses to tea farmers.

[0003] In addition, due to drought stress, the content of flavor quality components such as flavonoids and amino acids in the surviving tea trees decreased significantly, which not only affected the yield, but also caused the finished tea to taste bitter in the spring of the following year, which seriously reduced the quality of the tea.

[0004] In general, due to the impact of global warming in recent years, the decline in the economic value of tea trees caused by drought has become increasingly serious. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a tea signal peptide drought resistance inducer and its application, which improves the drought resistance of tea plants and increases the content of chlorophyll, carotenoids and other substances in tea plants.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a tea signal peptide drought resistance inducer, wherein the active substance comprises one or more of CLE25 signal peptide, methyl jasmonate and cis-3-hexene acetate;

[0008] The amino acid sequence of the CLE25 signal peptide is: RKVPNGPDPIHN.

[0009] Preferably, when the active substance is CLE25 signal peptide, the concentration of the CLE25 signal peptide is 100 μM.

[0010] Preferably, when the active substances are CLE25 signal peptide and methyl jasmonate, the concentration of the CLE25 signal peptide is 68.75 μM, and the concentration of the methyl jasmonate is 31.25 μM.

[0011] Preferably, when the active substances are CLE25 signal peptide and cis-3-hexenyl acetate, the concentration of the CLE25 signal peptide is 75.6 μM, and the concentration of the cis-3-hexenyl acetate is 24.4 μM.

[0012] The present invention also provides the use of the tea signal peptide drought resistance inducer described in the above technical solution in improving the drought resistance of tea plants.

[0013] Preferably, the chlorophyll content of tea leaves is increased.

[0014] Preferably, the carotenoid content of tea tree is increased.

[0015] Preferably, the water-soluble protein content of tea trees and the activity of enzymes related to antioxidant activity are increased.

[0016] Preferably, the content of tea tree catechins and free amino acids is increased.

[0017] Preferably, the application comprises: spraying the tea signal peptide drought resistance inducer on tea trees until water drips from the leaf surface, and spraying once.

[0018] The beneficial effects of the present invention are:

[0019] When the tea signal peptide drought-resistant inducer only has the CLE25 signal peptide, it is sprayed on the surface of tea leaves under drought stress. Compared with tea leaves under drought stress without spraying the drought-resistant agent, the total amount of pigments in the fresh leaves after spraying increases, wherein the chlorophyll a content increases by 18.61%, the chlorophyll b content increases by 11.76%, and the total amount of carotenoids increases by 22.58%. In addition, the total amount of water-soluble protein in the fresh leaf samples after induction increases by 18.20%, the activity of enzymes related to antioxidant activity increases significantly (increases by 35.16%), the total amount of catechins increases by 12.87%, and the total amount of free amino acids increases by 6.32%. The prepared drought-resistant inducer and its induction method can significantly improve the drought resistance of tea trees, prevent drought damage to tea trees, and enhance the recovery ability of tea trees after drought damage.

[0020] When the active substances are CLE25 signal peptide and methyl jasmonate, they are sprayed on the surface of tea leaves under drought stress. Compared with tea leaves under drought stress without spraying drought-resistant agents, the total amount of pigments in the fresh leaves after spraying increases, among which the chlorophyll a content increases by 28.13%, the chlorophyll b content increases by 16.36%, and the total amount of carotenoids increases by 24.18%. In addition, the total amount of water-soluble protein in the fresh leaf samples after induction increases by 20.10%, the activity of enzymes related to antioxidant activity increases significantly (increases by 45.28%), the total amount of catechins increases by 17.14%, and the total amount of free amino acids increases by 13.92%. The prepared drought-resistant inducer and its induction method can significantly improve the drought resistance of tea trees, prevent drought damage to tea trees, and enhance the recovery ability of tea trees after drought damage.

[0021] When the active substances are CLE25 signal peptide and cis-3-hexene acetate, they are sprayed on the surface of tea leaves under drought stress. Compared with tea leaves under drought stress without spraying drought-resistant agents, the total amount of pigments in the fresh leaves after spraying increases, among which the chlorophyll a content increases by 19.32%, the chlorophyll b content increases by 12.28%, and the total amount of carotenoids increases by 19.32%. In addition, the total amount of water-soluble protein in the fresh leaf samples after induction increases by 17.59%, the activity of enzymes related to antioxidant activity increases significantly (increases by 32.18%), the total amount of catechins increases by 11.16%, and the total amount of free amino acids increases by 8.97%. The prepared drought-resistant inducer and its induction method can significantly improve the drought resistance of tea trees, prevent drought damage to tea trees, and enhance the recovery ability of tea trees after drought damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The results are the differences in the contents of chlorophyll a (Chl a), chlorophyll b (chlb), total carotenoids and total pigments in tea leaves after drought stress and signal peptide drought resistance induction;

[0024] Figure 2 The results are the differences in the contents of water-soluble sugar and water-soluble protein in tea leaves after drought stress and signal peptide drought resistance induction;

[0025] Figure 3 The results are the differences in antioxidant enzyme activities in tea leaves after drought stress and signal peptide drought resistance induction;

[0026] Figure 4 The results are the differences in the contents of ester-type catechins in tea leaves after drought stress and signal peptide drought resistance induction;

[0027] Figure 5 These are the results of differences in the content of non-ester catechins in tea leaves after drought stress and signal peptide drought resistance induction. DETAILED DESCRIPTION

[0028] The invention provides a tea signal peptide drought resistance inducer, wherein the active substance comprises one or more of a CLE25 signal peptide, methyl jasmonate and cis-3-hexenyl acetate; the amino acid sequence of the CLE25 signal peptide is: RKVPNGPDPIHN (SEQ ID No. 1).

[0029] In the present invention, when the active substance is preferably only the CLE25 signal peptide, the concentration of the CLE25 signal peptide is preferably 100 μM.

[0030] In the present invention, when the active substance is preferably CLE25 signal peptide and methyl jasmonate, the concentration of the CLE25 signal peptide is preferably 68.75 μM, and the concentration of the methyl jasmonate is preferably 31.25 μM.

[0031] In the present invention, when the active substance is preferably CLE25 signal peptide and cis-3-hexenyl acetate, the concentration of the CLE25 signal peptide is preferably 75.6 μM, and the concentration of the cis-3-hexenyl acetate is preferably 24.4 μM.

[0032] The present invention has no special limitation on the preparation method of the tea signal peptide drought resistance inducer, and the preparation method only needs to be mixing the active substance with water.

[0033] The present invention also improves the application of the tea signal peptide drought-resistant inducer described in the above technical scheme in improving the drought resistance of tea trees. In the present invention, after the tea trees are sprayed with the tea signal peptide drought-resistant inducer after drought treatment, the chlorophyll content of the tea trees is increased, and the chlorophyll includes chlorophyll a and chlorophyll b. In the present invention, after the tea trees are sprayed with the tea signal peptide drought-resistant inducer after drought treatment, the carotenoid content of the tea trees is increased. In the present invention, after the tea trees are sprayed with the tea signal peptide drought-resistant inducer after drought treatment, the water-soluble protein content and the activity of enzymes related to antioxidant activity of the tea trees are increased. In the present invention, after the tea trees are sprayed with the tea signal peptide drought-resistant inducer after drought treatment, the catechin and free amino acid content of the tea trees are increased.

[0034] In the present invention, the application preferably comprises: spraying the tea signal peptide drought resistance inducer on tea trees until water drips from the leaf surface, and spraying once.

[0035] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] The tea trees grown with normal watering were CK; the tea trees without watering under drought stress were DS; the treatment group T1 in which CLE25 signal peptide was used as an inducer, and the concentration of CLE25 signal peptide in the inducer was 100 μM.

[0038] The tea trees used were 5-year-old Zhongcha 108 tea varieties (drought intolerant) planted in the greenhouse of the Tea Research Institute of the Chinese Academy of Agricultural Sciences. Tea seedlings with strong growth and uniform growth were selected and subjected to high temperature (40°C) and drought (20% PEG 6000) stress treatments. After 7 consecutive days of treatment, the T1 group was sprayed with foliar inducer until water dripped from the leaf surface. The spraying was performed once and drought culture was continued. The CK group was watered every 2 days, and the DS group was not watered and subjected to the same drought treatment.

[0039] After 28 days, the young leaves at the 2nd to 3rd leaf position of the new shoot were taken and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. Three biological replicates were set at each time point to measure the physiological indicators related to drought resistance. The results are shown in Figure 1-5 and Tables 2–7.

[0040] Example 2

[0041] The tea trees grown with normal watering were CK; the tea trees without watering under drought stress were DS; the treatment group T2 with CLE25 signal peptide + methyl jasmonate as inducer was set, and the concentration of CLE25 signal peptide in the inducer was 68.75 μM, and the concentration of methyl jasmonate was 31.25 μM.

[0042] The tea trees used were 5-year-old Zhongcha 108 tea varieties (drought intolerant) planted in the greenhouse of the Tea Research Institute of the Chinese Academy of Agricultural Sciences. Robust and uniform tea seedlings were selected for high temperature (40°C) and drought (20% PEG 6000) stress treatments. After 7 consecutive days of treatment, the T2 group was sprayed with foliar inducer until water dripped from the leaf surface. The spraying was repeated once and drought culture was continued. The CK group was watered every 2 days, and the DS group was not watered and subjected to the same drought treatment.

[0043] After 28 days, the young leaves at the 2nd to 3rd leaf position of the new shoot were taken and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. Three biological replicates were set at each time point to measure the physiological indicators related to drought resistance. The results are shown in Figure 1-5 and Tables 2–7.

[0044] Example 3

[0045] The tea trees grown with normal watering were CK; the tea trees without watering under drought stress were DS; the treatment group T3 with CLE25 signal peptide + cis-3-hexenyl acetate as inducers was set, and the concentration of CLE25 signal peptide in the inducer was 75.6 μM, and the concentration of cis-3-hexenyl acetate was 24.4 μM.

[0046] The tea trees used were 5-year-old Zhongcha 108 tea varieties (drought intolerant) planted in the greenhouse of the Tea Research Institute of the Chinese Academy of Agricultural Sciences. Tea seedlings with strong growth and uniform growth were selected and subjected to high temperature (40°C) and drought (20% PEG 6000) stress treatments. After 7 consecutive days of treatment, the T3 group was sprayed with foliar inducer until water dripped from the leaf surface. The spraying was performed once and drought culture was continued. The CK group was watered every 2 days, and the DS group was not watered and subjected to the same drought treatment.

[0047] After 28 days, the young leaves at the 2nd to 3rd leaf position of the new shoot were taken and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. Three biological replicates were set at each time point to measure the physiological indicators related to drought resistance. The results are shown in Figure 1-5 and Tables 2–7.

[0048] The optimal concentration of signal peptide for inducing drought-prone tea trees was screened under the same conditions as in Example 1, except for the concentration of signal peptide. The results are shown in Table 1.

[0049] Table 1 Screening of optimal concentrations of signal peptides for inducing drought-prone tea trees

[0050] Content (mg / g) CK DS 25 μM signal peptide 50 μM signal peptide 100 μM signal peptide 125 μM signal peptide Water soluble sugar 80.28±3.48 104.93±1.2 103.3±1.21 105.2±1.08 118.13±2.15 112.3±1.75 Water soluble protein 29.73±0.75 34.39±0.87 34.66±0.78 33.96±0.63 35.74±0.76 34.74±0.56 Total pigment 0.86±0.02 0.51±0.01 0.52±0.04 0.59±0.07 0.63±0 0.59±0.07

[0051] By comparing basic indicators, including differences in water-soluble sugar, water-soluble protein and total pigment content, the optimal concentration was screened out as 100 μM.

[0052] Table 2 Differences in free amino acid content in tea leaves after drought stress and signal peptide drought resistance induction (mg·g - 1 DW)

[0053]

[0054]

[0055] It can be concluded from the table above that drought stress caused the total amount of free amino acids to drop significantly to 11.71 mg / g, and the total amount of free amino acids in the sample after signal peptide (T1) induction treatment increased by 6.32% compared with the drought sample, the total amount of free amino acids in the sample after signal peptide / methyl jasmonate combination (T2) treatment increased by 13.92% compared with the drought sample, and the total amount of free amino acids in the sample after signal peptide / cis-3-hexene acetate combination (T3) treatment increased by 8.97% compared with the drought sample. Therefore, the free amino acid content level in fresh leaves was significantly restored after signal peptide drought resistance induction, which is conducive to quality improvement.

[0056] Table 3 Differences in the contents of abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and gibberellin (GA) in tea leaves after drought stress and signal peptide drought resistance induction (mg g -1 DW)

[0057] Plant hormones CK DS DS+T1 DS+T2 DS+T3 Abscisic acid 2444.7±19.5a 1047.82±108.1b 1447.1±36.2c 1384.0±23.2c 518.8±16.9d Abscisic acid glucose ester - 5644.10a - - 4792.9b Salicylic acid 52651±899a 58724±1468b 70603±1765c 62257±9d 69552±1910c Jasmonic acid 1281.7±31a 568.4±0.1b 585.7±14.6b 722.3±16.3c 252.6±22.3d Endogenous Methyl Jasmonate 359.4±13a 96.7±6b 92.2±2b 134.0±3c 15.2±4d Abscisic acid 8 42.5±0.4 151.5±1.4b 279.2±7.0c 144.2±4.9b 169.0±12.4d Abscisic acid 3 14.0±1.9a 35.8±0.7b 32.1±0.8b 40.0±3.5c 36.3±2.3cb Abscisic acid 29 186.5±3.5a 607±35.9b 544±13.6c - 645.7±16.1d

[0058] It can be concluded from the above table that drought stress causes significant changes in the content of different endogenous hormones in fresh tea leaves. After the signal peptide (T1) induction treatment, the content levels of endogenous hormones such as abscisic acid, salicylic acid and abscisic acid 8 in the sample were significantly higher than those in the drought untreated sample. After the signal peptide / methyl jasmonate combination (T2) treatment, the content levels of endogenous hormones such as abscisic acid, salicylic acid, methyl jasmonate and abscisic acid 3 in the sample were significantly higher than those in the drought untreated sample. After the signal peptide / cis-3-hexene acetate combination (T3) treatment, the content level of salicylic acid hormone in the sample was significantly higher than that in the drought untreated sample, and the content of abscisic acid glucose ester was at a high level. Therefore, after the signal peptide drought resistance induction, by regulating the content levels of endogenous drought resistance hormones such as abscisic acid, salicylic acid, and methyl jasmonate, it is beneficial to improve resistance.

[0059] Table 4 Chlorophyll, carotenoids and total pigment contents in samples after drought resistance treatment with different signal peptides

[0060] Content (mg / g) CK DS DS+T1 DS+T2 DS+T3 Chlorophyll a 0.51±0.01 0.32±0 0.38±0 0.44±0.01 0.39±0.03 Chlorophyll b 0.2±0.01 0.1±0.01 0.11±0 0.16±0 0.14±0.02 Carotenoids 0.15±0 0.09±0 0.11±0 0.12±0.01 0.13±0.02 Total pigment 0.86±0.02 0.51±0.01 0.6±0 0.72±0.02 0.66±0.07

[0061] It can be concluded from the above table that drought stress causes the total pigment content to drop significantly to 0.51 mg / g. The total pigment content in the samples after signal peptide (T1, T2 and T3) induction treatment is higher than that in the drought samples. Chlorophyll, carotenoids, etc. are key pigments for photosynthesis. Drought stress causes the content of these light-capturing pigments to decrease, which is not conducive to the survival of tea trees. Therefore, restoring the content level of these pigments through signal peptide induction is beneficial to improving the drought resistance of tea trees.

[0062] Table 5 Water-soluble sugar and water-soluble protein contents in samples after drought resistance treatment with different signal peptides

[0063] Content (mg / g) CK DS DS+T1 DS+T2 DS+T3 Water soluble sugar 80.28±3.48 104.93±1.2 117.13±2.45 102.2±1.24 190.72±1.19 Water soluble protein 29.73±0.75 34.39±0.87 34.74±0.87 39.66±0.99 38.74±0.08

[0064] It can be concluded from the above table that drought stress leads to an increase in the content of water-soluble sugars and water-soluble proteins. The total amount of these two components in the samples after signal peptide (T1, T2 and T3) induction treatment is significantly increased, which can enhance the plant's resistance to adversity. Therefore, signal peptide induction is beneficial to improving the drought resistance of tea trees.

[0065] Table 6 Results of enzyme activity determination of peroxidase, catalase and ascorbate peroxidase in samples after drought resistance treatment with different signal peptides

[0066] <![CDATA[Enzyme activity (U / g -1 min -1 )]]> CK DS DS+T1 DS+T2 DS+T3 Peroxidase (POD) 17.9±0.9 25.2±0.6 21.67±0.58 37.13±1.21 13.2±3.29 Catalase (CAT) 23.07±4.5 48.2±9.2 118.8±13.44 92.8±7.6 86.67±1.1 Ascorbate peroxidase (APX) 31.1±0.5 33.1±0.8 47.6±0.5 66.7±4.3 37±0.9

[0067] Superoxide dismutase, catalase and ascorbate peroxidase are key indicator enzymes that show the antioxidant level of plants. From the above table, it can be concluded that drought stress causes a slight increase in the activity of these oxidases, but it is not significant. After the signal peptide (T1, T2 and T3) induction treatment, the enzyme activities of superoxide dismutase, catalase and ascorbate peroxidase in the samples are significantly improved, enhancing the plant's resistance to adversity. Therefore, signal peptide induction is beneficial to the improvement of tea tree drought resistance.

[0068] Table 7 Contents of main catechins in samples after drought resistance treatment with different signal peptides

[0069] Content (mg / g) CK DS DS+T1 DS+T2 DS+T3 Epigallocatechin gallate 24.62±1.33 12.14±0.66 14.89±0.38 16.97±0.84 13.38±0.6 Epicatechin gallate 13.26±0.44 7.1±0.31 8.86±0.23 10.45±0.56 8.1±0.22 Epigallocatechin gallate 0.27±0.01 0.15±0.01 0.13±0.01 0.17±0.01 0.13±0.01 Epigallocatechin gallate 0±0 0±0 0±0 0±0 0±0 Epigallocatechin gallate 2.99±0.09 4.35±0.19 3.86±0.01 4±0.07 3.66±0.09 Epicatechin 4.74±0.07 6.16±0.15 6.4±0.07 6.35±0.22 5.75±0.07 Epigallocatechin gallate 0.9±0.14 1.34±0.07 1.32±0.03 1.18±0.04 1.05±0.08 Catechins 0.27±0.03 0.39±0 0.38±0.02 0.33±0.03 0.3±0.01 Total catechins 47.06±2.02 31.62±1.4 35.84±0.71 39.44±1.69 32.37±1.07

[0070] It can be concluded from the above table that drought stress caused the total amount of catechins to drop significantly to 31.62 mg / g, which was significantly lower than the 47.06 mg / g content in the fresh leaves of normal (CK) tea trees. After the signal peptide (T1) induction treatment, the total amount of catechins in the sample recovered to 35.84 mg / g compared with the drought sample, and the total amount of catechins in the sample treated with the signal peptide / methyl jasmonate combination (T2) recovered to 39.44 mg / g compared with the drought sample, and the total amount of catechins in the sample treated with the signal peptide / cis-3-hexene acetate combination (T3) recovered to 32.37 mg / g compared with the drought sample. Therefore, the free amino acid content level in the fresh leaves was significantly restored after the signal peptide drought resistance induction, which is conducive to quality improvement.

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tea signal peptide drought resistance inducer, It is characterized in that The active substance includes one or more of CLE25 signal peptide, methyl jasmonate and cis-3-hexenyl acetate; The amino acid sequence of the CLE25 signal peptide is: RKVPNGPDPIHN.

2. The tea signal peptide drought resistance inducer according to claim 1, It is characterized in that When the active substance is CLE25 signal peptide, the concentration of the CLE25 signal peptide is 100 μM.

3. The tea signal peptide drought resistance inducer according to claim 1, It is characterized in that When the active substances are CLE25 signal peptide and methyl jasmonate, the concentration of the CLE25 signal peptide is 68.75 μM, and the concentration of the methyl jasmonate is 31.25 μM.

4. The tea signal peptide drought resistance inducer according to claim 1, It is characterized in that When the active substances are CLE25 signal peptide and cis-3-hexenyl acetate, the concentration of the CLE25 signal peptide is 75.6 μM, and the concentration of the cis-3-hexenyl acetate is 24.4 μM.

5. Use of the tea signal peptide drought resistance inducer according to any one of claims 1 to 4 in improving the drought resistance of tea plants.

6. The use according to claim 5, It is characterized in that Increase the chlorophyll content of tea leaves.

7. The use according to claim 5, It is characterized in that Increase the carotenoid content of tea tree.

8. The use according to claim 5, It is characterized in that Improve the content of water-soluble protein and the activity of enzymes related to antioxidant activity in tea trees.

9. The use according to claim 5, It is characterized in that Increase the content of tea catechins and free amino acids.

10. The use according to claim 5, It is characterized in that The application comprises: spraying the tea signal peptide drought resistance inducer on tea trees until water drips from the leaf surface, and spraying once.

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