Leaf-directed delivery combined with permeation-promoting hypersensitive protein and its application

Abstract

This invention, belonging to the field of biotechnology, relates to a recombinant protein for foliar targeted delivery and permeation-enhancing hypersensitive protein. The recombinant protein consists of two components: a hypersensitive protein functional domain that promotes plant growth and a positively charged functional domain that binds to plant leaves. This novel hypersensitive protein can target foliar surfaces and promote its penetration, thereby significantly promoting plant growth. This reduces the application dosage and enhances its effectiveness, significantly promoting the commercialization of hypersensitive protein products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a leaf surface directed delivery combined with a permeation-promoting hypersensitive protein and an application thereof. Background Art

[0002] In 1992, researchers at Cornell University discovered and isolated a protein from the bacterium Erwinia amylovora that not only protects plants from disease but also promotes their growth. They named it "Harpin," or hypersensitive protein (Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science. 1992 Jul 3; 257(5066): 85-8). Harpin is a natural protein secreted by Gram-negative plant pathogens via the type III secretion pathway. As a major factor in stimulating hypersensitive responses in plants, it has multiple biological effects, including inducing strong disease resistance and promoting plant growth and development. It is a novel, safe, and highly effective plant immune elicitor.

[0003] Hypersensitive proteins are plant immune inducers that are harmless to humans and animals, environmentally friendly, and have been called "a green revolution for plant protection and agricultural product safety producers." Compared to the mechanisms of action of traditional pesticides, fertilizers, and fungicides, hypersensitive proteins offer a broad spectrum, low toxicity, no resistance risk or lag, and a long-lasting effect. They also exhibit synergistic or enhanced effects when mixed with other fungicides.

[0004] The application of hypersensitive protein has a different mechanism of action from traditional agrochemical products, and repeated use will not cause drug resistance; it activates the crop's own functions, has a significant yield-increasing effect, a long-lasting effect, a broad disease prevention spectrum, and reduces labor input and planting costs; the application method is simple, has good compatibility, and is easy to use: it can be used for foliar spraying, root irrigation, seed soaking, seed mixing, root dipping, etc.; it is suitable for spraying with small agricultural machinery, as well as large and medium-sized agricultural machinery and aircraft; when used for foliar spraying, it only needs to be sprayed, unlike traditional pesticides that must be sprayed on the entire plant; it can be mixed with pesticides and fertilizers; it is suitable for all crops; it is green and environmentally friendly.

[0005] Due to the high production cost and poor bioavailability of hypersensitive proteins, their application is greatly limited. Summary of the Invention

[0006] The invention forms a recombinant protein by fusing a binding domain that can target plant leaves with a hypersensitive protein.

[0007] The present invention provides a recombinant protein for leaf-directed delivery combined with a permeation-promoting hypersensitive protein, which consists of two parts: a protein functional domain that can promote plant growth and a positively charged protein functional domain that can be directed to bind to plant leaves. Preferably, the two parts are connected by a connecting peptide, and the specific connecting peptide is a rigid connecting peptide, for example, the rigid connecting peptide is EAAAK;

[0008] In addition, preferably, the positively charged protein functional domain capable of directionally binding to plant leaves is connected to the C-terminus of the protein functional domain capable of promoting plant growth by a connecting peptide.

[0009] In a preferred embodiment, the functional domain of the protein that can promote plant growth is a hypersensitive protein that stimulates plant growth and development; preferably, its amino acid sequence is: MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLA KSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA.

[0010] In another preferred embodiment, the amino acid sequence of the functional domain of the protein capable of directionally binding to plant leaves is AENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN.

[0011] The present invention also provides a nucleic acid encoding the recombinant protein, and an expression plasmid containing the nucleic acid, preferably a pET vector.

[0012] The present invention also provides a recombinant host bacteria containing the expression plasmid, preferably Escherichia coli.

[0013] The present invention particularly provides the use of the recombinant protein in promoting plant seed germination and plant growth.

[0014] Furthermore, the present invention also provides a method for promoting plant seed germination, which comprises incubating a solution containing the recombinant protein with the seeds and then accelerating germination, preferably with an incubation time of 0.5 to 3 hours; preferably, the plants are wheat, rice, tomato, pepper, cucumber, strawberry, watermelon, and tobacco.

[0015] The present invention also provides a method for promoting plant growth, which comprises spraying a solution containing the recombinant protein onto plants at the seedling stage, for example, at the 2-4 leaf stage; preferably, the plants are wheat, rice, tomato, pepper, cucumber, strawberry, watermelon, or tobacco.

[0016] Experiments have shown that the recombinant protein provided by the present invention can target plant leaves, thereby increasing its residence time on the leaf surface, compared with the original hypersensitive protein; at the same time, the binding domain targeted to the plant leaf surface is positively charged, which can effectively increase the probability of the new hypersensitive protein passing through the leaf barrier and entering the cell, thereby increasing its absorption rate and ultimately reducing the application amount of the new hypersensitive protein while enhancing its effect, which has strong promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 , Schematic diagram of recombinant hypersensitive protein

[0018] Figure 2 , protein EGFP and EGFP-LP expression results

[0019] Figure 3 Characterization of leaf surface binding protein EGFP and EGFP-LP

[0020] Figure 4 , purification results of recombinant hypersensitive protein

[0021] Figure 5 , characterization of wheat immuned by recombinant hypersensitive protein. Among them, A is the plant height phenotype of wheat, and B is the bar graph of the average plant height of wheat.

[0022] Figure 6 , fresh weight bar graph of wheat immunized with recombinant hypersensitive protein

[0023] Figure 7 , characterization of tobacco cells immunized with recombinant hypersensitive protein. A is the plant height phenotype of tobacco plants, and B is a bar graph of the average plant height of tobacco plants. DETAILED DESCRIPTION

[0024] The present invention is further described below with reference to specific examples in order to provide a better understanding of the present invention, but the present invention is not limited thereto.

[0025] Example 1: Construction of recombinant protein plasmid

[0026] 1. Construction of recombinant protein

[0027] The recombinant protein (Harpin Hrpz LP) has two main functional domains ( Figure 1): Harpin Hrpz, a hypersensitive protein that stimulates plant growth and development, whose amino acid sequence is shown in SEQ ID NO: 1 (MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVV VKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA); and leaf binding domain LP, whose amino acid sequence is shown in SEQ ID NO: 2 (A ENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN)(Dittrich J,Brethauer C,Gonchar enko L,etal.Rational Design Yields Molecular Insights on Leaf-Binding of AnchorPeptides[J].ACS applied materials&interfaces,2022,14(25):28412-28426.), can combine with the leaf wax part in the leaves to fix the protein on the leaf surface, increase the residence time of the recombinant protein on the leaf surface, and thus enhance the effect of the hypersensitive protein.LP is connected to the C-terminus of Harpin Hrpz protein by a rigid linker peptide EAAA K. The amino acid sequence of the recombinant protein Harpin Hrpz LP is SEQ ID NO3:MQSLS LNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSD AGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAAEAAAKEAAAKGAENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN.

[0028] 2. Construction of protein expression plasmid

[0029] Construction of plasmid pET-EGFP / pET-EGFP_LP: The synthetic gene sequence encoding protein pET-EGFP / pET-EGFP_LP was inserted into the pET-28a(+) plasmid between NcoI and BamHI to obtain plasmid pET-EGFP_LP / pET-EGFP.

[0030] The construction of plasmid pET-Harpin Hrpz / pET-Harpin Hrpz LP was the same as above.

[0031] Example 2: Functional characterization of LP leaf-binding peptides

[0032] 1) Transform the plasmid pET-EGFP_LP / pET-EGFP into Escherichia coli, screen for positive transformants, inoculate them into LB medium containing 50 mg / L kanamycin, and culture overnight at 37°C to obtain seed solution;

[0033] 2) The seed solution was transferred into TB medium containing 50 mg / L kanamycin at a 1% inoculum volume and cultured at 30°C until the OD600 reached 0.6-0.8. 1 mM IPTG was added to induce the expression of the recombinant protein. After culturing at 30°C for 6 hours, the cells were collected.

[0034] 3) Add protein lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.4) to resuspend the cells, lyse with an ultrasonic disruptor for 30 min, and centrifuge at 12,000 rpm for 30 min to collect the supernatant;

[0035] 4) In the experimental group, freshly picked leaves were immersed in a solution containing EGFP_LP protein (35.4 kDa) and incubated at room temperature for 5 min to allow the recombinant protein to fully bind to the leaves. In the control group, freshly picked leaves were immersed in a solution containing EGFP protein (28 kDa) (Heikal AA, Hess ST, Webb W W. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity[J]. Chemical Physics, 2001, 274(1):37-55.) and incubated at room temperature for 5 min.

[0036] 6) Remove the leaves and soak them in Tris-HCl buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.4). Oscillate at room temperature for 3 minutes to remove proteins not bound to the leaves. Repeat the same procedure for the control group.

[0037] 7) Take a portion of the treated leaves and examine them under a fluorescence microscope. After excitation (wavelength 488 nm), test whether the leaf surface emits fluorescence.

[0038] Based on the above experimental results, we can conclude that: Figure 2 As shown in Figure A, there is an obvious band between 20 and 31 kDa in the supernatant of cell lysate, and the fluorescent protein (EGFP) is 28 kDa, so the recombinant protein EGFP is successfully expressed. Figure 2 As shown in Figure B, the size of the recombinant protein in the supernatant of the cell lysate ranges from 30 to 41 kDa, the size of the recombinant protein EGFP_LP is 35.4 kDa, and the band is clear, so the recombinant protein EGFP_LP is successfully expressed.

[0039] Figure 3In the figure, A shows the brightfield image of the control group leaves, showing clear leaves; B shows the image of the control group leaves excited at 488 nm; C shows the image of the experimental group leaves, showing clear leaves; and D shows the image of the experimental group leaves excited at 488 nm. Under the same experimental conditions, after washing, leaves infiltrated with EGFP protein in the control group showed no fluorescence signal under 488 nm excitation, indicating that the fluorescent protein did not bind to the leaf surface. However, after washing, leaves infiltrated with EGFP_LP protein in the experimental group showed a strong fluorescence signal under 488 nm excitation, indicating that the EGFP_LP protein successfully bound to the leaf.

[0040] Example 3: Expression and purification of novel hypersensitive proteins in Escherichia coli

[0041] 1) Transform the plasmids pET28a(+)-Harpin Hrpz and pET28a(+)-Harpin-Hrpz LP into Escherichia coli. Screen for positive transformants and inoculate them into LB medium containing 50 mg / L kanamycin. Incubate overnight at 37°C to obtain seed solution.

[0042] 2) The seed solution was transferred into TB medium containing 50 mg / L kanamycin at a 1% inoculum volume and cultured at 37°C until the OD600 reached 0.6-0.8. 0.8 mM IPTG was added to induce the expression of the recombinant protein. After culturing at 37°C for 6 hours, the cells were collected.

[0043] 3) Add lysis buffer (20 mM Tris-HCl, 500 mM NaCl, pH 8.0) to resuspend the cells, lyse using an ultrasonic disruptor for 30 min, and centrifuge at 12,000 rpm for 30 min to collect the supernatant;

[0044] 4) The collected supernatant was filtered through a 0.22 μm membrane and purified using a nickel column to obtain Harpin Hrpz and harpinhrpz LP protein samples, respectively.

[0045] 5) Imidazole was removed by dialysis using dialysate (20 mM Tris-HCl, 500 mM NaCl, pH 8.0).

[0046] Depend on Figure 4 The results in Figure A show that the size of the purified recombinant protein is between 30 and 41 kDa, and the size of the recombinant protein Harpin Hrpz is 36.02 kDa with a relatively single band, so the expression and purification of the hypersensitive protein Harpin Hrpz was successful. Figure 4From the results in Figure B, we can see that the size of the purified protein is between 41 and 53 kDa, the size of the novel hypersensitive protein Harpin Hrpz LP is 42.36 kDa and the band is single, indicating that the novel hypersensitive protein Harpin Hrpz LP was successfully expressed and purified.

[0047] Example 4: Characterization of recombinant hypersensitive protein-immunized wheat

[0048] 1) Soak wheat seeds in tap water for 12 hours, remove them, and blot dry with absorbent paper. Select an equal number of plump seeds, add buffer (control group), and incubate with purified Harpin Hrpz and Harpin Hrpz LP protein samples (final protein concentration of 1 g / L) for 1 hour.

[0049] 2) Spread the seeds on moist cotton and incubate at room temperature for 5 days

[0050] 3) Calculate the germination rate of seeds.

[0051] 4) Continue culturing for 2 days and count the wheat plant heights.

[0052] 5) Use absorbent paper to absorb the moisture from the roots and weigh the total fresh weight using a balance.

[0053] As shown in Table 1, the germination rate of the control group was 86.36%, the germination rate of Harpin Hrpz and Harpin Hrpz LP was 96%, and the germination rate of Harpin Hrpz and Harpin Hrpz LP was increased by 9.64%.

[0054] From Table 2 and Figure 5 As shown in Figures A and B, the average plant height of the control group was approximately 1027 μm, the average plant height of Harpin Hrpz was approximately 1097 μm, and the average plant height of Harpin Hrpz LP was approximately 1110 μm, increases of 70 μm and 83 μm, respectively, and increases of 6.8% and 8%. This indicates that Harpin Hrpz significantly promotes wheat growth, and Harpin Hrpz LP can enhance the growth-promoting effect of Harpin Hrpz on wheat.

[0055] Table 1 Statistics of germination rate of recombinant hypersensitive immune wheat for 5 days

[0056]

[0057]

[0058] Table 2 Plant height measurement of recombinant hypersensitive immune wheat

[0059] test comparison Harpin Hrpz Harpin Hrpz LP 1 1014.51 1098.36 1169.72 2 1079.29 1161.55 1082.69 3 1005.46 1079.61 1092.8 4 1061.07 1085.42 1110.35 5 1078.88 1053.79 1105.7 6 1018.64 1053.15 1104.1 7 937.54 1154.03 1108.05 Average value (μm) 1027.91 1097.99 1110.49

[0060] From Table 3 and Figure 6 It can be seen that the total fresh weight of the control group was 2.372g, the total fresh weight of Harpin Hrpz was 4.129g, and the total fresh weight of Harpin Hrpz LP was 5.034g, with the total fresh weight increased by 74% and 112%, respectively. This shows that Harpin Hrpz can significantly increase the yield. Compared with Harpin Hrpz, Harpin Hrpz LP increased the fresh weight of the plant by 38%.

[0061] Table 3 Fresh weight of wheat immunized with recombinant hypersensitive protein

[0062] test comparison Harpin hrpz Harpin hrpz LP Fresh weight / (g) 2.37 4.13 5.03

[0063] In summary, it was shown that Harpin Hrpz LP was better than HarpinHrpz in promoting wheat plant height and increasing yield.

[0064] Example 5: Characterization of novel hypersensitive protein immunotobacco

[0065] 1) Mix vermiculite and nutrient soil in a ratio of 1:3 and moisten with water.

[0066] 2) Sow tobacco seeds on mixed soil, cover with a moisture-retaining transparent cover, and then culture at room temperature for two days.

[0067] 3) When the seeds germinate to the 2-leaf stage, spray the seeds with purified samples of Harpin Hrpz and Harpin Hrpz LP proteins (final protein concentration of 3 g / L) every three days for a total of three times.

[0068] 4) Cultivate the tobacco plants for another 12 days and count the plant heights.

[0069] From Table 4 and Figure 7 As shown in Figures A and B, the average plant height of the tobacco grown for 14 days was about 74.7 μm in the control group, about 101.37 μm in HarpinHrpz, and about 119.62 μm in HarpinHrpz LP, which were increased by 26.67 μm and 44.92 μm, respectively, and the plant height increased by 35.7% and 60.14%, respectively.

[0070] Table 4 Measurement of plant height of tobacco plants immunized with recombinant hypersensitive protein

[0071] test comparison Harpin Hrpz Harpin Hrpz LP 1 63.35 93.94 122.41 2 67.76 96.04 111.68 3 96.3 96.15 127.06 4 82.24 103.91 119.89 5 63.83 116.79 117.06 Average value (μm) 74.70 101.37 119.62

[0072] Table 4 Measurement of plant height of tobacco plants immunized with recombinant hypersensitive protein

[0073] test comparison Harpin Hrpz Harpin Hrpz LP 1 63.35 93.94 122.41 2 67.76 96.04 111.68 3 96.3 96.15 127.06 4 82.24 103.91 119.89 5 63.83 116.79 117.06 Average value (μm) 74.70 101.37 119.62

[0074] This shows that both Harpin Hrpz and Harpin Hrpz LP can significantly promote tobacco growth, and the promoting effect of Harpin Hrpz LP on tobacco growth is significantly better than that of Harpin Hrpz.

Claims

1. A method for promoting plant seed germination or plant growth by foliar targeted delivery of a recombinant protein combined with a permeability-promoting hypersensitive protein; wherein the plant is wheat or tobacco; The recombinant protein for leaf-directed delivery combined with permeation-promoting hypersensitive protein consists of two parts: a protein functional domain that can promote plant growth and a positively charged protein functional domain that can be directed to bind to plant leaves; The protein functional domain capable of promoting plant growth is a hypersensitive protein that stimulates plant growth and development; The protein functional domain capable of promoting plant growth and the positively charged protein functional domain capable of directionally binding to plant leaves are connected by a connecting peptide, and the positively charged protein functional domain capable of directionally binding to plant leaves is connected to the C-terminus of the protein functional domain capable of promoting plant growth by the connecting peptide; The connecting peptide is EAAAK; The amino acid sequence of the functional domain of the protein that can promote plant growth is: MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA; The amino acid sequence of the protein functional domain that can directionally bind to plant leaves is AENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN.

2. A method for promoting plant seed germination, characterized in that: The solution containing the recombinant protein for leaf-directed delivery combined with the permeability-promoting hypersensitive protein is incubated with the seeds, and then the seeds are germinated; The recombinant protein for leaf-directed delivery combined with permeation-promoting hypersensitive protein consists of two parts: a protein functional domain that can promote plant growth and a positively charged protein functional domain that can be directed to bind to plant leaves; The protein functional domain capable of promoting plant growth is a hypersensitive protein that stimulates plant growth and development; The protein functional domain capable of promoting plant growth and the positively charged protein functional domain capable of directionally binding to plant leaves are connected by a connecting peptide, and the positively charged protein functional domain capable of directionally binding to plant leaves is connected to the C-terminus of the protein functional domain capable of promoting plant growth by the connecting peptide; The connecting peptide is EAAAK; The amino acid sequence of the functional domain of the protein that can promote plant growth is: MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA; The amino acid sequence of the protein functional domain that can bind to plant leaves is AENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN; The plant is wheat or tobacco.

3. The method according to claim 2, wherein The incubation time is 0.5 to 3 hours.

4. A method for promoting plant growth, characterized in that: spraying a solution containing a recombinant protein for leaf-directed delivery and binding to a permeation-promoting hypersensitive protein onto the seedling stage of the plant; The recombinant protein for leaf-directed delivery combined with a permeation-promoting hypersensitive protein consists of two parts: a protein functional domain that can promote plant growth and a positively charged protein functional domain that can bind to plant leaves in a directed manner. The two parts are connected by a connecting peptide, and the positively charged protein functional domain that can bind to plant leaves in a directed manner is connected to the C-terminus of the protein functional domain that can promote plant growth by a connecting peptide. The connecting peptide is EAAAK; The amino acid sequence of the functional domain of the protein that can promote plant growth is: MQSLSLNSSSLQTPAMALVLVRPEAETTGSTSSKALQEVVVKLAEELMRNGQLDDSSPLGKLLAKSMAADGKAGGGIEDVIAALDKLIHEKLGDNFGASADSASGTGQQDLMTQVLNGLAKSMLDDLLTKQDGGTSFSEDDMPMLNKIAQFMDDNPAQFPKPDSGSWVNELKEDNFLDGDETAAFRSALDIIGQQLGNQQSDAGSLAGTGGGLGTPSSFSNNSSVMGDPLIDANTGPGDSGNTRGEAGQLIGELIDRGLQSVLAGGGLGTPVNTPQTGTSANGGQSAQDLDQLLGGLLLKGLEATLKDAGQTGTDVQSSAAQIATLLVSTLLQGTRNQAAA; The amino acid sequence of the protein functional domain that can bind to plant leaves is AENLYFQGDSHEERHHGRHGHHKYGRKFHEKHHSHRGYRSNYLYDN; The plant is wheat or tobacco.

5. The method according to claim 4, wherein The plant seedling stage is the plant seedling 2-4 leaf stage.

Images