Use of gene osvde to improve photosynthetic efficiency in plants

Abstract

The present application belongs to the field of agricultural biotechnology, and relates to a use of a violaxanthin de-epoxidase VDE in improving photosynthetic rate and biomass of plants. Overexpression of the violaxanthin de-epoxidase VDE in plants can improve the photosynthetic rate and biomass of the plants, and is helpful for improving plant traits and cultivating new plant varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to the use of a chlorophyll decyclooxygenase (VDE) in improving plant photosynthetic rate and biomass. Background Technology

[0002] Photosynthesis provides food and energy for almost all life and is the most important chemical reaction on Earth. Through photosynthesis, plants can use light energy in chloroplasts to split water to release oxygen (photochemical reaction or light reaction) and fix CO2 to generate organic matter (carbon assimilation reaction or carbon reaction). Light energy is a necessary condition for photosynthesis, but excessive light energy can also cause varying degrees of photo-oxidative damage to plants. When the light intensity received by plant leaves exceeds their own carbon assimilation capacity, photosynthetic pigments will be overactivated, and the electron transport chain will be in an over-reduced state. In order to balance the redox state of the photosystem, maintain the normal rate of the electron transport chain, and avoid cell damage caused by the large production of reactive oxygen species, plants have evolved a series of complex photoprotection mechanisms. The most important of these mechanisms is the nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Baker R N. Chlorophyll Fluorescence: A Probe of Photosynthesis In Vivo[J]. Annual Review of Plant Biology,2008,59(0):89-113.).

[0003] NPQ refers to the biochemical process in which excess light energy is dissipated as heat in the light-harvesting complex of photosystem II, aiming to reduce the production of peroxides in plants and thus avoid photo-oxidative damage (Shi Y, Ke X, Yang X, et al. Plants response to light stress[J]. J Genet Genomics, 2022.). NPQ includes several mechanisms such as energy-dependent quenching (qE), photoinhibitory quenching (qI), zeaxanthin-dependent quenching (qZ), state transition-dependent quenching (qT), and sustained Chl fluorescence quenching (qH) (PSL, HST, JSB, et al. Into the Shadows and Back into Sunlight: Photosynthesis in Fluctuating Light. [J]. Annual review of plant biology, 2022, 73(1): 617-648.). Under low light conditions, the proton concentration gradient (ΔpH) across the thylakoid membrane is low, and the PSII antenna system is in a light-trapping state. When light intensity suddenly increases, the proton concentration in the thylakoid lumen rises rapidly, creating a large ΔpH across the membrane, which quickly activates the fastest-responding part of NPQ—qE (Goss R, Lepetit B. Biodiversity of npq[J]. J Plant Physiol, 2015, 172: 13-32.). qE includes two key components: the xanthophyll cycle and the S subunit (PsbS) of the photosystem II protein complex.The high concentration of protons caused by high light intensity can not only activate violaxanthin de-epoxidase (VDE), catalyzing the rapid formation of Zea from violaxanthin (Vio), but also bind to and protonate the PsbS protein, thereby promoting the transition of the PSII antenna system from a light-harvesting state to a light-protecting state (Li XP, Bjorkman O, Shih C, et al. Apigment-binding protein essential for regulation of photosynthetic light harvesting[J]. Nature, 2000, 403(6768):391-395.). qE can be rapidly activated within half a second to cope with sudden fluctuations in light intensity, thus protecting plants and enabling them to survive better in natural environments with constantly fluctuating light intensity.

[0004] Violaxanthin decyclooxidase (VDE), a key enzyme involved in the NPQ process, is responsible for catalyzing the conversion of violaxanthin (Vio) into antheraxanthin (Ant) and zeaxanthin (Zea). Zea, a carotenoid, is synthesized due to VDE activation caused by a decrease in the pH of the thylakoid cavity and is located in the light-harvesting complex of PSII and PSI. Only a small portion of Vio in plants is converted into Ant and Zea, and the specific ratio varies depending on the plant's growth environment and species (Demmig-Adams B, Stewart JJ, Lopez-Pozo M, et al. Zeaxanthin, a molecule for photoprotection in many different environments[J]. Molecules, 2020, 25(24).).

[0005] Although studies have reported that VDE can enhance photosynthesis in tobacco (Kromdijk J, Glowacka K, Leonelli L, et al. Improving photosynthesis and crop productivity by accelerating recovery from photoprotection[J]. Science, 2016, 354(6314):857-861.), subsequent research on VDE has mainly focused on overexpressing VDE from different species in plants such as tobacco, Arabidopsis, or tomato using transgenic methods, with particular emphasis on the effects of VDE overexpression on resistance to abiotic stress. This is because Zea plays an important role in resistance to abiotic stress. For example, expression of VDE in cucumber and wolfberry in Arabidopsis improved the plant's tolerance to strong light and drought, respectively (Guan C, Ji J, Zhang X, et al. Positive feedback regulation of a lycium chinense-derived vde gene by drought-induced endogenous aba, and over-expression of this vde gene improve drought-induced photo-damage inarabidopsis[J]. J Plant Physiol, 2015, 175: 26-36.; Li X, Zhao W, Sun X, et al. Molecular cloning and characterization of violaxanthin deep-epoxidase (csvde) in cucumber[J]. PLoS One, 2013, 8(5): e64383.), while expression of ChVDE enhanced the tolerance of transgenic Arabidopsis to drought and salt stress (Sun LN, Wang F, Wang JW, et al. Overexpression of the chvde gene, encoding a violaxanthin de-epoxidase, improves tolerance to drought and salt stress in transgenic arabidopsis[J]. 3Biotech, 2019, 9(5):197.).Overexpression of peanut VDE in tobacco can alleviate photoinhibition of photosystem II under stress (Yang S, Meng DY, Hou LL, et al. Peanut violaxanthin deep-epoxidase alleviates the sensitivity of psiiphotoinhibition to heat and high irradiance stress in transgenic tobacco[J]. Plant Cell Rep,2015,34(8):1417-1428.).

[0006] Patent document CN103602729A discloses that the rice chlorophyll decyclooxygenase gene OsVDE2 (NCBI accession number NP_001052592) enhances rice's resistance to photoinhibition and plays an important role in photosensitivity. However, it does not disclose the effects of overexpressing this OsVDE2 gene on rice's agronomic traits. Patent document CN109207508A discloses that Arabidopsis thaliana... The expression of the photosensitizing protein PsbS, the zeaxanthin decyclooxygenase VDE, and the zeaxanthin cyclooxygenase ZEP genes from Arabidopsis thaliana in plants can improve photosynthetic efficiency and crop yield, but their individual expression does not have this effect. Patent document CN109477118A discloses a method for increasing biomass production and / or carbon fixation and / or growth in plants when the photosynthetic system II subunit S (PsbS) from Arabidopsis thaliana is expressed in plants in combination with zeaxanthin cyclooxygenase (ZEP) and zeaxanthin decyclooxygenase (VDE), or a combination of PsbS and ZEP, or a combination of ZEP and VDE. However, the individual expression of PsbS, ZEP, or VDE does not have this effect.

[0007] Based on information currently available, the impact of VDE on crops remains unclear. Summary of the Invention

[0008] In our research on the mechanism of rice photosynthesis, our research group screened a gene in the Xiushui 134 rice variety that positively regulates the photosynthetic rate and biomass through gene homology comparison. This gene was identified as the OsVDE gene (NCBI accession number XP_015636342.1). We verified the function of the OsVDE gene through transgenic experiments. The experiments showed that overexpression of the OsVDE gene accelerates photosynthetic induction (the gradual increase in photosynthetic rate caused by increased light intensity), improves the photosynthetic efficiency of rice leaves (the amount of carbon dioxide fixed by the plant per unit time), and increases biomass (the dry weight of all stems, leaves, and panicles above ground). Based on this discovery, this invention includes the following technical solution.

[0009] A method for increasing the photosynthetic rate and / or biomass of plants, characterized by overexpressing violaxanthin de-epoxidase (VDE) in plants.

[0010] Preferably, in the above method, violet decyclooxygenase VDE is independently overexpressed in plants.

[0011] The phrase "independent overexpression in plants" means that the VDE gene is not co-overexpressed in plants with other genes related to the NPQ biochemical process / pathway, such as the photosynthetic system II subunit S (PsbS) gene and the zeaxanthin cyclooxygenase (ZEP) gene.

[0012] The aforementioned flavin decyclooxygenase (VDE) can be selected from the following groups: flavin decyclooxygenase from Arabidopsis thaliana (NCBI accession number NP_001321301.1), flavin decyclooxygenase from maize (NCBI accession number NP_001147756.1), flavin decyclooxygenase OsVDE from rice (NCBI accession number XP_015636342.1), flavin decyclooxygenase from soybean (NCBI accession number NP_001241404.1), and flavin decyclooxygenase from rapeseed (NCBI accession number XP_013641072.1), etc.

[0013] In a preferred embodiment, the above-mentioned chlorophyll decyclooxidase (VDE) is the rice-derived chlorophyll decyclooxidase OsVDE (NCBI accession number XP_015636342.1) with an amino acid sequence as shown in SEQ ID NO:1, or a conserved variant polypeptide thereof. The conserved variant polypeptide is a polypeptide that has more than 95% homology with VDE, preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, more preferably more than 99% homology, and has VDE function, i.e., an OsVDE isoenzyme.

[0014] MMPRQCGNRALLAEGSSTVVVVHGRKTRGGISTVTTSSRRRSHGGVRYHRCCPPRAHLWRKDHLPLHHAKISARCSEIKAHTVLQGSDALSSIREWSRSHLVTMTGLVACAVLVVPSA DAVDALKTCTCLLKECRIELAKCIANPSCAANVACLNTCNNRPDETECQIKCGDLFENTVVDEFNECAVSRKKCVPQKSDVGEFPVPDPSALVKNFNMADFNGKWYISSGLNPTFDTF DCQLHEFRVEGDKLIANLTWRIRTPDSGFFTRTAIQRFVQDPAQPAILYNHDNEFLHYQDDWYIISSKVENKEDDYIFVYYRGRNDAWDGYGGAVLYTRSKVVPESIVPELEERAAKSV GRDFSTFIRTDNTCGPEPPLVERIEKTVEQGEKTIIREVQEIEGEIEGEVKELEEEEVTLFKRLTDGLMEVKQDLMNFFQGLSKEEMELLDQMNMEATEVEKVFSRALPIRKLR(SEQ ID NO: 1).

[0015] The nucleotide sequence of the expression gene OsVDE of the chlorophyll decyclooxygenase OsVDE (NCBI accession number XP_015636342.1) is shown in SEQ ID NO:2.

[0016] The aforementioned plants can be crops, preferably grasses. These crops include, but are not limited to, rice, wheat, corn, soybeans, barley, oats, rye, sorghum, cotton, vegetables, and cruciferous plants (such as rapeseed and Arabidopsis thaliana), with rice being the preferred crop.

[0017] In one embodiment, overexpression of chlorophyll decyclooxygenase (VDE) in plants is achieved by the following method:

[0018] A. The gene encoding chlorophyll decyclooxygenase (VDE), such as the gene OsVDE with the nucleotide sequence SEQ ID NO:2, is cloned into a plasmid vector, preferably into a plasmid vector suitable for expression in Agrobacterium, to form a recombinant plasmid, i.e., a VDE overexpression vector. Then, plant cells or tissues are transformed using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrocoagulation, or Agrobacterium-mediated transformation. The transformed plant tissues are then cultured into plants, preferably through Agrobacterium-mediated transformation, to obtain transgenic plants overexpressing VDE; or

[0019] B. By using gene editing technology, the gene encoding chlorophyll decyclooxygenase (VDE), for example, the gene OsVDE with the nucleotide sequence SEQ ID NO:2, is cloned into a plant chromosome to obtain transgenic plants overexpressing chlorophyll decyclooxygenase (VDE), such as OsVDE; or

[0020] C. The encoding genes of chlorophyll decyclooxygenase (VDE) already existing in the plant genome, such as the OsVDE gene, are placed under the regulation of a functionally enhanced promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the rice-derived ubiquitin gene promoter, i.e., the Ubi promoter. The nucleotide sequence of the Ubi promoter derived from Nipponbare rice is shown in SEQ ID NO:3.

[0021] Preferably, the plasmid vector in step A above is selected from binary Agrobacterium vectors and vectors that can be used for plant micro-bombardment.

[0022] Optionally, the plasmid vector mentioned in step A above is selected from the following group: pHB-YFP, pHB-FLAG, pBin19, pUN1301, fluorescent reporter vector pGreenII0800-LUC, pCAMBIA3300, pCAMBIA1301, pCAMBIA2301, pBI121, pTF102, etc., which are vectors used for plant transgenic purposes or modified vectors.

[0023] The aforementioned Agrobacterium can be selected from Agrobacterium tumefaciens, Agrobacterium EHA105, and Agrobacterium GV3101. For example, the recombinant plasmid can be transferred into the Agrobacterium strain using a freeze-thaw method to form a microbial engineered bacterium.

[0024] The gene editing technology described in step B above can be selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-Cas12a system, CRISPR-BEST system, and MuGENT.

[0025] In one embodiment, step C above involves assembling the Ubi promoter from Nipponbare rice with the OsVDE gene to form a VDE expression cassette / frame (i.e., Ubi+VDE expression cassette), then cloning one or more copies of the VDE expression cassette / frame into an expression vector to form a VDE recombinant expression vector, and using transgenic technology to introduce the recombinant expression vector into a plant, such as rice, to obtain transgenic plants that overexpress VDE.

[0026] Preferably, the nucleotide sequence of the coding region CDS of the above-mentioned chlorophyll decyclooxidase VDE is SEQ ID NO:2.

[0027] This invention is the first to demonstrate that overexpressing chlorophyll decyclooxygenase (VDE) alone in rice, without overexpressing it together with other genes in the NPQ pathway such as PsbS and / or ZEP, can improve photosynthetic rate and plant biomass. Therefore, it can simplify the operation of constructing transgenic plants with high photosynthetic rate and / or biomass, reduce the development cost of transgenic plants, and promote crop improvement and the breeding of new plant varieties with excellent agronomic traits. Attached Figure Description

[0028] Figure 1 This demonstrates the construction and validation of OsVDE-overexpressing transgenic rice. Among them, A: Pro Ubi - Schematic diagram of the VDE vector structure; B: Gel electrophoresis image of OsVDE-FLAG fusion protein expression detected by Western blotting using Anti-FLAG antibody. OE-VDE: Transgenic rice overexpressing VDE; WT: Wild type; C: Statistical bar chart showing the RNA expression level of the OsVDE gene (n=3) detected by RT-qPCR, with OsActin1 as the internal reference gene. Error bars represent standard deviation (SD). Asterisks indicate that the transgenic OE-VDE-1, OE-VDE-3, and OE-VDE-5 showed significant differences compared to WT using t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n represents the number of independent biological samples; D: Phenotypes of wild-type (WT) and VDE-overexpressing (OE-VDE) lines. The photos show rice at the tillering stage, 60 days after transplanting in the field (Shanghai Songjiang Experimental Field, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences).

[0029] Figure 2The chart shows the statistical bar graphs of changes in the content of lutein cycling pigments and other major pigments in transgenic plants overexpressing OsVDE. A: Changes in the content of violaxanthin and zeaxanthin in the lutein cycle in leaves of rice overexpressing VDE protein, expressed as mean ± variance (n=6); B: Changes in the content of chlorophyll a (Chla) and chlorophyll b (Chlb) in leaves of rice overexpressing VDE protein, expressed as mean ± variance (n=6); C: Changes in the de-epoxy state (DES) of the lutein cycle and the total amount (VAZ) of violaxanthin, antheraxanthin, and zeaxanthin in leaves of rice overexpressing VDE protein, expressed as mean ± variance (n=6); D: Changes in the content of lutein and neoxanthin in leaves of rice overexpressing VDE protein, expressed as mean ± variance (n=6). An asterisk indicates that the transgenic OE-VDE-1, OE-VDE-3, and OE-VDE-5 showed significant differences compared to WT using t-tests, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 3 The results of field agronomic traits comparison between OE-VDE overexpressing transgenic plants and WT wild-type plants are shown. A: Plant height, values ​​are expressed as mean ± variance (n≥40); B: Number of panicles, values ​​are expressed as mean ± variance (n≥40); C: Biomass, values ​​are expressed as mean ± variance (n≥40); D: Grain weight, values ​​are expressed as mean ± variance (n≥40); E: Leaf nitrogen content, values ​​are expressed as mean ± variance (n=5); F: Specific leaf weight, values ​​are expressed as mean ± variance (n=5). Experimental materials for AD were obtained from harvested rice in the field. Experimental materials for EF were obtained from the last fully expanded leaf of rice in the heading stage in the field. n: Biological replicates. For Figures A, B, C, D, E, and F, the t-test showed significant differences between genes OE-VDE-1, OE-VDE-3, and OE-VDE-5 and WT, *P<0.05.

[0031] Figure 4 The comparison of photosynthetic parameters based on chlorophyll fluorescence between OE-VDE overexpressing transgenic plants and WT wild-type plants under different growth conditions is shown. AD: Dynamic response curve of NPQ in rice under normal light conditions; growth environment: 400 μmol·m⁻². -2 ·s -1 The temperature was 28℃, and measurements were taken using a Dual-PAM-100 (WALZ, Inc., Germany). Data are expressed as mean ± standard deviation (n = 10). EH: Dynamic response curve of NPQ in rice treated under high light for three days. Growth environment: 1000 μmol·m -2 ·s-1 The temperature was 28℃, and measurements were taken using a Dual-PAM-100 (WALZ, Inc., Germany). Data are expressed as mean ± standard deviation (n = 11). IL: Dynamic response curve of NPQ in field-grown rice, measured using a Dual-PAM-100 (WALZ, Inc., Germany). Data are expressed as mean ± standard deviation (n ≥ 7). Non-photochemical quenching: NPQ; Quantum yield of PSII for regulated energy dissipation: Y(NPQ); Actual photoluminescence quantum yield of PSII: Y(II); Quantum yield of PSII for non-regulated energy dissipation: Y(NO). All experimental samples were the last fully expanded leaf of rice at the tillering stage, and underwent dark adaptation for more than 2 hours. Chlorophyll fluorescence dynamic curves were then measured using a Dual-PAM-100, initially providing 10 min of 1300 μmol·m⁻¹. -2 ·s -1 Then, the environment was kept in darkness for another 6 minutes, and a value was recorded every 15 seconds. For Figure AL, the asterisks indicate that the transgenic OE-VDE-1, OE-VDE-3, and OE-VDE-5 were significantly different from WT, as shown by the t-test (p < 0.05). The black lines represent the corresponding time period on the horizontal axis.

[0032] Figure 5 The photosynthetic induction and chlorophyll fluorescence response curves of OE-VDE overexpressing transgenic plants and WT wild-type plants after transition from low light to high light are shown. A: Net photosynthetic assimilation rate was measured using a LI-6800 (Li-Cor, Inc., USA), and data are presented as mean ± standard deviation (n=6); B: 1-qL was measured using a Dual-PAM-100 (WALZ, Inc., Germany), and data are presented as mean ± standard deviation (n=6); C: Stomatal conductance (g) s The detection of (A) was performed using a LI-6800 (Li-Cor, Inc., USA), and the data are expressed as mean ± standard deviation (n=6); D: PSII actual photon yield (Y(II)) was measured using a Dual-PAM-100 (WALZ, Inc., Germany), and the data are expressed as mean ± standard deviation (n=6); E: PSII quantum yield for non-regulated energy dissipation (Y(NO)) was measured using a Dual-PAM-100 (WALZ, Inc., Germany), and the data are expressed as mean ± standard deviation (n=6); (F) PSII quantum yield for regulated energy dissipation (Y(NPQ)) was measured using a Dual-PAM-100 (WALZ, Inc., Germany), and the data are expressed as mean ± standard deviation (n=6). The experimental sample was artificially grown rice in the tillering stage. The last fully expanded leaf was selected and first treated with 100 μmol·m -2 ·s -1After 4 min of light intensity treatment, gas exchange and fluorescence-related parameters were recorded, and then the sample was transferred to a 2000 μmol·m⁻¹ light source. -2 ·s -1 Under light intensity for 10 minutes, gas exchange parameters were recorded every 3 seconds, and fluorescence parameters were recorded every 12 seconds. After measuring the relevant data under light, the plants were subjected to dark treatment for more than 2 hours before measuring Fo and Fm. An asterisk indicates a significant difference compared to WT (*p < 0.05), and the black line represents a time period corresponding to the horizontal axis.

[0033] Figure 6 This shows the relative carbon assimilation of OE-VDE overexpressing transgenic plants compared to WT wild-type plants after transitioning from low to high light conditions, based on... Figure 5 The net photosynthetic rate data of wild-type and OE-VDE overexpressing plants in Figure A were obtained by statistical analysis. n≥6, asterisks indicate significant differences compared to WT, and t-test showed *p<0.05. Detailed Implementation

[0034] Our study confirms that overexpression of the OsVDE gene in rice, such as Xiushui 134, accelerates photosynthetic induction (the gradual increase in photosynthetic rate caused by increased light intensity), improves the photosynthetic efficiency of rice leaves (the amount of carbon dioxide fixed by the plant per unit time), and increases plant biomass (the dry weight of all stems, leaves, and panicles above ground). This suggests that the OsVDE gene can be used alone to improve the agronomic traits of wild plants and to breed new transgenic plant varieties with high photosynthetic efficiency. Moreover, overexpression of the OsVDE gene in wild plants can achieve the same or similar technical effects without the participation of other related genes in the NPQ pathway, such as PsbS and / or ZEP genes, thus eliminating the cumbersome operations of cloning and assembling / fusion of these NPQ-related genes. This is extremely beneficial for the construction of VDE-overexpressing transgenic plants (hereinafter referred to as OE-VDE) and helps to improve the success rate of construction.

[0035] As used in this article, the term "wild-type" refers to native plants, such as rice, that have not undergone genetic engineering or mutagenesis.

[0036] Correspondingly, the terms “transgenic plant,” “genetically engineered plant,” and “(plant) mutant” in this article have the same meaning, referring to plants that have been genetically modified from wild-type plants, especially OE-VDE plants with increased light energy utilization and / or biomass.

[0037] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0038] In this document, for the sake of simplicity, the name of a protein, such as OsVDE, and its encoding gene (DNA), OsVDE, are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or category of chlorophyll decyclooxygenase, VDE refers to a protein; when used as a gene description, it refers to the gene encoding that protein.

[0039] Those skilled in the art will expect that conserved variant polypeptides / isoenzymes with high homology (identity) to the amino acid sequence SEQ ID NO:1, such as more than 95% of the flavin decyclooxygenase OsVDE, will have the same function.

[0040] As used herein, the term "conserved variant protein" means a protein that substantially retains the same biological function or activity as the protein. The variant specifically refers to minor amino acid mutations, such as (1) proteins formed by substitution, deletion, or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the OsVDE amino acid sequence, and possessing the function of wild-type OsVDE; (2) proteins having more than 95%, more preferably 98%, more preferably 99% identity with the OsVDE amino acid sequence, and possessing the function of wild-type OsVDE; or (3) proteins formed by adding a tag sequence to the N or C terminus of the OsVDE amino acid sequence, or by adding a signal peptide sequence to its N terminus. Such fragments, derivatives, and analogs are well known to those skilled in the art in accordance with the teachings herein. The term "mutation" includes, but is not limited to, substitution, deletion, insertion, or chemical modification of amino acid residues, preferably positive mutations, i.e., functionally enhanced mutations. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of non-conservative and conserved substitutions. A “conservative” amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in proteins with amino acids defined in the same or similar amino acid categories. However, as used herein, a conserved mutation does not include hydrophilic-hydrophilic, hydrophobic-hydrophobic, hydroxyl-containing-hydroxyl-containing, or small residue-small residue substitutions if a conserved mutation can be alternatively expressed as aliphatic-to-aliphatic, nonpolar-to-nonpolar, polar-to-polar, acidic-to-acidic, basic-to-basic, aromatic-to-aromatic, or restriction-restriction-restriction-residue substitutions. Common cases of conserved substitutions known in the art include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed, for example, according to the table below.

[0041] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0042] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a protein with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0043] "Deletion" refers to a modification of a protein by removing one or more amino acids from a reference protein. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference protein sequence, while preserving OsVDE activity and / or the modified properties of engineered OsVDE. Deletions can target the interior and / or ends of the protein. In various embodiments, deletions can comprise continuous segments or can be discontinuous.

[0044] "Insertion" refers to a modification of a protein by adding one or more amino acids from a reference protein. In some embodiments, modified engineered OsVDEs include inserting one or more amino acids into a naturally occurring OsVDE and inserting one or more amino acids into the amino acid sequence of another modified OsVDE. Insertions can be internal to the protein, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions can be continuous amino acid segments or separated by one or more amino acids in a naturally occurring protein.

[0045] This invention also includes analogs of the claimed protein OsVDE. These analogs may differ from the natural SEQ ID NO:1 by differences in amino acid sequence, by differences in modifications that do not affect the sequence, or both. These protein analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known biochemical techniques. Analogs also include those having residues different from the natural L-amino acid (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0046] The construction of the aforementioned transgenic plants can be achieved using traditional Agrobacterium-mediated transformation with recombinant plasmids or gene editing technology.

[0047] In a specific implementation, a Ubi+VDE expression cassette and nucleic acid construct, or expression construct, are constructed based on the gene OsVDE with SEQ ID NO:2 and the Ubi promoter with SEQ ID NO:3. Then, a Ubi+VDE expression plasmid is constructed, and finally, the Ubi promoter and VDE as exogenous genes are expressed in wild plants.

[0048] The coding sequence of the protein VDE or fragments thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, conventional techniques can be used to first obtain the VDE gene from genomic DNA, and then primers can be designed according to the nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, to amplify the VDE gene from the genomic DNA.

[0049] As used herein, the terms "expression cassette," "expression box," "gene expression cassette," or "nucleic acid construct" refer to a gene expression system containing all the necessary elements required for expressing the target protein VDE. Typically, it includes the following elements: a Ubi promoter, a gene sequence encoding the protein, a tag protein, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc.; these elements are operatively linked. In this invention, the preferred Ubi promoter for regulating VDE gene expression is polynucleotide SEQ ID NO:3.

[0050] As used herein, the term "expression construct" or "expression building block" refers to a recombinant DNA molecule that may contain one or more gene expression cassettes. These "constructs" are typically contained within an expression vector (plasmid vector).

[0051] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, the Ubi promoter region is placed at a specific position relative to the target gene OsVDE nucleic acid sequence SEQ ID NO:2, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0052] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the protein VDE. The nucleic acid constructs can be manipulated according to the different expression vectors or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0053] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequences SEQ ID NO:2-3 of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Specific expression of the OsVDE gene of the present invention is typically achieved by operably linking the nucleic acid sequence SEQ ID NO:2 of the present invention to the Ubi promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0054] Gene knock-in vectors can be used to integrate the Ubi+VDE described herein into regions of interest in the host genome. Typically, in addition to the polynucleotide sequence described herein, gene knock-in vectors may also contain 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequence described herein, and 3' homologous arms.

[0055] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin resistance, or chloramphenicol for *E. coli*, *Agrobacterium*, etc.

[0056] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0057] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0058] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0059] 1) Provide Agrobacterium carrying an expression vector, said expression vector containing the coding sequence of the Ubi promoter and the protein OsVDE;

[0060] 2) Contact plant cells, tissues, or organs with Agrobacterium in step 1) to transfer the coding sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0061] 3) Select plant cells or tissues into which the coding sequence has been transferred;

[0062] 4) Regenerate plants from the plant cells or tissues in step 3).

[0063] The method described herein can be used to construct transgenic plants with different uses.

[0064] We identified transcription factors that play a key role in chloroplast development regulation through transcriptome sequencing and gene regulatory network construction, and verified their functions through transgenic experiments. Among these transcription factors, we found that overexpression of OsVDE using the Ubi promoter accelerates photosynthetic induction (the gradual increase in photosynthetic rate caused by increased light intensity), improves photosynthetic efficiency in rice leaves (the amount of carbon dioxide fixed by the plant per unit time), and increases biomass (the dry weight of all aboveground parts, including stems, leaves, and panicles).

[0065] The methods used in this study for transgenic vector design, data analysis, and experimental verification are reasonable and can serve as a reference for research on other biological processes.

[0066] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0067] Example

[0068] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0069] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0070] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0071] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0072] The primer synthesis and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0073] Some of the PCR primers used in the examples are listed in Table 1 below.

[0074] Table 1. Primer sequences used for gene identification and quantitative PCR primer sequences used for gene expression determination.

[0075]

[0076] Note: In primer names, "-F" indicates forward and "-R" indicates reverse.

[0077] Example 1: Construction and validation of VDE-overexpressing transgenic Xiushui 134 rice

[0078] The transgenic Xiushui 134 rice overexpressing VDE was constructed by the following steps:

[0079] 1. Plasmid Pro Ubi -VDE build

[0080] 1.1 Gene Cloning

[0081] 1.1.1 The genome of Japanese rice (Oryza sativa L.) was extracted, and the promoter Ubi sequence was cloned.

[0082] Rice leaves were picked, ground into powder with liquid nitrogen, and the rice genome was extracted using a plant genomic DNA extraction kit (DP321) (Tiangen Biotech (Beijing) Co., Ltd.).

[0083] PCR primers (5'-3'):

[0084] Forward 1300-Ubi-F:

[0085] GCTATGACATGATTACGAATTCGTCGTGCCCCTCTCTAGAGA;

[0086] Reverse 1300-Ubi-R

[0087] TCCCCGGGTACCGAGCTCAAGTAACACCAAACAACAGG.

[0088] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0089] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 2 min 10 s, 35 cycles; 68℃ for 10 min.

[0090] The gene fragment amplified by PCR was sequenced and verified, confirming that the Ubi sequence is SEQ ID NO:3, and the verification was correct.

[0091] 1.1.2 RNA was extracted from Xiushui 134 rice and the VDE sequence was cloned by RT-PCR.

[0092] Wild-type Xiushui 134 rice leaves were collected, ground into powder using liquid nitrogen, and total RNA was extracted from Arabidopsis thaliana using the GeneJET Plant RNA Purification Kit (Thermo Scientific, catalog number K0802). The extracted RNA was then directly reverse transcribed to produce cDNA.

[0093] Using a reverse transcription kit ( One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No.: AT311) reverse transcription to produce cDNA.

[0094] PCR primers (5'-3'):

[0095] Forward VDE-Ft:

[0096] AGCTCGGTACCCGGGGATCCATGATGCCGCGGCAGTGCGG;

[0097] Reverse VDE-Rt: AGGTCGACTCTAGAGGATCCCCTTAGTTTCCTTATTGGCA.

[0098] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0099] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 2 min, 35 cycles; 68℃ for 10 min.

[0100] Sequencing of the gene fragment amplified by RT-PCR confirmed that the VDE sequence is SEQ ID NO:2, and the verification was correct.

[0101] 1.2 Construction of plant carriers

[0102] 1.2.1 The 2000bp fragment of the promoter Ubi sequence SEQ ID NO:3 was amplified by PCR using the fidelity enzyme kod-FX-Neo, and the sequence was confirmed to be correct.

[0103] PCR primers (5'-3'):

[0104] Forward 1300-Ubi-F:

[0105] GCTATGACATGATTACGAATTCGTCGTGCCCCTCTCTAGAGA;

[0106] Reverse 1300-Ubi-R

[0107] TCCCCGGGTACCGAGCTCAAGTAACACCAAACAACAGG.

[0108] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0109] PCR conditions: 94℃ for 2 min, 98℃ for 10 s, 58℃ for 30 s, 68℃ for 2 min, 35 cycles, 68℃ for 10 min.

[0110] Sequencing of the gene fragment amplified by RT-PCR confirmed that the Ubi sequence is SEQ ID NO:3, confirming the correctness of the verification.

[0111] 1.2.2 The Ubi promoter sequence was ligated into the plant expression vector pCAMBIA1300-FLAG-Tnos using the EcoRI restriction site to form the vector pCAMBIA1300-Ubi-FLAG-Tnos.

[0112] Use the restriction enzyme EcoRI-HF (NEB, catalog number: R3101).

[0113] Enzyme digestion conditions: 37℃ for 1.5h, 85℃ for 25min.

[0114] Using the ClonExpress II One Step Cloning Kit (Novizan, item number: C112-01)

[0115] Recombination reaction conditions: 37℃ for 30 min, and immediately placed on ice to cool after the reaction is completed.

[0116] 1.2.3 The VDE sequence was ligated into the plant expression vector pCAMBIA1300-Ubi-FLAG-Tnos using the SacI restriction site to form the vector pCAMBIA1300-Ubi-VDE-FLAG-Tnos.

[0117] Use the restriction enzyme SacI-HF (NEB, catalog number: R3156).

[0118] Enzyme digestion conditions: 37℃ for 1.5h, 85℃ for 25min.

[0119] Using the ClonExpress II One Step Cloning Kit (Novizan, item number: C112-01)

[0120] Recombination reaction conditions: 37℃ for 30 min, and immediately placed on ice to cool after the reaction is completed.

[0121] Take 1 μg of the correctly sequenced plasmid pCAMBIA1300-Ubi-VDE-FLAG-Tnos and TOP10 heat-shock competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1010), incubate on ice for 30 minutes, heat shock at 42°C for 5 minutes, place on ice for 2 minutes, add 200 μL of LB medium, and incubate at 37°C on a shaker for 1 hour. Spread all bacterial cells onto kanamycin-resistant LB agar plates and incubate upside down at 37°C for 1 day.

[0122] Select colonies for PCR identification.

[0123] PCR primers (5'-3'):

[0124] Forward VDE-Fm: GGAGCTTTTGGATCAGATGA;

[0125] Reverse 1300-FLAG-R: CGTCATCGTCCTTGTAATCG.

[0126] After the PCR reaction is completed, the PCR products are identified by agarose gel electrophoresis. If there are bands of the correct size, the corresponding number of bacteria is a positive clone, which is then sent to the company for sequencing verification.

[0127] After the above steps, the overexpression plasmid Pro was constructed. ubi -VDE.

[0128] 2. Construction of VDE-overexpressing transgenic rice

[0129] 2.1 Plasmid Proubi -VDE-transferred Agrobacterium

[0130] 2.1.1 Take 1 mg of the correctly sequenced plasmid Pro constructed above. Ubi -VDE and Agrobacterium EHA105 competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1001) were incubated on ice for 30 minutes, flash-frozen in liquid nitrogen for 5 minutes, heat-shocked at 37°C for 5 minutes, placed on ice for 5 minutes, and then 1 mL of LB medium was added. The cells were then incubated on a shaker at 28°C for 4 hours. All bacterial cells were plated on kanamycin-resistant and rifamycin-resistant LB agar plates and incubated at 28°C for 2-4 days to obtain engineered Agrobacterium.

[0131] 2.2 Rice transformation was performed using Agrobacterium-mediated callus infection.

[0132] The obtained Agrobacterium-mediated transformation was performed at Wuhan Boyuan Biotechnology Co., Ltd., resulting in transgenic overexpressing rice plants. After resistance screening and PCR verification, three homozygous T3 lines were obtained, labeled OE-VDE-1, OE-VDE-3, and OE-VDE-5, respectively. These are three distinct lines corresponding to different insertion sites. Because vector insertion is random, having the same phenotype at all three insertion sites ensures the stability and consistency of gene expression.

[0133] 3. Investigation of VDE gene expression level, protein content, and plant phenotype in transgenic rice plants.

[0134] 3.1 Assessment of VDE Transcriptional Expression Levels in Transgenic Materials

[0135] RNA was extracted from Xiushui 134 rice, and the expression level of VDE was identified by qPCR. The steps are as follows:

[0136] Wild-type Xiushui 134 rice and transgenic Xiushui 134 rice were harvested, ground into powder using liquid nitrogen, and total RNA was extracted from Xiushui 134 rice using the GeneJET Plant RNA Purification Kit (Thermo Scientific, catalog number K0802). The extracted RNA was then directly reverse transcribed to produce cDNA.

[0137] Using a reverse transcription kit ( One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No.: AT311) for reverse transcription to produce cDNA.

[0138] The CFX 96 system (Bio-Rad) was used to perform real-time qPCR using Taq Pro Universal SYBR qPCR Master Mix (Novizan, catalog number Q712-02).

[0139] qPCR program: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, 35 cycles.

[0140] See Figure 1 Compared to the wild type (WT), the expression level of VDE in transgenic materials was significantly increased. Figure 1 (C)

[0141] 3.2 Assessment of VDE protein expression levels in transgenic materials

[0142] 3.2.1 Extraction of total protein samples from transgenic materials

[0143] Extract the last fully expanded leaf from the tillering stage of Xiushui 134 rice and cut a 10cm section. 2 Fresh leaves were placed in a 2mL centrifuge tube containing steel balls. After being frozen in liquid nitrogen, the leaves were ground into powder, and 400μL of SDS protein extraction buffer and 10mM DTT were added. An appropriate amount of protease inhibitor was added as needed. The sample was thoroughly shaken and heated in a 95℃ metal bath for 5 minutes. After centrifugation at 13000rpm for 5 minutes, the supernatant was transferred to a new 1.5mL centrifuge tube to obtain the total protein sample. The sample was quantified using a BCA protein quantification kit (catalog number: 20201ES, Yisheng Biotechnology Co., Ltd.), and then the target protein was detected by Western blotting (WB) using a precast gel (catalog number: MA0298, Dalian Meilun Biotechnology Co., Ltd.).

[0144] Table 2. SDS protein extract formulation:

[0145]

[0146] 3.2.2 Western blot assay

[0147] After standardizing the protein sample loading amount, conduct the experiment according to the following steps:

[0148] (1) SDS-PAGE electrophoresis: electrophoresis was performed at 90V (constant voltage) for 30 min in the electrophoresis buffer, followed by electrophoresis at 120V (constant voltage) for 30 min.

[0149] (2) Transfer: Place the black fiber pad, white filter cotton, PVDF membrane (0.45μm) activated with methanol, protein gel, white filter cotton and black fiber pad in sequence on the transfer sandwich clamp, and place the sandwich clamp in the electrophoresis tank (protein gel near the negative electrode of the power supply, PVDF membrane near the positive electrode of the power supply), put in an ice pack, add an appropriate amount of transfer buffer, and transfer the membrane at 200mA (constant current) for 90min.

[0150] (3) Blocking: After removing the PVDF membrane, confirm that the protein marker has been correctly transferred to the membrane, soak it in TBST, and wash it 3 times on a shaker (100-120 rpm) for 5 minutes each time. Then add an appropriate amount of blocking solution and block it at room temperature for 1 hour on a shaker (50-60 rpm) or overnight at 4°C.

[0151] (4) Primary antibody incubation: Wash the membrane with TBST for 5 min (100-120 rpm), then add the primary antibody that has been diluted with blocking buffer in advance (generally diluted 1000-3000 times), and place it on a shaker (50-60 rpm) at 4°C overnight or at room temperature for 60-90 min.

[0152] (5) Secondary antibody incubation: Wash the membrane three times with TBST for 5 minutes each time on a shaker with a speed of 100-120 rpm; then add the secondary antibody diluted with blocking buffer (generally diluted 3000-10000 times), and adjust the shaker speed to 50-60 rpm, and incubate at room temperature for 60 minutes.

[0153] (6) Color development: Turn on the Tianneng chemiluminescence gel imaging system and computer software in advance, and pre-cool the instrument; wash the membrane 3 times (100-120 rpm), 5 min each time with TBST; at the same time, take 500 μL each of ECL color development solution A and B into a new 2 mL centrifuge tube and mix well; after the PVDF membrane is cleaned, place it on the lid of a square culture dish and evenly wet the PVDF membrane with color development solution; then expose and image, and save the image.

[0154] (7) Ponceau S staining: Place the PVDF membrane with the image taken in an antibody incubator and wash it several times with ddH2O to ensure that the chromogenic solution is washed away. Then, place the washed PVDF membrane in Ponceau S staining solution for 2-3 minutes. After that, remove the PVDF membrane and continue washing it with ddH2O on a shaker until the red background is completely removed. Finally, take a picture using a Tianneng chemiluminescence gel imaging system or a mobile phone (placed on a piece of white paper). If protein quantification is not required, Ponceau S staining is not necessary.

[0155] Antibodies used: Anti-FLAG (catalog number: 2368S, ABclonal); HRP Goat Anti-RabbitlgG (H+L) (catalog number: AS014, ABclonal).

[0156] like Figure 1 As shown, compared to the wild type (WT), the expression level of VDE protein in the transgenic material was significantly increased ( Figure 1 (B)

[0157] 4. Phenotypic characteristics of WT and OE-VDE lines at the tillering stage

[0158] See Figure 1 In Figure D, the left image shows the phenotype of wild-type plants at 60 days of tillering, while the right image shows the phenotype of transgenic plants at 60 days of tillering.

[0159] Example 2: Changes in the content of lutein cycling pigments and other major pigments in OsVDE-overexpressing transgenic plants

[0160] 1. Pigment extraction from genetically modified materials

[0161] Select the last fully unfolded leaf of the rice plant during the tillering stage, and cut a 2cm section from the middle of the leaf. 2 The long region was placed in a 2mL centrifuge tube with steel balls, frozen in liquid nitrogen, and then ground into powder. 1mL of pre-cooled 80% acetone (protected from light) was added, mixed well, and then placed on a shaker at 4℃ and 100rpm. After 3 hours, the sample was centrifuged at 12000rpm for 5 minutes (4℃). The supernatant was aspirated with a syringe and filtered through a 0.22μm nylon filter into a new 2mL sample vial. The filtered sample was then directly subjected to subsequent high-performance liquid chromatography (HPLC) separation experiments.

[0162] Formula for calculating the deep-epoxidation state (DES) of the lutein cycle:

[0163] DES(%)=(Zea+0.5*Ant) / (Zea+Ant+Vio).

[0164] 2. Pigment liquid chromatography detection of transgenic materials

[0165] (1) Pre-treat the chromatographic column and equilibrate for 10 min. Mobile phase: 100% methanol; flow rate: 1 mL / min.

[0166] (2) Sample detection. Mobile phase: 100% methanol; flow rate: 1 mL / min; column temperature: 25℃; injection volume: 1 μL; detection wavelength: 440 nm.

[0167] (3) Rinse the column with 10% methanol solution for 20 min, then rinse with 90% methanol solution for 20 min, and finally rinse with 100% methanol solution for 20 min. The flow rate is set to 1 mL / min.

[0168] The HPLC method for pigment separation in this experiment was provided by Beijing Spectrum Technology Co., Ltd.

[0169] See Figure 2 Compared to wild-type (WT), the contents of Zea and Ant in OE-VDE overexpression plants were significantly increased, the contents of Vio were significantly decreased, the contents of VAZ were significantly increased, and the DES value, i.e., the de-epoxylation state of the xanthophyll cycle, was increased. Higher DES can alleviate photoinhibition under strong light. Zeaxanthin is a key component involved in NPQ induction. Its increased content will accelerate NPQ induction, but it may also lead to a slowdown in the NPQ relaxation rate due to excessive accumulation (Murchie EH, Ruban AV. Dynamic non-photochemical quenching in plants: From molecular mechanism to productivity[J]. Plant J,2020,101(4):885-896.). The contents of other major pigments, chlorophyll a (Chla), chlorophyll b (Chlb), xanthophyll, and neoxanthin, did not change significantly compared to WT and OE-VDE overexpression materials.

[0170] Example 3: Comparison of field agronomic traits between VDE-overexpressing transgenic plants and WT wild-type plants.

[0171] 1. Assessment of plant height, number of ears, biomass, and yield of transgenic plants.

[0172] The material evaluation steps are as follows:

[0173] At harvest, 12 rice plants were randomly selected from 5 plots of each line. To avoid boundary effects, edge samples were omitted in all experiments. First, each panicle was bagged and cut at the base. Then, the plant height and panicle number of each individual rice plant were measured. Next, the panicles and stems of each plant were separated. The stems were blanched at 110℃ for 1 hour, then dried at 70℃ for 3 days, while the panicles were dried at 40℃ for 7 days until the sample dry weight reached a constant. Finally, the aboveground biomass and panicle weight of each individual rice plant were measured.

[0174] See Figure 3In the Chinese AD, compared with the wild type (WT), the transgenic lines OE-VDE-3 and OE-VDE-5 showed a significant increase in plant height, a significant increase in the number of panicles (effective tillers) in all lines, a significant increase in biomass in all lines, with an increase of about 11-16%, and a significant increase in yield in the transgenic line OE-VDE-5. All of these indicate that overexpression of VDE increases rice biomass (dry weight of all stems, leaves and panicles above ground).

[0175] 2. Evaluation of nitrogen content and specific leaf weight in transgenic plants

[0176] The material evaluation steps are as follows:

[0177] Determination of specific leaf weight (SLW) and leaf nitrogen content (LNC): (1) Take the newest fully expanded leaf at the heading stage with a diameter of 10 cm, measure the leaf width at both ends of the leaf, and calculate the leaf area; (2) Put the leaf sample into an oven, fix it at 110℃ for 1 h, and then dry it at 70℃ for 3 days until the dry weight of the sample is constant; (3) Weigh the leaf weight using an electronic balance and calculate the specific leaf weight (SLW = leaf weight / leaf area); (4) Grind the dried sample into powder using a high-throughput tissue grinder, and weigh 3-5 mg of the sample using a micro and ultramicro balance, and measure the LNC using a vario isotope CUBE elemental analyzer (elementar, Germany).

[0178] See Figure 3 Compared to the wild-type (WT), the nitrogen content in the leaves of the transgenic line OE-VDE showed no significant difference, indicating that VDE overexpression does not affect nitrogen use. Specific leaf weight data showed that, except for OE-VDE-1, which showed a decrease in specific leaf weight, there were no significant differences among the other transgenic lines, indicating that VDE overexpression does not affect leaf structure. In summary, VDE overexpression significantly increases rice biomass without affecting normal plant growth and development.

[0179] Example 4: Comparison of photosynthetic parameters based on chlorophyll fluorescence between VDE-overexpressing transgenic plants and WT wild-type plants under different growth conditions

[0180] The photosynthetic parameters of chlorophyll fluorescence were detected using the following method.

[0181] Measuring instrument: Dual-PAM-100 (WALZ, Inc., Germany).

[0182] Detection steps: Set the actinic light intensity (PAR) to 1300 μmol·m -2 ·s -1Light induction for 10 min, followed by darkness (relaxation time) for 6 min, with saturation pulses applied every 15 s and corresponding chlorophyll fluorescence parameters (F, F', Fm', and Fo') recorded. After measuring the relevant data under light, the plants were subjected to dark treatment for more than 2 h, and Fo and Fm were measured again. The formula for calculating fluorescence parameters (Kramer DM, Johnson G, Kiirats O, et al. New fluorescence parameters for the determination of qa redox state and excitation energy fluxes[J]. Photosynth Res, 2004, 79(2):209):

[0183] NPQ=Fm / Fm'-1

[0184] Y(II)=(Fm'-F') / Fm'

[0185] Y(NO) = F / Fm'

[0186] Y(NPQ)=F / Fm'-F / Fm

[0187] 1-qL=1-(Fm'-F') / (Fm'-Fo')-Fo' / F'

[0188] Fo'≈Fo / (Fv / Fm+Fo / Fm')

[0189] See results Figure 4 The NPQ dynamic curves of rice plants grown under normal greenhouse light showed that, in the first 40–80 seconds, the NPQ response rate of the OE-VDE line was faster than that of the WT line; however, after 80 seconds, there was no significant difference between the OE-VDE and WT lines, and the NPQ relaxation process was not affected. Analysis of other fluorescence parameters revealed that, compared to wild-type rice, the OE-VDE lines had higher Y(NPQ) and lower Y(NO), but Y(II) remained unchanged. Figure 4 AD). The unchanged Y(II) indicates that the electron transport rate was not affected. Next, the same chlorophyll fluorescence induction curve measurements were performed on plants treated with high light in a greenhouse and plants grown in the field; the results were consistent with those under normal light. Figure 4(EI). In summary, VDE overexpression does indeed accelerate the induction of NPQ in rice. Furthermore, while accelerating the NPQ induction process, VDE overexpression did not affect the NPQ maximum value or relaxation process. This transgenic material overexpressing VDE showed accelerated NPQ induction but no difference in the NPQ maximum value or relaxation process, making it an excellent material for studying the effects of changes in the NPQ induction process on photosynthetic efficiency, biomass, and yield.

[0190] Example 5: Comparison of photosynthetic induction and chlorophyll fluorescence response curves of VDE-overexpressing transgenic plants and WT wild-type plants after transition from low light to high light.

[0191] The photosynthetic parameters of chlorophyll fluorescence were detected using the following method.

[0192] Measuring instrument: Dual-PAM-100 (WALZ, Inc., Germany).

[0193] Detection steps: Set the actinic light intensity (PAR) to 100 μmol·m -2 ·s -1 Photoinduction for 4 min, followed by induction with 2000 μmol·m -2 ·s -1 Under light intensity for 10 minutes, a saturation pulse was applied every 12 seconds, and the corresponding chlorophyll fluorescence parameters (F, F', Fm', and Fo') were recorded. After measuring the relevant data under light, the plants were subjected to dark treatment for more than 2 hours before Fo and Fm were measured again.

[0194] Formulas for calculating fluorescence parameters (Kramer DM, Johnson G, Kiirats O, et al. New fluorescence parameters for the determination of qa redox state and excitation energy fluxes[J]. Photosynth Res, 2004, 79(2):209):

[0195] NPQ=Fm / Fm'-1

[0196] Y(II)=(Fm'-F') / Fm'

[0197] Y(NO) = F / Fm'

[0198] Y(NPQ)=F / Fm'-F / Fm

[0199] 1-qL=1-(Fm'-F') / (Fm'-Fo')-Fo' / F'

[0200] Fo'≈Fo / (Fv / Fm+Fo / Fm')

[0201] Perform the gas exchange experiment as follows.

[0202] Measuring instrument: LI-6800 (Li-Cor, Inc., USA)

[0203] Detection procedure: Set the CO2 concentration in the leaf chamber to 400 ppm and the flow rate to 500 μmol·s. -1 Temperature 28℃, humidity 65%, light intensity: 100 μmol·m -2 ·s -1 Photoinduction for 4 min, followed by induction with 2000 μmol·m -2 ·s -1 Under light intensity for 10 minutes, gas exchange parameters (A, g) were recorded every 3 seconds. s ).

[0204] See Figure 5 From low light (100 μmol·m -2 ·s -1 ) Towards high light (2000 μmol·m -2 ·s -1 Conversion process (simplified to L) 100 -H 2000 The dynamic changes in photosynthetic response of transgenic materials were investigated. The results showed that during the light conversion process of L100-H2000, all three OE-VDE plants exhibited a faster photosynthetic induction rate. In the early stage of light induction (6-249s) of L100-H2000, the leaf CO2 assimilation rate (A) of the OE-VDE line was significantly higher than that of WT.

[0205] See Figure 6 The results showed that, during the light conversion process of L100-H2000, the relative carbon assimilation of OE-VDE plants was significantly increased by 20%–23% compared to WT. Starting from 108s, the g content of all OE-VDE lines increased significantly. s (Stomatal conductance is an indicator of the degree of stomatal opening. The opening and closing state of stomata directly affects the CO2 concentration in the leaves, and thus affects the rate of photosynthesis.) It is also significantly higher than that of the wild type, which is conducive to the leaves absorbing more CO2.

[0206] Although the above embodiments only use rice as an example to illustrate the technical solution of the present invention, the technical solution of the present invention is also applicable to other plant species such as rice, corn, wheat, soybeans, vegetables, and other crops. Therefore, without departing from the spirit of the present invention, those skilled in the art can make various modifications or alterations to the present invention, and equivalent forms of such modifications or alterations should also fall within the scope of the present invention.

[0207] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are prior art or common general knowledge.

Claims

1. A method for increasing the photosynthetic rate and / or biomass of plants, characterized in that, To induce plants to overexpress chlorophyll decyclooxygenase (VDE).

2. The method as described in claim 1, characterized in that, Violet decyclooxygenase (VDE) is independently overexpressed in plants.

3. The method as described in claim 1, characterized in that, The flavin decyclooxygenase (VDE) is selected from the following group: flavin decyclooxygenase from Arabidopsis thaliana (NCBI accession number NP_001321301.1), flavin decyclooxygenase from maize (NCBI accession number NP_001147756.1), flavin decyclooxygenase OsVDE from rice (NCBI accession number XP_015636342.1), flavin decyclooxygenase from soybean (NCBI accession number NP_001241404.1), and flavin decyclooxygenase from rapeseed (NCBI accession number XP_013641072.1).

4. The method as described in claim 1, characterized in that, The chlorophyll decyclooxidase (VDE) is the rice-derived chlorophyll decyclooxidase OsVDE (NCBI accession number XP_015636342.1, nucleotide sequence of the expression gene OsVDE is SEQ ID NO:2) or its conserved variant polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

1. The conserved variant polypeptide is a polypeptide that has more than 95% homology with VDE and has VDE function, namely, an OsVDE isoenzyme.

5. The method as described in claim 1, characterized in that, The plant is an agricultural crop, selected from rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, and cruciferous plants, with rice being the preferred crop.

6. The method as described in claim 1, characterized in that, The following method was used to achieve overexpression of chlorophyll decyclooxidase (VDE) in plants: A. The gene encoding chlorophyll decyclooxygenase (VDE) is cloned into a plasmid vector to form a recombinant plasmid, i.e., a VDE overexpression vector. Then, plant cells or tissues are transformed by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrocoagulation or Agrobacterium-mediated transformation. The transformed plant tissues are then cultured into plants to obtain transgenic plants that overexpress VDE. or B. By cloning the gene encoding violet decyclooxygenase (VDE) into a plant chromosome using gene editing technology, transgenic plants overexpressing violet decyclooxygenase (VDE) can be obtained; or C. Place the gene encoding viniferin decyclooxygenase (VDE), which is already present in the plant genome, under the regulation of an enhanced promoter.

7. The method as described in claim 5, characterized in that, The plasmid vector mentioned in step A is selected from binary Agrobacterium vectors and vectors that can be used for plant micro-bombardment.

8. The method as described in claim 5, characterized in that, The Agrobacterium species were selected from Agrobacterium tumefaciens, Agrobacterium EHA105, and Agrobacterium GV3101.

9. The method as described in claim 5, characterized in that, The gene editing technology described in step B is selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-Cas12a system, CRISPR-BEST system, and MuGENT.

10. The method as described in claim 5, characterized in that, Step C involves assembling the Ubi promoter from Nipponbare rice with the OsVDE gene to form a VDE expression cassette / frame, i.e., the Ubi+VDE expression cassette. Then, one or more copies of the VDE expression cassette / frame are cloned into an expression vector to form a VDE recombinant expression vector. Using transgenic technology, this recombinant expression vector is introduced into plants, such as rice, to obtain transgenic plants that overexpress VDE.

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