Mutant, combined protein, gene, vector, cell, composition and method for improving photosynthesis efficiency of crops

Abstract

The invention relates to the field of plant genes, and discloses a mutant, a combined protein, a gene, a vector, a cell, a composition and a method for improving photosynthesis efficiency of crops. The mutant is a mutant of at least one enzyme selected from the group consisting of an OsSPS2 enzyme, an OsSPS3 enzyme, an AtSPS1 enzyme, an AtSPS2 enzyme, an OsFBN5 enzyme and an AtFBN5 enzyme. The mutant and the gene provided by the invention can be used for improving the photosynthesis efficiency of crops.

Description

Technical Field

[0001] This invention relates to the field of plant genes, specifically to mutants, combinatorial proteins, genes, vectors, cells, compositions, and methods for improving crop photosynthetic efficiency. Background Technology

[0002] The SPS gene, or Solanesyl diphosphate synthase gene, is found in many plants and encodes a key enzyme, SPS, in the plastoquinone (PQ-9) biosynthetic pathway. The product catalyzed by this enzyme can provide isopentenyl side chains for PQ-9 and play an important biological function.

[0003] Fibrillin 5 (FBN5), a protein that interacts with SPS, is encoded by the FBN5 gene. FBN5 promotes SPS activity through its interaction with SPS. However, current research has not explored the interaction between SPS and FBN5 and its impact on photosynthetic efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a new mutant and / or gene that can improve the photosynthetic efficiency of crops.

[0005] Currently, no literature reports specific sites that can affect the catalytic activity of SPS or the interaction between SPS and FBN5. To regulate the interaction between SPS and FBN5 in various crops and thus influence photosynthetic efficiency, this invention designs SPS and FBN5 mutants from different species, aiming to provide potential candidate genes for improving crop photosynthetic efficiency.

[0006] This invention reveals the functions of SPS and FBN5 mutants in plant photosynthesis and their potential applications through research on them.

[0007] To achieve the above objective, a first aspect of the present invention provides a mutant, which is a mutant of at least one enzyme selected from OsSPS2, OsSPS3, OsFBN5, AtSPS1, AtSPS2, and AtFBN5; the amino acid sequence of the OsSPS2 enzyme is shown in SEQ ID NO. 1; the amino acid sequence of the OsSPS3 enzyme is shown in SEQ ID NO. 2; the amino acid sequence of the OsFBN5 enzyme is shown in SEQ ID NO. 3; the amino acid sequence of the AtSPS1 enzyme is shown in SEQ ID NO. 4; the amino acid sequence of the AtSPS2 enzyme is shown in SEQ ID NO. 5; and the amino acid sequence of the AtFBN5 enzyme is shown in SEQ ID NO. 6; the mutation site of the mutant includes at least one of the following: a: At least one of the following positions in SEQ ID NO. 1—positions 121, 122, 123, 126, 161, 168, 172, 177, 178, 190, 230, 238, 239, 254, 295, 309, and 341—has mutated; b: At least one of the following positions in SEQ ID NO. 2 is mutated: position 132, position 133, position 134, position 137, position 172, position 179, position 183, position 188, position 189, position 201, position 241, position 249, position 250, position 265, position 306, position 320, and position 352. c: At least one of the following positions in SEQ ID NO. 3 is mutated: position 156, position 162, position 163, and position 190; d: At least one of the following positions in SEQ ID NO. 4 is mutated: position 180, position 181, position 193, and position 344. e: At least one of the following positions in SEQ ID NO. 5 is mutated: position 191, position 192, position 204, and position 355; f: At least one of the 153rd and 159th positions of SEQ ID NO. 6 is mutated.

[0008] SEQ ID NO. 1: FISHERMAN PERISVSSLLEVVSDLLKLNNNLKSLVGAENPVLVSAAEQIFGAGGKRLRPALVFLVSRATAELAGLLELTTTEHQRLAEIIEMIHTASLIHDDVIDDSGMRRGKETIHQLYGTRVAVLAGDFFAQSSWFLANLENIEVIKLISIKDFASQDFASGE QASTLFDCDVTLDDYLLKSYKTASLSRSAAIFSGVSTTICEQMYEYGRNLGLSFQVVDDILDFTQSAEQLGKPAGSDLAKGNLTAPVIFALQDEPKLREIIDSEFSESDSLATAIDLVHRSGGIRRAQELAKEKGDLALQCLQCLFRSTQSQNQLQLQLQD

[0009] SEQ ID NO. 2: MAATIPDAATGVGVAERISVSSLLEVVADDLLKLNNNLKSLVGAENPVLVSAAEQIFGAGGKRLRPALVFLVSRATAELAGLLELTTTEHQRLAEIIEMIHTASLIHDDVIDDSGMRRGKETIHQLYGTRVAVLAGDFFAQSSWFLANEVIKASVIKDFSKS QASTLFDCDITLDDYLLKSYKTASLIAASTRSAAIFSGVSTAICEQMYEYGRNLGLSFQVVDDILDFTQSAEQLGKPAGSDLAKGNLTAPVIFALQDEPQLREIIDSEFSETNSLATAIELVHRSGGIKRAHELAREKGEIQCLPRSEQSLQNLEVLEDLE

[0010] SEQ ID NO. 3: MARAVGSYEAALGDAKDALYAALEGMNRGIFGMTSEKRSEIHALVELLESKNPTPEPTDKLQDKVDGCWRLVYSTISILGKKRTKLGLRDFISLGDFQMIDV KEEKAVNVIKFSARALKILSGQLTIEASYKITTKTKVDITLDSSTITPDQLMNIFQKNYDMLLAIFNPEGWLEITYVDESLRIGRDDKANIFVLERADPSEV。

[0011] SEQ ID NO. 4: MFDLKQESKQPISLVTLFELVAVDLQTLNDNLLSIVGAENPVLISAAEQIFGAGGKRMRPGLVFLVSHATAELAGLKELTEHRRLAEIIEIHTASLIHDDVLDESDMRRGKETVHELFGTRVAVLAGDFMFAQASWYLANLENLEVIKLISQVIKDFASGEIKQASSLFDCDTKLDEYLLKSFYKTASLVAASTKGAAIFSRVEPDVTEQMYEFGKNLGLSFQIVDDILDFTQSTEQLGKPAGSDLAKGNLTAPVIFALEREPRLREIIESEFCEAGSLEEAIEAVTGGGIKRAQELAREKADDAIKNLQCLPRSGFRSALEDMVLYNLERID.

[0012] SEQ ID NO. 5 MAVPAKSKENSLVNGIGQDQTVMLNLRQESRKPISLETLFEVVADDLQRLNDNLSLIVGAENPVLISAAEQIFASAGGKRMRPGLVFLVSRATAELAGLKELTVEHRRLGEIIEMIHTASLIHDDVLDESDMRRGRETVHELFGTRVAVLAGDFMFAQASWYLANLENLEVIKLISQVIKDFASGEIKQASSLFDCDVKLDDYMLKSYYKTASLVAASTKGAAIFSKVESKVAEQMYQFGKNLGLSFQVVDDILDFTQSTEQLGKPAANDLAKGNITAPVIFALENEPRLREIIESEFCEPGSLEEAIEVRNRGGIKKAQELAKEKAELALKNLNCLPRSGFRSALEDMVMFNLERID.

[0013] SEQ ID NO. 6: MCSSPNEQQQDEEQEQEQEEITVSHIKEELYEALKGINRGIFGVKSDKKTEIEGLVKLLECRNPTPEPTGELDKIGGCWKLIYSTITVLGSKRTKLGLRDFVSLGDL LQQIDIAQGKTVHVLKFDVRGLNLLDGEFRIVASFKISSKSSVEITYESSTIKPDQLMNIFRKNMDLLLGIFNPEGLFEISYLDEDLQVGRDGKGNVFVLERIEKP.

[0014] According to the preferred embodiment 1, the mutant is a single mutant.

[0015] In a preferred embodiment 1, more preferably, the amino acid sequence of the mutant includes at least one of the following amino acid sequences: X1: The amino acid sequence shown in SEQ ID NO. 1 has at least one of the following mutations: G at position 121 is mutated to W or A, G at position 122 is mutated to W, K at position 123 is mutated to A, R at position 126 is mutated to A, H at position 161 is mutated to A, D at position 168 is mutated to A, D at position 172 is mutated to A, R at position 177 is mutated to A, R at position 178 is mutated to A, R at position 190 is mutated to A, E at position 230 is mutated to A, F at position 238 is mutated to A, D at position 239 is mutated to A, K at position 254 is mutated to A, D at position 295 is mutated to A, K at position 309 is mutated to A, E at position 341 is mutated to A, D, R, Y or Q; X2: The amino acid sequence shown in SEQ ID NO. 2 has at least one of the following mutations: G at position 132 is mutated to W or A, G at position 133 is mutated to W or A, K at position 134 is mutated to A, R at position 137 is mutated to A, H at position 172 is changed to A, D at position 179 is mutated to A, D at position 183 is mutated to A, R at position 188 is mutated to A, R at position 189 is mutated to A, R at position 201 is mutated to A, E at position 241 is mutated to A, F at position 249 is mutated to A, D at position 250 is mutated to A, K at position 265 is mutated to A, D at position 306 is mutated to A, K at position 320 is mutated to A, E at position 352 is mutated to A, D, R, Y or Q; X3: The amino acid sequence shown in SEQ ID NO. 3 has at least one of the following mutations: R at position 156 is mutated to D or E, R at position 162 is mutated to D or E, D at position 163 is mutated to A, and R at position 190 is mutated to E. X4: At least one of the following mutations occurs in the amino acid sequence shown in SEQ ID NO. 4: R at position 180 is mutated to A, R at position 181 is mutated to A, R at position 193 is mutated to A, and E at position 344 is mutated to A, D, R, Y or Q. X5: The amino acid sequence shown in SEQ ID NO. 5 has at least one of the following mutations: R at position 191 is mutated to A, R at position 192 is mutated to A, R at position 204 is mutated to A, and E at position 355 is mutated to A, D, R, Y or Q. X6: At least one of the following mutations occurs in the amino acid sequence shown in SEQ ID NO. 6: R at position 153 is mutated to D or E, or R at position 159 is mutated to D or E.

[0016] According to the preferred embodiment 2, the mutant is a multiple mutant.

[0017] In a preferred embodiment 2, more preferably, the amino acid sequence of the mutant includes at least one of the following amino acid sequences: Y1: The amino acid sequence shown in SEQ ID NO. 1 contains at least two of the following mutations: G at position 121 is mutated to W or A, G at position 122 is mutated to W, K at position 123 is mutated to A, R at position 126 is mutated to A, H at position 161 is mutated to A, D at position 168 is mutated to A, D at position 172 is mutated to A, R at position 177 is mutated to A, R at position 178 is mutated to A, R at position 190 is mutated to A, E at position 230 is mutated to A, F at position 238 is mutated to A, D at position 239 is mutated to A, K at position 254 is mutated to A, D at position 295 is mutated to A, K at position 309 is mutated to A, and E at position 341 is mutated to A, D, R, Y or Q. Y2: The amino acid sequence shown in SEQ ID NO. 2 has at least two of the following mutations: G at position 132 is mutated to W or A, G at position 133 is mutated to W or A, K at position 134 is mutated to A, R at position 137 is mutated to A, H at position 172 is mutated to A, D at position 179 is mutated to A, D at position 183 is mutated to A, R at position 188 is mutated to A, R at position 189 is mutated to A, R at position 201 is mutated to A, E at position 241 is mutated to A, F at position 249 is mutated to A, D at position 250 is mutated to A, K at position 265 is mutated to A, D at position 306 is mutated to A, K at position 320 is mutated to A, and E at position 352 is mutated to A, D, R, Y, or Q. Y3: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 3: R at position 156 is mutated to D or E, R at position 162 is mutated to D or E, D at position 163 is mutated to A, and R at position 190 is mutated to E. Y4: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 4: R at position 180 is mutated to A, R at position 181 is mutated to A, R at position 193 is mutated to A, and E at position 344 is mutated to A, D, R, Y, or Q. Y5: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 5: R at position 191 is mutated to A, R at position 192 is mutated to A, R at position 204 is mutated to A, and E at position 355 is mutated to A, D, R, Y or Q. Y6: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 6: the R at position 153 is mutated to D or E, and the R at position 159 is mutated to D or E.

[0018] The phrase "at least two of the following mutations occur in the amino acid sequence as shown in the present invention" means that at least two sites (or three, four, five, etc.) in the amino acid sequence are mutated. The specific sites and types of mutations can be selected from at least two of the cases listed below. For example, when two mutations occur in the amino acid sequence as shown in SEQ ID NO. 1, it could be a mutation of G to W at position 121 and a mutation of G to W at position 122; or a mutation of G to A at position 121 and a mutation of G to W at position 122; or a mutation of G to W at position 121 and a mutation of K to A at position 123; and so on.

[0019] A second aspect of the present invention provides a combinatorial protein comprising a Z1 protein and a Z2 protein; wherein the Z1 protein is selected from at least one of the following: a mutant with a mutation at position 341 in the amino acid sequence shown in SEQ ID NO. 1; a mutant with a mutation at position 352 in the amino acid sequence shown in SEQ ID NO. 2; a mutant with a mutation at position 344 in the amino acid sequence shown in SEQ ID NO. 4; or a mutant with a mutation at position 355 in the amino acid sequence shown in SEQ ID NO. 5; wherein the Z2 protein is selected from at least one of the following: an OsFBN5 enzyme; a mutant with a mutation at position 156 or 162 in the amino acid sequence shown in SEQ ID NO. 3; or a mutant with a mutation at position 153 or 159 in the amino acid sequence shown in SEQ ID NO. 6.

[0020] The present invention defines a mutant in which the E at position 341 of the amino acid sequence shown in SEQ ID NO. 1 is mutated as OsSPS2_E341.

[0021] The present invention defines a mutant in which the E at position 352 of the amino acid sequence shown in SEQ ID NO. 2 is mutated as OsSPS3_E352.

[0022] The present invention defines a mutant in which the E at position 344 of the amino acid sequence shown in SEQ ID NO. 4 is mutated as AtSPS1_E344.

[0023] The present invention defines a mutant in which the E at position 355 of the amino acid sequence shown in SEQ ID NO. 5 is mutated as AtSPS2_E355.

[0024] The present invention defines a mutant in which the R at position 156 of the amino acid sequence shown in SEQ ID NO. 3 is mutated as OsFBN5_R156.

[0025] The present invention defines a mutant in which the R at position 162 of the amino acid sequence shown in SEQ ID NO. 3 is mutated as OsFBN5_R162.

[0026] The present invention defines a mutant in which the R at position 153 of the amino acid sequence shown in SEQ ID NO. 6 is mutated as AtFBN5_R153.

[0027] The present invention defines a mutant in which the R at position 159 of the amino acid sequence shown in SEQ ID NO. 6 is mutated as AtFBN5_R159.

[0028] The present invention defines the mutant with E mutated to D at position 352 in the amino acid sequence shown in SEQ ID NO. 2 as OsSPS3_E352D.

[0029] Preferably, the combined protein comprises OsSPS3_E352D and OsFBN5. The amino acid sequence of OsSPS3_E352D is shown in SEQ ID NO. 7.

[0030] SEQ ID NO. 7: MAATIPDAAATGVGVAERISVSSLLEVVADDLLKLNNNLKSLVGAENPVLVSAAEQIFGAGGKRLRPALVFLVSRATAELAGLLELTTEHQRLAEIIEMIHTASLIHDDVIDDSGMRRGKETIHQLYGTRVAVLAGDFMFAQSSWFLANLENIEVIKLISQVIKDFASGEIK QASTLFDCDITLDDYLLKSYYKTASLIAASTRSAAIFSGVSTAICEQMYEYGRNLGLSFQVVDDILDFTQSAEQLGKPAGSDLAKGNLTAPVIFALQDEPQLREIIDSDFSETNSLATAIELVHRSGGIKRAHELAREKGEIAIQSLQCLPRSEFRSTLENMVKYNLERID.

[0031] In this invention, compared with the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, the mutation sites of the above mutants and the codons corresponding to the mutated amino acids are shown in Table 1.

[0032] Table 1

[0033] A third aspect of the present invention provides a gene encoding the mutant described in the first aspect or the combinatorial protein described in the second aspect, wherein the nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the mutant described in the first aspect or the combinatorial protein described in the second aspect.

[0034] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 is as shown in SEQ ID NO. 8.

[0035] SEQ ID NO. 8

[0036] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is as shown in SEQ ID NO. 9.

[0037] SEQ ID NO. 9:

[0038] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 3 is as shown in SEQ ID NO. 10.

[0039] SEQ ID NO. 10 .

[0040] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 4 is as shown in SEQ ID NO. 11.

[0041] SEQ ID NO. 11

[0042] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 5 is as shown in SEQ ID NO. 12.

[0043] SEQ ID NO. 12

[0044] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 6 is as shown in SEQ ID NO. 13.

[0045] SEQ ID NO. 13 .

[0046] A fourth aspect of the present invention provides an expression vector containing the gene described in the third aspect of the present invention.

[0047] A fifth aspect of the present invention provides a transgenic cell containing the expression vector described in the fourth aspect.

[0048] A sixth aspect of the present invention provides a composition comprising, as an active ingredient, the mutant described in the first aspect or the combinatorial protein described in the second aspect.

[0049] The seventh aspect of the present invention provides the use of the mutants described in the first aspect, the combinatorial proteins described in the second aspect, the genes described in the third aspect, the expression vectors described in the fourth aspect, the transgenic cells described in the fifth aspect, and the compositions described in the sixth aspect in improving crop photosynthetic efficiency.

[0050] An eighth aspect of the present invention provides a method for improving the photosynthetic efficiency of crops, the method comprising: transferring the expression vector described in the fourth aspect into a target crop, such that the target crop expresses the mutant described in the first aspect or the combinatorial protein described in the second aspect; or... The method includes: transferring the expression vector described in the fourth aspect into a target crop through hybridization, transfer, or backcrossing, so that the target crop expresses the mutant described in the first aspect or the combinatorial protein described in the second aspect.

[0051] By applying the genes, and / or proteins, and / or cells, and / or vectors, and / or compositions of this invention that affect SPS catalysis, SPS-FBN5 interaction, and / or for crop improvement, the catalytic efficiency of SPS can be increased, thereby enhancing the plant's ability to regulate photosynthetic-related responses. This optimization not only enhances the plant's adaptability to environmental changes but also improves crop growth rate and health, providing a new technical means for crop improvement.

[0052] This invention makes a significant contribution to stabilizing and / or increasing crop yields.

[0053] The crops described in this invention include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, and algae, as well as clones and plant parts used for asexual reproduction (e.g., cuttings, layering, young shoots, rhizomes, underground stems, clumps, root necks, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" further encompasses the whole plant, plant parents and offspring, and plant parts, including seeds, branches, stems, leaves, roots (including tubers), flowers, florets, fruits, fleshy stems, peduncles, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of the plant, as well as tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores. Plants described in this invention particularly include rice, wheat, corn, sorghum, barley, soybean, sunflower, rapeseed, alfalfa, cotton, tomato, potato, or tobacco. Detailed Implementation

[0054] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0055] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available products; room temperature refers to 25±2℃.

[0056] Preparation method of 50×TAE solution: Weigh 242 g of Tris and 18.6 g of EDTA into a 1 L beaker, add about 800 mL of deionized water, stir, add 57.1 mL of glacial acetic acid, dissolve completely, adjust the pH to 8.3 with NaOH, and then add deionized water to make up to 1 L. Store at room temperature.

[0057] Preparation method of 1×TAE solution: Take 20mL of 50×TAE solution and add deionized water to make up to 1L.

[0058] Example 1 Construction of expression carrier (1) Amplification of the target gene NdeI and KpnI-HF restriction sites were added to both ends of the nucleotide sequences shown in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13, respectively; NdeI and KpnI-HF restriction sites were added to both ends of the nucleotide sequence of the mutant gene; the gene was synthesized by Wuhan Baijiesi Biotechnology Co., Ltd. using whole-genome synthesis technology.

[0059] Using the aforementioned whole-gene synthesis fragments as templates, wild-type and mutant gene sequences of SPS or FBN5 were obtained by PCR and ligated into the corresponding vectors. The PCR reaction system, PCR program settings, and primers used in the PCR reaction are shown in Tables 2, 3, and 4, respectively. Table 2

[0060] Table 3

[0061] The extension time at 72°C depends on the specific fragment length and the efficiency of the enzyme used. Steps 2-4 in the above procedure are repeated for 25 cycles.

[0062] Table 4

[0063] Note: Amplification Us SPS2 enzyme, Us SPS3 enzyme, To SPS1 enzyme, To SPS2 enzyme, Us FBN5 enzyme and To The primers used to amplify the mutant gene of FBN5 enzyme were respectively... Us SPS2 enzyme, Us SPS3 enzyme, To SPS1 enzyme, To SPS2 enzyme, Us FBN5 enzyme and To The primers used for the FBN5 enzyme gene are the same.

[0064] (2) Nucleic acid agarose gel electrophoresis and gel recovery of PCR products a. Weigh 1 g of agarose into an Erlenmeyer flask, add 100 mL of 1×TAE solution, heat in a microwave oven until the agarose particles are completely dissolved, cool until it is not hot to the touch, add 5 µL of ethidium bromide solution, mix well, and pour into a gel casting tank with a comb already placed in it, and let it cool and solidify. b. Before use, remove the comb, put the gel into the electrophoresis tank, add 1×TAE solution; add the sample into the well, close the lid and start electrophoresis, and stop electrophoresis when the bromophenol blue moves to 1 / 3 of the way from the bottom; c. Cut the target band and place it in a clean 1.5 mL EP tube for gel recovery; the gel recovery kit was purchased from TIANGEN (catalog number DP209); add 300 µL of sol solution PN (provided in the kit) for every 100 mg of gel and incubate in a 50°C water bath, inverting the tube constantly to help dissolve the gel. d. Add the solution from c to the pretreated adsorption column (provided by the kit), let it stand at room temperature for 5 min to allow it to fully bind, and then centrifuge at 12000 rpm for 1 min; e. Pour out the liquid in the collection tube and repeat step d once to increase the product recovery rate; f. Add 500 µL of wash solution PW (provided in the kit) to the purification column and centrifuge at 12000 rpm for 1 min; repeat this step once. g. Centrifuge the adsorption column at 13000 rpm for 2 min to remove as much PW solution as possible; h. Place the purification column into a new EP tube, heat at 65°C for 10 min, and allow the PW solution to evaporate completely; i. Add 50 µL of elution solution (provided in the kit) to the center of the adsorption column, place on a 65°C heating plate for 2 min, and centrifuge at 13000 rpm for 2 min to elute the DNA; repeat this step once. j. Finally, the recovered PCR products were stored at -20°C.

[0065] (3) Enzyme digestion reaction After electrophoresis confirmed the correct DNA fragment size, the pET15b empty vector and PCR product were double-digested with enzymes. NdeI restriction enzyme (with 10×rCutsmart solution) was purchased from New England BioLabs, catalog number R0111V, and KpnI-HF restriction enzyme (with 10×rCutsmart solution) was purchased from New England BioLabs, catalog number R3142V. The enzyme digestion system is shown in Table 5. Table 5

[0066] After incubation at 37°C for 3 hours, electrophoresis was performed and the enzyme was recovered using a gel extraction kit. To improve the efficiency of vector cleavage, an additional 1.5 μL of restriction endonuclease was added and the digestion time was extended by 5 hours.

[0067] (4) Connection reaction The recovered gene fragments were ligated according to the following system, using T4 DNA ligase (purchased from New England BioLabs, catalog number #M0202S, containing T4 DNA ligase buffer), and then incubated at 16°C for at least 5 hours. The ligation system is shown in Table 6: Table 6

[0068] (5) Transformation After ligation, the ligation product was transformed into E. coli JM109 competent cells (purchased from Promega, product number ST1105) and cultured. The specific steps were as follows: a. Take out a tube of competent cells and place it on ice to thaw for 10 min; b. Add the ligation product to the competent cells, mix well, and incubate on ice for 30 min; c. Heat shock at 42℃ for 90 s, and then place on ice for 2 min; d. Add 200 μL of Luria-Bertani (LB) medium (antibiotic-free) to the tube and incubate at 37℃ and 220 rpm for 30 min; bring the solid LB plate with the corresponding antibiotic to room temperature for preheating; e. Take out the bacterial culture, spread it evenly on the plate, and incubate at 37℃ for 16 h.

[0069] (6) Sequencing Some single clones were randomly selected from the plates cultured for 16 h and transferred to a clean plate. The plates were then cultured at 37°C for 5 h and sent to Wuhan Baijies Biotechnology Co., Ltd. for sequencing. After the sequencing results were returned, the sequences were compared to confirm the correct clones.

[0070] Example 2 Expression and purification of target proteins (1) Transformation The correctly sequenced plasmids were transformed into E. coli BL21(DE3) cells (purchased from Kangti Life Technology Co., Ltd., product number KTSM109) using the same transformation method as above.

[0071] (2) Expression First, the protein was pre-prepared to determine the optimal expression conditions and purification strategy. Once the conditions were established, large-scale extraction was then performed. Protein expression was conducted in *E. coli* BL21(DE3) cells (purchased from Kangti Life Science Co., Ltd., product number KTSM109), following the procedure below: a. Pick a single colony from a plate cultured for 16 h and add it to 100 mL of LB medium (containing 100 μg / mL ampicillin). Incubate at 37℃ and 220 rpm for 5 h, until obvious turbidity is observed; b. Expand the bacterial culture from step a to 2 L of LB medium (containing 100 μg / mL ampicillin) at a 1:100 volume ratio and incubate at 37℃ and 220 rpm for 3 h; when the OD600 value reaches 0.7, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM for induction, and the induction time is 4 h; c. Centrifuge at 4℃ and 5000 rpm for 10 min, collect the bacteria, and use them for protein purification.

[0072] (3) Purification a. Resuspend the collected *E. coli* in cell lysis solution at a volume ratio of 30 mL:1 L to the LB medium used to collect the *E. coli*. Stir on a magnetic stirrer for 20 min to homogenize the cells. During this time, add the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF, final concentration 1 mM), lysozyme for lysing bacterial cell walls (final concentration 40 μg / mL), deoxyribonuclease I for degrading nucleic acids (final concentration 1 μg / mL), and cofactor MgCl2 (final concentration 10 mM). Then, perform high-pressure disruption. b. After high-pressure disruption, centrifuge at 13500 rpm and 4℃ for 45 min, collect the supernatant for affinity chromatography; protein purification is performed in a constant temperature room at 4℃. c. First, pour the supernatant from centrifugation into a prepared Ni column for thorough binding; then, wash with 20 mL of imidazole elution solution (containing 10 mM imidazole) to remove contaminating proteins; then wash with 50 mL of imidazole elution solution (containing 30 mM imidazole) to remove contaminating proteins; finally, elute the target protein with 50 mL of imidazole elution solution (containing 250 mM imidazole), wherein the imidazole elution solution contains, in addition to imidazole, 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 100 mM NaCl, with a pH of 7.5; then analyze the results by SDS-PAGE. d. The protein obtained after affinity chromatography was diluted and loaded onto an ion exchange column. After loading, a linear gradient elution (NaCl from 0 to 1 M) was performed using 500 mL of NaCl-free buffer (20 mM HEPES, pH 7.5) and 500 mL of NaCl-containing buffer (20 mM HEPES, 1000 mM NaCl, pH 7.5). This step was performed using a protein purification instrument. The results were then analyzed by SDS-PAGE, and proteins with good properties and purity were pooled and concentrated for the next step of molecular sieve chromatography. e. Before molecular sieve chromatography, centrifuge at 13500 rpm for 10 min to remove some of the precipitated protein and impurities, then use a syringe to transfer the sample into the loading loop; then elute the protein with elution buffer (containing 100 mM NaCl, 20 mM HEPES, pH 7.5), collecting 0.5 mL of sample per tube; then analyze the protein using SDS-PAGE, pool the proteins that need to be preserved, and then aliquot and freeze; for proteins used for activity assays, add glycerol to a final concentration of 30 vol% for preservation.

[0073] Test Example 1 Catalytic activity study of SPS Assuming all proteins are at the same concentration, the catalytic efficiency of each protein on the substrate was tested using a microplate reader. The specific testing steps are as follows: a. Preparation of buffer solutions, substrates, and cofactors: Tris buffer solution for activity assay: First, prepare a 2 M stock solution, adjust its pH to 8.0 with hydrochloric acid, dilute to 100 mM before use, add MgCl2 (final concentration 10 mM) and Triton X-100 filtered through a 0.22 μm filter membrane (the volume of Triton X-100 is 0.05% of the total volume of Tris buffer solution). Preparation of substrates (IPP, GGPP): First, prepare a stock solution of 20 mM IPP and 5 mM GGPP with deionized water, and dilute it with activity assay buffer before use. Preparation of inorganic pyrophosphatase: Prepare a concentration of 100 U / mL with deionized water, and dilute with activity assay buffer before use. Store at -80 ℃. Preparation of malachite green colorimetric solution: Mix reagent A and reagent B from the malachite green phosphate detection kit (purchased from Beyotime, catalog number #S0196M) at a volume ratio of 2:1 to prepare the colorimetric solution for later use.

[0074] b. During the test, first add the activity test buffer, inorganic pyrophosphatase (final concentration 10 mU / mL), SPS enzyme (final concentration 0.007 mg / mL), and finally add the substrate (final concentration of IPP 50 μM, final concentration of GGPP 25 μM). Finally, fix the total volume to 100 μL with the activity test buffer and react at 30℃ for 10 min. The addition of SPS enzyme refers to the addition of one of the following: OsSPS2 enzyme, OsSPS3 enzyme, AtSPS1 enzyme, AtSPS2 enzyme, or SPS mutant. When testing the activity of FBN5 or FBN5 mutants, add FBN5 protein or FBN5 mutant to the enzyme reaction system at a final concentration of 0.0012 mg / mL.

[0075] c. Add 35 μL of malachite green colorimetric solution to each reaction system, mix well, and let stand for 30 min. Malachite green and phosphate ions form a colored complex under specific conditions, and the absorbance of this complex can be detected at a wavelength of 630 nm.

[0076] d. The absorbance of the reaction system at 630 nm was measured using an ELISA reader, and the amount of product generated was calculated based on the phosphate standard curve (i.e., control) to evaluate the catalytic activity of each protein. Each sample was repeated three times.

[0077] This test case examined the catalytic efficiency of SPS mutants, wild-type SPS enzymes interacting with FBN5 mutants, and SPS mutants interacting with FBN5 or FBN5 mutants.

[0078] Normal reaction (A1): refers to the reaction in which substrate, inorganic pyrophosphatase and wild-type SPS enzyme are added to the enzyme reaction system, and the normal catalytic reaction and the further conversion of the product generated by the inorganic pyrophosphatase into phosphate are carried out. Therefore, the UV absorbance at 630 nm reflects the catalytic ability of the wild-type enzyme for the substrate.

[0079] Control (A0): This refers to the enzyme reaction system in which substrate and wild-type enzyme are added, but inorganic pyrophosphatase is not added. Normal catalytic reaction occurs, but the product is not further converted into phosphate. Therefore, the UV absorbance at 630 nm is regarded as the amount of phosphate when the product is not further converted.

[0080] Experimental group (A2): In the enzyme reaction system, substrate, inorganic pyrophosphatase, SPS enzyme (wild-type SPS enzyme or SPS mutant), and FBN5 enzyme (wild-type FBN5 enzyme or FBN5 mutant) or two enzymes or SPS mutants were added. Normal catalytic reactions and the further conversion of the product by the inorganic pyrophosphatase into phosphate groups occurred. Therefore, the UV absorbance at 630 nm reflects the catalytic efficiency of the mutant on the substrate. The formula for calculating the catalytic efficiency is (A2-A0) / (A1-A0)×100%.

[0081] To compare the catalytic efficiency of wild-type and various mutants, the concentrations of wild-type SPS enzyme, wild-type FBN5, and different mutant proteins were adjusted to be consistent before being added to the reaction system for testing and comparison.

[0082] The catalytic efficiency of the normal reaction was defined as 100%, thus the relative catalytic efficiencies of each mutant sample were obtained, and the results are shown in Table 7. As can be seen from the results in Table 7, wild-type OsFBN5 significantly promotes the catalytic activity of SPS. Furthermore, it was found that compared to wild-type OsSPS3, mutating E to D at position 352 of OsSPS3 further enhances the catalytic efficiency of OsSPS3_E352D with OsFBN5.

[0083] The Michaelis constant (Km) is a core parameter in enzyme kinetics, used to characterize the affinity between an enzyme and its substrate (specifically, enzymes following Michaelis-Menten kinetics). It is defined as the substrate concentration required for the enzyme-catalyzed reaction rate to reach half of its maximum reaction rate (Vmax). Generally, a lower Km value indicates a lower substrate concentration required for the enzyme to reach half-saturation, suggesting a higher affinity between the enzyme and its substrate. Under specific environmental conditions (such as constant pH, temperature, and ionic strength), Km can be considered a characteristic constant. The formula for calculating Km is as follows, where Km is called the Michaelis constant, Vmax is the maximum reaction rate, and [S] is the substrate concentration.

[0084]

[0085] Therefore, the physical meaning of Km value is the substrate concentration (i.e., Km) when the reaction rate V reaches 1 / 2 of Vmax. The unit is generally mol / L, which is determined only by the properties of the enzyme and is not related to the enzyme concentration.

[0086] The transformation number (kcat) is another key kinetic parameter, calculated by dividing Vmax by the total enzyme concentration. kcat reflects the maximum rate at which the enzyme catalyzes the conversion of substrate to product under saturated conditions, and its unit is s⁻¹ or min⁻¹. This parameter can be intuitively understood as the maximum number of substrate molecules converted by each enzyme active site per unit time; therefore, a higher kcat value indicates a stronger intrinsic catalytic ability of the enzyme. The formula for calculating kcat is as follows, where kcat is called the transformation number, Vmax is the reaction rate when the enzyme is saturated with substrate, and [E]t is the enzyme concentration.

[0087]

[0088] The kcat / Km ratio is widely considered a core indicator for evaluating enzyme catalytic efficiency. This specific constant combines the enzyme's catalytic rate (kcat) and substrate affinity (Km): a high kcat value and a low Km value together result in a high kcat / Km ratio, signifying efficient catalysis. When an enzyme has multiple potential substrates, its kcat / Km value can be used to identify the enzyme's optimal substrate (i.e., the substrate with the highest ratio). In protein engineering and directed evolution studies, comparing the kcat / Km values ​​of wild-type and mutant enzymes allows for precise quantification of the improvement or change in the catalytic efficiency of modified enzymes.

[0089] In the Michaelis-Menten kinetics experiment, each 100 μL SPS reaction system contained 0.007 mg / mL of purified wild-type SPS enzyme or SPS mutant enzyme, and 10 mU·mL⁻¹ was added. -1Inorganic pyrophosphatase was used as the coupling enzyme. The substrate (GGPP) concentration gradients were 0 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, 9 μM, 10 μM, and 15 μM. The reaction was initiated by adding 150 μM IPP as the corresponding substrate, incubated for 3 minutes, and then terminated by adding 35 μL of freshly prepared malachite green chromogenic solution, followed by standing at room temperature for 30 minutes. The amount of inorganic phosphate released was detected at 630 nm using an Agilent BioTek Cytation 5 microplate reader. All experiments were performed in triplicate. The SPS and co-incubation reaction systems with FBN5 were similar to the SPS reaction system, except that wild-type FBN5 or a mutant FBN5 enzyme with a final concentration of 0.0012 mg / mL was added to a 100 μL SPS reaction system.

[0090] The enzyme kinetics results are shown in Table 7. Table 7 shows that the kcat / Km ratio of OsSPS3 for GGPP is 4.78 min. -1 ·μM -1 However, after the addition of its binding protein OsFBN5, this value increased to 18.07 min. -1 ·μM -1 The catalytic efficiency was increased by approximately 3.8 times. This result clearly demonstrates that OsFBN5 can significantly enhance the catalytic activity of OsSPS3. Furthermore, after mutating E to D at position 352 of OsSPS3, the kcat / Km ratio was increased to 21.40 min compared to wild-type OsSPS3. -1 ·μM -1 This indicates that OsFBN5 can further enhance the catalytic efficiency of OsSPS3_E352D.

[0091] Table 7

[0092] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A mutant, characterized in that, This mutant is selected from Os SPS2 enzyme, Os SPS3 enzyme, Os FBN5 enzyme, At SPS1 enzyme, At SPS2 enzyme and At A mutant of at least one of the FBN5 enzymes; Os The amino acid sequence of the SPS2 enzyme is shown in SEQ ID NO. 1; Os The amino acid sequence of the SPS3 enzyme is shown in SEQ ID NO. 2; Os The amino acid sequence of the FBN5 enzyme is shown in SEQ ID NO. 3; At The amino acid sequence of the SPS1 enzyme is shown in SEQ ID NO. 4; At The amino acid sequence of the SPS2 enzyme is shown in SEQ ID NO. 5; At The amino acid sequence of the FBN5 enzyme is shown in SEQ ID NO. 6; The mutation site of the mutant includes at least one of the following: a: At least one of the following positions in SEQ ID NO. 1—positions 121, 122, 123, 126, 161, 168, 172, 177, 178, 190, 230, 238, 239, 254, 295, 309, and 341—has mutated; b: At least one of the following positions in SEQ ID NO. 2 is mutated: position 132, position 133, position 134, position 137, position 172, position 179, position 183, position 188, position 189, position 201, position 241, position 249, position 250, position 265, position 306, position 320, and position 352. c: At least one of the following positions in SEQ ID NO. 3 is mutated: position 156, position 162, position 163, and position 190; d: At least one of the following positions in SEQ ID NO. 4 is mutated: position 180, position 181, position 193, and position 344. e: At least one of the following positions in SEQ ID NO. 5 is mutated: position 191, position 192, position 204, and position 355; f: At least one of the 153rd and 159th positions of SEQ ID NO. 6 is mutated.

2. The mutant according to claim 1, characterized in that, The mutant is a single mutant; Preferably, the amino acid sequence of the mutant includes at least one of the following amino acid sequences: X1: The amino acid sequence shown in SEQ ID NO. 1 has at least one of the following mutations: G at position 121 is mutated to W or A, G at position 122 is mutated to W, K at position 123 is mutated to A, R at position 126 is mutated to A, H at position 161 is mutated to A, D at position 168 is mutated to A, D at position 172 is mutated to A, R at position 177 is mutated to A, R at position 178 is mutated to A, R at position 190 is mutated to A, E at position 230 is mutated to A, F at position 238 is mutated to A, D at position 239 is mutated to A, K at position 254 is mutated to A, D at position 295 is mutated to A, K at position 309 is mutated to A, E at position 341 is mutated to A, D, R, Y or Q; X2: The amino acid sequence shown in SEQ ID NO. 2 has at least one of the following mutations: G at position 132 is mutated to W or A, G at position 133 is mutated to W or A, K at position 134 is mutated to A, R at position 137 is mutated to A, H at position 172 is changed to A, D at position 179 is mutated to A, D at position 183 is mutated to A, R at position 188 is mutated to A, R at position 189 is mutated to A, R at position 201 is mutated to A, E at position 241 is mutated to A, F at position 249 is mutated to A, D at position 250 is mutated to A, K at position 265 is mutated to A, D at position 306 is mutated to A, K at position 320 is mutated to A, E at position 352 is mutated to A, D, R, Y or Q; X3: The amino acid sequence shown in SEQ ID NO. 3 has at least one of the following mutations: R at position 156 is mutated to D or E, R at position 162 is mutated to D or E, D at position 163 is mutated to A, and R at position 190 is mutated to E. X4: At least one of the following mutations occurs in the amino acid sequence shown in SEQ ID NO. 4: R at position 180 is mutated to A, R at position 181 is mutated to A, R at position 193 is mutated to A, and E at position 344 is mutated to A, D, R, Y or Q. X5: The amino acid sequence shown in SEQ ID NO. 5 has at least one of the following mutations: R at position 191 is mutated to A, R at position 192 is mutated to A, R at position 204 is mutated to A, and E at position 355 is mutated to A, D, R, Y or Q. X6: At least one of the following mutations occurs in the amino acid sequence shown in SEQ ID NO. 6: R at position 153 is mutated to D or E, or R at position 159 is mutated to D or E.

3. The mutant according to claim 1, characterized in that, The mutant is a multiple mutant; Preferably, the amino acid sequence of the mutant includes at least one of the following amino acid sequences: Y1: The amino acid sequence shown in SEQ ID NO. 1 contains at least two of the following mutations: G at position 121 is mutated to W or A, G at position 122 is mutated to W, K at position 123 is mutated to A, R at position 126 is mutated to A, H at position 161 is mutated to A, D at position 168 is mutated to A, D at position 172 is mutated to A, R at position 177 is mutated to A, R at position 178 is mutated to A, R at position 190 is mutated to A, E at position 230 is mutated to A, F at position 238 is mutated to A, D at position 239 is mutated to A, K at position 254 is mutated to A, D at position 295 is mutated to A, K at position 309 is mutated to A, and E at position 341 is mutated to A, D, R, Y or Q. Y2: The amino acid sequence shown in SEQ ID NO. 2 has at least two of the following mutations: G at position 132 is mutated to W or A, G at position 133 is mutated to W or A, K at position 134 is mutated to A, R at position 137 is mutated to A, H at position 172 is mutated to A, D at position 179 is mutated to A, D at position 183 is mutated to A, R at position 188 is mutated to A, R at position 189 is mutated to A, R at position 201 is mutated to A, E at position 241 is mutated to A, F at position 249 is mutated to A, D at position 250 is mutated to A, K at position 265 is mutated to A, D at position 306 is mutated to A, K at position 320 is mutated to A, and E at position 352 is mutated to A, D, R, Y, or Q. Y3: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 3: R at position 156 is mutated to D or E, R at position 162 is mutated to D or E, D at position 163 is mutated to A, and R at position 190 is mutated to E. Y4: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 4: R at position 180 is mutated to A, R at position 181 is mutated to A, R at position 193 is mutated to A, and E at position 344 is mutated to A, D, R, Y, or Q. Y5: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 5: R at position 191 is mutated to A, R at position 192 is mutated to A, R at position 204 is mutated to A, and E at position 355 is mutated to A, D, R, Y or Q. Y6: At least two of the following mutations occur in the amino acid sequence shown in SEQ ID NO. 6: the R at position 153 is mutated to D or E, and the R at position 159 is mutated to D or E.

4. A combinatorial protein, characterized in that, This combined protein includes Z1 protein and Z2 protein; The Z1 protein is selected from at least one of the following: a mutant with a mutation at position 341 in the amino acid sequence shown in SEQ ID NO. 1, a mutant with a mutation at position 352 in the amino acid sequence shown in SEQ ID NO. 2, a mutant with a mutation at position 344 in the amino acid sequence shown in SEQ ID NO. 4, or a mutant with a mutation at position 355 in the amino acid sequence shown in SEQ ID NO. 5; The Z2 protein is selected from Os At least one of the following: FBN5 enzyme, a mutant with a mutation at position 156 or 162 in the amino acid sequence shown in SEQ ID NO. 3, or a mutant with a mutation at position 153 or 159 in the amino acid sequence shown in SEQ ID NO.

6.

5. A gene encoding a mutant as described in any one of claims 1-3 or a combinatorial protein as described in claim 4, characterized in that, The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the mutant described in any one of claims 1-3 or the combinatorial protein described in claim 4.

6. An expression carrier, characterized in that, The expression vector contains the gene described in claim 5.

7. A transgenic cell, characterized in that, The transgenic cell contains the expression vector as described in claim 6.

8. A composition, characterized in that, The composition contains the mutant as described in any one of claims 1-3 or the combined protein as described in claim 4 as the active ingredient.

9. The use of the mutant according to any one of claims 1-3, the combinatorial protein according to claim 4, the gene according to claim 5, the expression vector according to claim 6, the transgenic cell according to claim 7, and the composition according to claim 8 in improving crop photosynthetic efficiency.

10. A method for improving crop photosynthetic efficiency, characterized in that, The method includes: transferring the expression vector of claim 6 into a target crop, such that the target crop expresses the mutant of any one of claims 1-3 or the combinatorial protein of claim 4; or, The method includes: transferring the expression vector containing the expression vector of claim 6 into a target crop through hybridization, transfer, or backcrossing, so that the target crop expresses the mutant of any one of claims 1-3 or the combinatorial protein of claim 4; or, This method includes: using CRISPR / Cas gene editing methods to edit the target crop... SPS Genes and / or FBN5 Genes are modified to make the target crop express the mutant of any one of claims 1-3 or the combinatorial protein of claim 4.

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