Application of transgreen peptide in relieving iron deficiency chlorosis of citrus leaf

Abstract

The present application relates to the technical field of citrus cultivation, and particularly relates to application of a green transition peptide in relieving iron deficiency chlorosis of citrus leaves, and application of the green transition peptide in a product for relieving iron deficiency chlorosis of citrus leaves. An amino acid sequence of the green transition peptide is NTLISKVLNKGDAFVFPIGLIHFQFNIGK. The green transition peptide is applied to promote absorption and transport of iron elements by citrus plants, the green transition peptide and EDDHA-iron can play a synergistic effect, relieve iron deficiency chlorosis of citrus, increase iron content and chlorophyll content of leaves, improve photosynthetic rate, increase fruit content, and improve fruit quality.

Description

Technical Field

[0001] This invention relates to the field of citrus cultivation technology, and in particular to the application of transgreen peptides in alleviating iron deficiency chlorosis in citrus leaves. Background Technology

[0002] Citrus is an important economic fruit tree in my country and globally, but its growth and development are highly sensitive to iron. Iron is a key micronutrient element for chlorophyll synthesis, photosynthesis, and the activity of various enzymes in plants. In my country, citrus is mostly grown in mountainous and hilly areas with poor soil, especially in calcareous soils. The alkaline soil causes available iron to be easily fixed as insoluble ferric oxides, preventing the citrus roots from absorbing and utilizing it, leading to iron deficiency chlorosis. Typical symptoms include interveinal chlorosis in young leaves, yellowing and whitening of the leaf tissue, and in severe cases, complete leaf whitening and premature leaf drop, weakened tree vigor, and decreased fruit quality, causing significant economic losses to the industry.

[0003] Currently, the main treatments on the market for iron deficiency chlorosis in citrus are traditional iron fertilizers such as ferrous sulfate, chelated iron such as EDTA-iron, and EDDHA-iron, which are applied through root irrigation or foliar spraying. However, existing technologies still have common problems such as low iron availability, severe soil solidification, and short duration of effectiveness. Summary of the Invention

[0004] To address the aforementioned technical problems, embodiments of the present invention provide the application of transgreen peptides in alleviating iron deficiency chlorosis in citrus leaves.

[0005] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions: This invention provides the application of transgreen peptide in alleviating iron deficiency chlorosis in citrus leaves, wherein the amino acid sequence of the transgreen peptide is NTLISKVLNKGDAFVFPIGLIHFQFNIGK.

[0006] In some embodiments, the transgreen peptide is used in products for alleviating iron deficiency chlorosis in citrus leaves.

[0007] In some embodiments, the product is a trace element liquid fertilizer.

[0008] In some embodiments, the trace element liquid fertilizer comprises the following raw materials: 0.75-1.25 g / L of green peptide, 50-90 g / L of EDDHA-iron, 10-15 g / L of EDTA-copper, 4-6 g / L of manganese sulfate, 15-25 g / L of chelated boron, 3-5 g / L of ammonium molybdate, 150-250 g / L of urea, 30-35 g / L of EDTA-magnesium, and 30 g / L of oleamidopropyl betaine, wherein the green peptide is the aforementioned green peptide.

[0009] In some embodiments, the preparation method of the trace element liquid fertilizer includes the following steps: dissolving 0.75-1.25 g / L of transgreen peptide, 50-90 g / L of EDDHA-iron, 10-15 g / L of EDTA-copper, 4-6 g / L of manganese sulfate, 15-25 g / L of chelated boron, 3-5 g / L of ammonium molybdate, 150-250 g / L of urea, 30-35 g / L of EDTA-magnesium, and 30 g / L of oleamidopropyl betaine in water, and diluting to obtain the trace element liquid fertilizer.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention relates to a transgreen peptide that promotes the absorption and transport of iron in citrus plants. This transgreen peptide and EDDHA-iron have a synergistic effect, which can alleviate iron deficiency chlorosis in citrus, increase the iron content and chlorophyll content of leaves, improve the photosynthetic rate, increase fruit content, and improve fruit quality. Attached Figure Description

[0011] Figure 1 The expression of the RT1 gene after treatment of citrus leaves with ten candidate precursor peptides; Figure 2 The determination of iron content in citrus leaves after treatment with ten candidate precursor peptides; Figure 3 This shows the expression of the RT1 gene after the green peptide mutation. Figure 4 The results show the iron content in the leaves after the green peptide mutation. Figure 5 These are actual images of the leaf color change in Examples 1-3, Comparative Examples 1-2, and the water control group. Figure 6a Curves showing the trend of iron content changes in leaves after treatment with different concentrations of chlorogenic peptide. Figure 6b Curves showing the trend of total chlorophyll content in leaves after treatment with different concentrations of chlorogenic peptide; Figure 7 These are actual images of the leaf color change in Examples 4-6, Comparative Examples 3-4, and the water control group. Figure 8a Curves showing the trend of iron content changes in leaves after treatment with different concentrations of EDDHA-iron. Figure 8b Curves showing the trend of total chlorophyll content in leaves after treatment with different concentrations of EDDHA-iron. Figure 9 These are actual photos of the leaf color change in Example 7 and Comparative Examples 5-6; Figure 10 The images show actual photos of the leaf color change in Example 8 and Comparative Example 7. Detailed Implementation

[0012] The technical solutions in some embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments provided in this disclosure are within the scope of protection of this disclosure.

[0013] ① Initial database construction: 18,696 citrus protein sequences with a length of less than or equal to 250 amino acids and their corresponding CDS sequences were obtained from the CPBD citrus database.

[0014] ② Domain pre-screening: The transmembrane helical structure was predicted using the DeepTMHMM algorithm, and proteins containing transmembrane domains were removed, retaining 17,004 candidate proteins without transmembrane structures. The SignalP-6.0 tool was used to predict secretory signal peptides (confidence threshold set to ≥0.9), and 964 potential active peptide precursors with high confidence signal peptides were further screened. Among the 964 potential active peptide precursors with high confidence signal peptides, sequences with endoplasmic reticulum retention signals (K / HDEL) at the C-terminus were searched and deleted, finally yielding 962 potential active peptide precursors with high confidence signal peptides.

[0015] ③ Iron absorption association analysis: Transcriptome data of citrus iron absorption were processed using the SRAtoFastq and Kallisto plugins of TBtools software; 26 core candidate genes that were differentially expressed during iron absorption were screened (screening criteria: |log2FC|≥1 and p<0.05), and 26 core regulatory genes that were highly associated with the iron absorption process were obtained.

[0016] ④ Conserved domain crossover analysis: The protein sequences corresponding to the above 26 genes were submitted to the alfalfa database (MtSSPdb). Non_SSP and Putative_SSP type proteins were screened by BlastP alignment. Only high confidence matching sequences with E values ​​<1e-10 were retained. Conserved motif clustering analysis was performed on these sequences using the MEME tool to explore conserved motifs that may be related to iron binding or signal transduction.

[0017] ⑤ Functional activity blind screening: Based on the results of conserved domain analysis, 10 candidate precursor peptide sequences were selected and synthesized by a company (GenScript Biotech Co., Ltd.); functional verification was carried out through foliar spraying experiments on citrus seedlings: qRT-PCR was used to detect changes in the expression level of the key iron transport gene RT1, and HPLC was used to quantitatively analyze the accumulation of iron in the leaves.

[0018] Screening revealed that only the small peptide with the sequence NTLISKVLNKGDAFVFPIGLIHFQFNIGK (the greening peptide) significantly activated RT1 gene expression (upregulated by 79.2%) and promoted leaf iron accumulation (upregulated by 78.53%), thereby promoting the greening of citrus leaves. The activation and promotion effects of the other nine peptides were all below 50% (e.g., ...). Figure 1 and Figure 2 (As shown).

[0019] ⑥ Key structure verification: Polar amino acids in this sequence were interchanged, and nonpolar amino acids were replaced with glycine. After mutation, the function of alleviating iron deficiency jaundice was lost. NTLISKVLNKGDAFVFPIGLIHFQFNIGK was mutated to GTLISDVLGKGDAFVFPIGLIDFGFNIGD. The key structural sites are 1 N, 5 K, 8 N, 21 H, 23 Q, 28 K, 6 amino acids. Figure 3 and Figure 4 ).

[0020] Field efficacy trial 1 Example 1

[0021] 0.75g of green peptide, 51g of EDDHA-iron, 12.6g of EDTA-copper, 5g of manganese sulfate, 15g of chelated boron, 3g of ammonium molybdate, 150g of urea, 30g of EDTA-magnesium, and 30g of oleic acid amyl betaine are added to 1L of water and diluted 500 times before use.

[0022] Example 2

[0023] 1g of green peptide, 51g of EDDHA-iron, 12.6g of EDTA-copper, 5g of manganese sulfate, 15g of chelated boron, 3g of ammonium molybdate, 150g of urea, 30g of EDTA-magnesium, and 30g of oleic acid amyl betaine are added to 1L of water and diluted 500 times before use.

[0024] Example 3

[0025] 1.25g of green peptide, 51g of EDDHA-iron, 12.6g of EDTA-copper, 5g of manganese sulfate, 15g of chelated boron, 3g of ammonium molybdate, 150g of urea, 30g of EDTA-magnesium, and 30g of oleic acid amyl betaine are added to 1L of water and diluted 500 times before use.

[0026] Comparative Example 1 0.5g of green peptide, 51g of EDDHA-iron, 12.6g of EDTA-copper, 5g of manganese sulfate, 15g of chelated boron, 3g of ammonium molybdate, 150g of urea, 30g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0027] Comparative Example 2 1.5g of green peptide, 51g of EDDHA-iron, 12.6g of EDTA-copper, 5g of manganese sulfate, 15g of chelated boron, 3g of ammonium molybdate, 150g of urea, 30g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0028] Experiment location: Renshou County, Meishan City Experimental methods: This fertilizer efficacy test included 6 treatments (including Examples 1-3, Comparative Examples 1 and 2, and a water control group). Each treatment was diluted 500 times and sprayed evenly on the leaves of citrus trees. 3L of the solution was sprayed on each tree. Each treatment was replicated by four plots with a plot area of ​​60.6 m². The plots were arranged in a randomized block design, and a protection row was set between plots.

[0029] Experimental crop: Mandarin orange The experiment was conducted during the spring shoot growth period, when the new shoots are growing rapidly and have a high demand for iron, and when the new leaves show signs of iron deficiency chlorosis.

[0030] Results survey: After the application of the pesticide, the iron content, total chlorophyll content, net photosynthetic rate, total sugar in the fruit, and sugar-acid ratio in the fruit were investigated.

[0031] The specific survey results (i.e., the effects of different concentrations of green peptide micronutrient liquid fertilizer on Papa oranges) are shown in Table 1 below: Water comparison 22.9 0.22 2.3 98.9 26 Example 1 87.2 0.96 10.1 106.7 30 Example 2 115.6 1.44 13.3 115.1 33 Example 3 126.3 1.54 14.4 121 35 Comparative Example 1 46.6 0.41 5.7 102.2 28 Comparative Example 2 115.3 1.38 12.5 114.6 31 Note: The water control and Comparative Example 1 are typical symptoms of iron deficiency chlorosis in citrus, including reduced chlorophyll in the leaves, loss of green between the veins, and the leaf color changing from green to yellow to white depending on the severity of the symptoms.

[0032] From Table 1, Figure 5 and Figures 6a-6b It was found that different concentrations of greening peptide could increase the iron content of citrus leaves. The iron content of leaves treated with 0.5g, 0.75g, 1g, 1.25g, and 1.5g of greening peptide micronutrient fertilizer were 46.6 mg / kg, 87.2 mg / kg, 115.6 mg / kg, 126.3 mg / kg, and 115.3 mg / kg, respectively. Compared with the water control treatment of 22.9 mg / kg, these were increases of 23.7 mg / kg, 64.3 mg / kg, 92.7 mg / kg, 103.4 mg / kg, and 92.4 mg / kg, respectively. All of these treatments alleviated the symptoms of iron deficiency chlorosis in citrus leaves to varying degrees and turned the leaves green. However, the greening rate slowed down at 1.25g / L, and when the concentration was further increased to 1.5g / L, an inhibitory effect was produced, and the greening effect of the leaves weakened.

[0033] Field efficacy trial 2 Example 4

[0034] 0.76g of green peptide, 50g of EDDHA-iron, 14g of EDTA-copper, 5.5g of manganese sulfate, 20g of chelated boron, 4g of ammonium molybdate, 200g of urea, 33g of EDTA-magnesium, and 30g of oleic acid amyl betaine are added to 1L of water and diluted 500 times before use.

[0035] Example 5

[0036] 0.76g of green peptide, 70g of EDDHA-iron, 14g of EDTA-copper, 5.5g of manganese sulfate, 20g of chelated boron, 4g of ammonium molybdate, 200g of urea, 33g of EDTA-magnesium, and 30g of oleic acid amyl betaine are added to 1L of water and diluted 500 times before use.

[0037] Example 6

[0038] 0.76g of green peptide, 90g of EDDHA-iron, 14g of EDTA-copper, 5.5g of manganese sulfate, 20g of chelated boron, 4g of ammonium molybdate, 200g of urea, 33g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0039] Comparative Example 3 0.76g of green peptide, 30g of EDDHA-iron, 14g of EDTA-copper, 5.5g of manganese sulfate, 20g of chelated boron, 4g of ammonium molybdate, 200g of urea, 33g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0040] Comparative Example 4 0.76g of green peptide, 110g of EDDHA-iron, 12.6g of EDTA-copper, 5.5g of manganese sulfate, 20g of chelated boron, 4g of ammonium molybdate, 200g of urea, 33.1g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0041] Experiment location: Renshou County, Meishan City Experimental methods: This fertilizer efficacy test included 6 treatments (including Examples 4-6, Comparative Examples 3 and 4, and a water control group). Each treatment was diluted 500 times and sprayed evenly on the leaves of citrus trees. 3L of the solution was sprayed on each tree. Each treatment was replicated by four plots with a plot area of ​​60.6 m². The plots were arranged in a randomized block design, and protection rows were set between plots.

[0042] Experimental crop: Mandarin orange The experiment was conducted during the spring shoot growth period, when the new shoots are growing rapidly and have a high demand for iron, and when the new leaves show signs of iron deficiency chlorosis.

[0043] Results survey: After the application of the pesticide, the iron content, total chlorophyll content, net photosynthetic rate, total sugar in the fruit, and sugar-acid ratio in the fruit were investigated.

[0044] The specific survey results (i.e., the effects of different concentrations of EDDHA-iron micronutrient liquid fertilizer on Papa Tangerines) are shown in Table 2 below: Water comparison 21.7 0.28 2.7 99.3 26 Example 4 99.9 1.12 12.6 115.6 32 Example 5 128.9 1.53 14.7 120.1 35 Example 6 130.5 1.68 16.4 125.2 36 Comparative Example 3 59.8 0.55 7.7 109.3 30 Comparative Example 4 116 1.33 10.2 116.3 33 Note: The water control shows typical symptoms of iron deficiency chlorosis in citrus, including reduced chlorophyll in the leaves and interveinal chlorosis. The leaf color changes from yellow to white depending on the severity of the symptoms. Comparative example 4 shows symptoms of pesticide damage, where excessive iron concentration causes necrosis at the leaf edges, resulting in a scorched appearance.

[0045] From Table 2, Figure 7 and Figures 8a-8b It was found that different concentrations of EDDHA-iron could increase the iron content of citrus leaves. The iron content of leaves treated with 30g, 50g, 70g, 90g, and 110g of EDDHA-iron was 59.8 mg / kg, 99.9 mg / kg, 128.9 mg / kg, 130.5 mg / kg, and 116 mg / kg, respectively, which were 38.1 mg / kg, 78.2 mg / kg, 107.2 mg / kg, 108.8 mg / kg, and 94.3 mg / kg higher than the 21.7 mg / kg of the water control. All of these treatments alleviated the symptoms of iron deficiency chlorosis in citrus leaves to varying degrees and turned the leaves green. However, the greening rate slowed down at 90g / L. When the concentration was further increased to 110g / L, phytotoxicity occurred, and the edges of the citrus leaves turned brown and scorched.

[0046] Field efficacy trial 3 Example 7

[0047] 1.1g transgreen peptide, 71g EDDHA-iron, 14.5g EDTA-copper, 6g manganese sulfate, 25g chelated boron, 5g ammonium molybdate, 220g urea, 34g EDTA-magnesium, 30g oleamide-propyl betaine, add to 1L of water and dilute 500 times before use.

[0048] Comparative Example 5 1.1g of green peptide, 14.5g of EDTA-copper, 6g of manganese sulfate, 25g of chelated boron, 5g of ammonium molybdate, 220g of urea, 34g of EDTA-magnesium, and 30g of oleamidopropyl betaine are added to 1L of water and diluted 500 times before use.

[0049] Comparative Example 6 71g of EDDHA-iron, 14.5g of EDTA-copper, 6g of manganese sulfate, 25g of chelated boron, 5g of ammonium molybdate, 220g of urea, 34g of EDTA-magnesium, and 30g of oleamidopropyl betaine are diluted to 1L of water and used after dilution 500 times.

[0050] Experiment location: Renshou County, Meishan City Experimental Methods: This fertilizer efficacy test included three treatments (including Example 7, Comparative Example 5, and Comparative Example 6). Each treatment was diluted 500 times and sprayed evenly onto the leaves of citrus trees. 3L of solution was applied to each tree. Each treatment is repeated in four cells, each cell area is 60.6 m², arranged in a randomized block layout, with guard rows between cells.

[0051] Experimental crop: Mandarin orange The experiment was conducted during the spring shoot growth period, when the new shoots are growing rapidly and have a high demand for iron, and when the new leaves show signs of iron deficiency chlorosis.

[0052] Results survey: After the application of the pesticide, the iron content, total chlorophyll content, net photosynthetic rate, total sugar in the fruit, and sugar-acid ratio in the fruit were investigated.

[0053] The specific survey results (i.e., the role of the micronutrient liquid fertilizer composed of transgreen peptide and EDDHA-iron in Papa oranges) are shown in Table 3 below: Example 7 139.2 1.81 19.9 135.7 38 Comparative Example 5 114.6 1.44 13.3 114.4 32 Comparative Example 6 122.8 1.56 14.6 121.6 34 From Table 3 and Figure 9 It can be seen that in Example 7, the combination of 1.1 g / L chlorogenic peptide and 71 g / L LEDDHA-iron showed the best greening effect on iron-deficient yellowing citrus leaves, with an iron content of 139.2 mg / kg, a chlorophyll content of 1.81 mg / g, and a net photosynthetic rate of 19.9 μmol / m. -2 .s -1 The total sugar content of the fruit was 135.7 g / kg, and the sugar-acid ratio was 38%. The comparative formulations of 51.1 g / L transgreen peptide and 671 g / L LEDDHA-iron could not achieve the same effect as the compound formulation.

[0054] Field efficacy trial 4 Example 8

[0055] 1.2g transgreen peptide, 40g EDDHA-iron, 14g EDTA-copper, 5.5g manganese sulfate, 20g chelated boron, 4g ammonium molybdate, 200g urea, 33g EDTA-magnesium, 30g oleamide-propyl betaine, add to 1L of water and dilute 500 times before use.

[0056] Comparative Example 7 1.2g small peptide, 40g EDDHA-iron, 14g EDTA-copper, 5.5g manganese sulfate, 20g chelated boron, 4g ammonium molybdate, 200g urea, 33g EDTA-magnesium, 30g oleic acid amyl betaine, make up to 1L of water, dilute 500 times before use.

[0057] Experiment location: Renshou County, Meishan City Experimental methods: This fertilizer efficacy test included two treatments (including Example 8 and Comparative Example 7). Each treatment was diluted 500 times and sprayed evenly on the leaves of citrus trees. 3L of the solution was sprayed on each tree. Each treatment was replicated by four plots with a plot area of ​​60.6 m². The plots were arranged in a randomized block design and a protection row was set between plots.

[0058] Experimental crop: Mandarin orange The experiment was conducted during the spring shoot growth period, when the new shoots are growing rapidly and have a high demand for iron, and when the new leaves show signs of iron deficiency chlorosis.

[0059] Results survey: After the application of the pesticide, the iron content, total chlorophyll content, net photosynthetic rate, total sugar in the fruit, and sugar-acid ratio in the fruit were investigated.

[0060] The specific survey results (i.e., the roles of different peptides in micronutrient liquid fertilizers in Papa Tangerines) are shown in Table 4 below: Example 8 120.1 1.46 13.7 114.9 33.3 Comparative Example 7 56.8 0.704 7.9 115 30 From Table 4, Figure 2 and Figure 10 It can be seen that, among the ten small peptides screened in the early stage, except for the greening peptide, the g small peptide, which significantly increased the iron content in leaves, still did not show the effect of alleviating iron deficiency chlorosis in citrus in the field experiment. The control group 7 treated with g small peptide still had obvious phenotypes of interveinal chlorosis and leaf mesophyll yellowing.

[0061] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of transgreen peptide in alleviating iron deficiency chlorosis in citrus leaves, characterized in that, The amino acid sequence of the green peptide is NTLISKVLNKGDAFVFPIGLIHFQFNIGK.

2. The application according to claim 1, characterized in that, The application of the transgreen peptide in products used to alleviate iron deficiency chlorosis in citrus leaves.

3. The application according to claim 2, characterized in that, The product is a liquid fertilizer containing trace elements.

4. The application according to claim 3, characterized in that, The trace element liquid fertilizer comprises the following raw materials: The green peptide is composed of 0.75-1.25 g / L of transgreen peptide, 50-90 g / L of EDDHA-iron, 10-15 g / L of EDTA-copper, 4-6 g / L of manganese sulfate, 15-25 g / L of chelated boron, 3-5 g / L of ammonium molybdate, 150-250 g / L of urea, 30-35 g / L of EDTA-magnesium, and 30 g / L of oleamidopropyl betaine, wherein the transgreen peptide is the transgreen peptide described in claim 1.

5. The application according to claim 4, characterized in that, The preparation method of the trace element liquid fertilizer includes the following steps: dissolving 0.75-1.25 g / L of transgreen peptide, 50-90 g / L of EDDHA-iron, 10-15 g / L of EDTA-copper, 4-6 g / L of manganese sulfate, 15-25 g / L of chelated boron, 3-5 g / L of ammonium molybdate, 150-250 g / L of urea, 30-35 g / L of EDTA-magnesium, and 30 g / L of oleamidopropyl betaine in water, and diluting to obtain the trace element liquid fertilizer.

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