New function of pil13 in improving photosynthesis and plant height and molecular identification reagent thereof

Abstract

The application provides a new function of PIL13 for improving photosynthesis and plant height and a molecular identification reagent thereof. A transcription factor playing a pivotal role in transcription of photosynthesis genes is mined through transcriptome sequencing and construction of a gene regulation network. The PIL13 gene is obtained through screening and verification from the transcription factors, and the PIL13 gene can improve the leaf photosynthetic rate of gramineous plants, improve the light energy utilization rate, and improve the plant height. The method of construction, analysis and experimental verification of the gene regulation network of the application is reasonable, not only provides a new target for improvement of plant photosynthesis traits, but also can be used as a reference for research on gene regulation networks of other biological processes.

Description

Technical Field

[0001] This invention belongs to the field of plant photosynthesis research. More specifically, this invention relates to a novel function of PIL13 in enhancing plant photosynthesis and plant height, and its molecular identification reagent. Background Technology

[0002] Photosynthesis is the process by which green plants use light energy to assimilate the carbon dioxide and water they absorb into organic matter. In photosynthesis, green plants use chloroplasts to convert carbon dioxide and water into energy-storing organic matter, releasing oxygen in the process.

[0003] Research on crop yield improvement mainly focuses on the following aspects: increasing crop sources, i.e., enhancing crop photosynthesis; increasing the size of the crop sink; and improving the ability of crop photosynthetic products to be transported from the source to the sink. Among these, increasing the sink capacity and improving the ability of photosynthetic products to be transported to the sink are effective breeding approaches.

[0004] Photosynthesis is fundamental to crop yield. Under suitable conditions, the water and carbon dioxide required for photosynthesis are not lacking, and some energy conversion steps in the process are relatively efficient. However, the conversion and fixation of light energy into chemical energy involves a series of complex enzymatic reactions, which are easily affected by unsuitable external factors such as temperature and light intensity, significantly reducing light energy utilization efficiency. For example, the light energy utilization rates of major crops such as rice and wheat are low, far from the theoretical values. Therefore, breeding varieties with strong stress resistance is one of the important ways to improve crop photosynthesis. Chloroplasts are the site of plant photosynthesis, which is crucial for crop yield. Therefore, chloroplast development and its regulatory mechanisms are among the key factors affecting the yield of grain crops such as rice.

[0005] In conjunction with existing plant transgenic engineering technologies, a deeper understanding of chloroplast development and regulation mechanisms is needed to find new methods and approaches to improve plant photosynthetic efficiency, providing ideas and technical guidance for high-yield crop breeding.

[0006] Despite numerous efforts to increase crop yields and improve crop quality, effective methods are still lacking. For example, in rice, a major food crop, many high-yield cultivars, especially super hybrid rice and large-ear, large-grain varieties, suffer from incomplete grain filling, which significantly hinders further increases in rice yield.

[0007] Therefore, there is an urgent need in this field to find effective means to solve the problem of incomplete grain filling in crops, so as to further improve crops and achieve increased crop yield and quality. Summary of the Invention

[0008] The purpose of this invention is to provide a novel function of PIL13 in enhancing plant photosynthesis and plant height, as well as a molecular identification reagent.

[0009] In a first aspect of the invention, an application of hub transcription factor PIL13 is provided for use as a regulatory target in gramineous plants to promote photosynthesis and increase plant height, or for the preparation of plants with increased photosynthesis and plant height; wherein the regulation is to increase the expression level or activity of hub transcription factor PIL13.

[0010] In another preferred embodiment, the promotion of photosynthesis includes: increasing the photosynthetic rate of the leaves.

[0011] In another preferred embodiment, the promotion of photosynthesis includes: increasing the expression of photosynthetic genes.

[0012] In another preferred embodiment, the photosynthetic genes include: Lhca6, Os03g0279950_PsbP, FNR, CYP20-2, or Os02g0744000.

[0013] In another preferred embodiment, a regulatory molecule that increases the expression level or activity of hub transcription factor PIL13 is used to promote photosynthesis and increase plant height; preferably, the regulatory molecule includes (but is not limited to): the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13 (such as gDNA), and an expression construct (such as an expression vector) containing a hub transcription factor PIL13 expression cassette; preferably, the expression cassette further includes (suitable for expression) a promoter and / or terminator; preferably, the regulatory molecule is exogenously introduced into the plant.

[0014] In another preferred embodiment, in the breeding of grass plants, plants with high expression levels or activity of the hub transcription factor PIL13 are selected as preferred plants for cultivation to obtain superior plants with strong photosynthesis and increased plant height; preferably, the traits of the superior plants are passed on to offspring plants.

[0015] In another preferred embodiment, the hub transcription factor PIL13 is selected from the group consisting of: (a) a protein with an amino acid sequence as shown in SEQ ID NO:2; (b) a derivative or active fragment having the function of the protein with a sequence that is ≥85% homology to the amino acid sequence shown in SEQ ID NO:2 (e.g., ≥88%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98%, or ≥99% homology); and (c) a derivative or active fragment having the function of the protein with the amino acid sequence shown in (a) by substitution, deletion, or addition of one or more (e.g., 1-20, 1-10, 1-5, 1-3, or 1-2) amino acid residues.

[0016] In another aspect of the present invention, a method for promoting photosynthesis and increasing plant height in grass plants is provided, comprising: (a) using hub transcription factor PIL13 as a regulatory target to increase the expression level or activity of the transcription factor in grass plants; or (b) in grass plant breeding, screening plants with high expression levels or activity of hub transcription factor PIL13, and cultivating them as preferred plants to obtain superior plants with strong photosynthesis and increased plant height.

[0017] In another preferred embodiment, in (a), the regulatory molecule used in the method includes (but is not limited to): the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13 (e.g., gDNA), and an expression construct (e.g., an expression vector) containing a hub transcription factor PIL13 expression cassette; preferably, the expression cassette further includes (suitable for expression) a promoter and / or terminator; preferably, the regulatory molecule is exogenously introduced into the plant.

[0018] In another preferred embodiment, the promotion of photosynthesis includes: increasing the photosynthetic rate of leaves and / or increasing the expression of photosynthetic genes; preferably, the photosynthetic genes include: Lhca6, Os03g0279950_PsbP, FNR, CYP20-2 or Os02g0744000.

[0019] In another aspect of the invention, the use of hub transcription factor PIL13 is provided for analyzing photosynthetic traits, plant height traits, and / or leaf traits in grasses; preferably, the analysis includes early analysis, such as analysis performed during the seed or seedling stage of grasses.

[0020] In another preferred embodiment, when analyzing the traits of grass plants, the hub transcription factor PIL13 in the plant (including (but not limited to) its seeds or seedlings) is analyzed. If the expression of hub transcription factor PIL13 is significantly high, then the grass plant has optimized traits. The optimized traits include: promoted photosynthesis and increased plant height. Preferably, the promoted photosynthesis includes: increased leaf photosynthetic rate and / or increased expression of photosynthetic genes.

[0021] In another preferred embodiment, primers with the sequences shown in SEQ ID NO:7 and SEQ ID NO:8 are used to detect the expression level of hub transcription factor PIL13.

[0022] In another preferred embodiment, the grass is a grass that has a photosynthetic mechanism (including the presence of chloroplasts) and expresses the hub transcription factor PIL13 or its homologs (homologous genes / proteins).

[0023] In another preferred embodiment, the grasses include (but are not limited to): plants of the genus Oryza.

[0024] In another preferred embodiment, the grasses include (but are not limited to): rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), wheat (Triticum aestivum), rye (Secalecereale), oats (Avena sativa L.), sorghum (Sorghum bicolor), millet (Setaria italica), foxtail millet (Panicum miliaceum), and short-stalked grass (Brachypodium distachyum).

[0025] In another aspect of the invention, a cell, tissue, or organ of a grass plant is provided, including increasing the expression level or activity of hub transcription factor PIL13 as a regulatory target; preferably, the regulation is implemented using the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13 (such as gDNA), or an expression construct (such as an expression vector) containing a hub transcription factor PIL13 expression cassette.

[0026] In another preferred embodiment, the grass cells, tissues, or organs do not have the ability to reproduce into adult grasses.

[0027] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0028] Figure 1 Verification of pRbcS2-OsPIL13-FLAG transgenic rice.

[0029] (A) The RNA expression level of the OsPIL13 gene was detected by RT-qPCR. OsActin1 was used as an internal reference gene. The data in the figure are mean ± standard error. Biological replication n = 3. * indicates p < 0.05 (two-tailed t test).

[0030] (B) The expression of OsPIL13-FLAG fusion protein was detected by Western blotting using Anti-FLAG antibody.

[0031] Among them, the latest fully expanded leaves of rice 48 days after transplanting were used as samples for RT-qPCR and Western blot experiments.

[0032] Figure 2 Effects of OsPIL13 transcription factor overexpression on rice plant height.

[0033] (A) Comparison of plant height between pRbcS2-OsPIL13-FLAG transgenic plants and wild-type plants. The photos were taken during the grain-filling stage (79 days after transplanting).

[0034] (B) Statistical chart comparing the plant height of pRbcS2-OsPIL13-FLAG transgenic plants with that of wild-type plants.

[0035] The data in the figure are mean ± standard deviation. The biological replicates were n = 36–45 (12–15 replicates / plot × 3 plots), and the measurement time was 90–100 days after transplanting.

[0036] * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. The p-value is obtained from a two-tailed t-test.

[0037] Figure 3 The effect of overexpression of OsPIL13 transcription factor on photosynthetic gene expression.

[0038] The data were obtained from RNA-seq analysis. RNA was extracted from the latest fully expanded leaves of rice plants 48 days after transplanting for RNA-seq. The figure shows the log2 fold change in the expression level of photosynthetic genes in OsPIL13-overexpressing rice lines relative to wild-type.

[0039] * indicates that the corrected p-value is <0.05. Both the log2 change factor and the corrected p-value are calculated using the DESeq2 package in R.

[0040] Figure 4 Effects of overexpression of hub transcription factor (OsPIL13) on photosynthetic rate of leaves under saturated light.

[0041] The data in the figure are mean ± standard deviation. In 2022, the biological replicates were n = 15 (5 replicates / plot × 3 plots), and in 2024, the biological replicates were n = 5 to 6 (1 to 2 replicates / plot × 3 plots).

[0042] * indicates p < 0.05, *** indicates p < 0.001, and the p-value is obtained by a two-tailed t-test. Detailed Implementation

[0043] The inventors identified transcription factors that play a pivotal role in the transcription of photosynthetic genes through transcriptome sequencing and the construction of gene regulatory networks. The functions of these transcription factors were verified through transgenic experiments. From these transcription factors, the PIL13 gene was obtained through extensive research and screening. This gene can improve the photosynthetic rate of leaves in grasses, increase light energy utilization, and enhance plant height.

[0044] The methods for constructing, analyzing, and experimentally verifying the gene regulatory network in this invention are reasonable. They not only provide new targets for improving photosynthetic traits in plants, but can also serve as a reference for research on gene regulatory networks in other biological processes.

[0045] hub transcription factor PIL13

[0046] In this invention, unless otherwise specified, the hub transcription factor PIL13 refers to a protein having the sequence SEQ ID NO:2 or its encoding gene, and also includes sequence variations having the same function as the hub transcription factor PIL13 protein. The encoding gene of hub transcription factor PIL13 can be gDNA or cDNA, and may also contain a promoter. The sequence of the encoding gene also includes sequences degenerate with those provided in this invention.

[0047] Variations of the hub transcription factor PIL13 polypeptide include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the hub transcription factor PIL13 polypeptide (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:2; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the hub transcription factor PIL13 polypeptide is also included in this invention.

[0048] Polypeptides derived from species other than rice that share high homology with the sequence shown in SEQ ID NO:2, or that play the same or similar roles in the same or similar regulatory pathways, are also included in this invention. That is, the “hub transcription factor PIL13” also includes its homologs (homologous genes / proteins). It should be understood that although the present invention preferably studies the hub transcription factor PIL13 obtained from the specific species rice, other polypeptides or genes obtained from other species that are highly homologous to the hub transcription factor PIL13 (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0049] Applications of modifying plant photosynthesis or plant height phenotypes

[0050] As used herein, "plant" refers to a plant that possesses a photosynthetic mechanism. Those skilled in the art know that the components of a plant's photosynthetic mechanism are common, and plants with a photosynthetic mechanism share common characteristics, including the presence of many conserved genes or regulatory elements in their genomes that regulate gene transcription and expression. As a preferred embodiment of the invention, the plant may be a monocotyledonous plant; more preferably, the plant is a grass (Poaceae), such as rice, wheat, barley, corn, or sorghum.

[0051] As used in this article, “non-reproductive material” refers to a biological material that does not have the characteristic of using photosynthesis to synthesize carbohydrates and proteins from inorganic substances such as water, carbon dioxide and inorganic salts to sustain its life.

[0052] As used in this article, the terms "mechanism", "pathway", "signaling pathway" and "regulatory pathway" are interchangeable.

[0053] Based on the inventor's new discovery, an application of hub transcription factor PIL13 is provided, which can be used as a regulatory target in grass plants to promote photosynthesis and increase plant height, or to prepare plants with increased photosynthesis and plant height; wherein, the regulation is to increase the expression level or activity of hub transcription factor PIL13.

[0054] Once the function of the hub transcription factor PIL13 is known, its expression or activity can be regulated using various methods familiar to those skilled in the art, which are readily achievable based on the scheme disclosed in this invention.

[0055] In this invention, the regulatory molecules of the hub transcription factor PIL13 protein or its encoding gene include promoters, agonists, upregulators, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression. Any substance that can increase the activity of hub transcription factor PIL13, improve the stability of hub transcription factor PIL13, upregulate the expression of hub transcription factor PIL13, or increase the effective duration of hub transcription factor PIL13 can be used in this invention as a substance useful for upregulating hub transcription factor PIL13.

[0056] The regulatory molecules can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. The hub transcription factor PIL13 protein or its regulatory molecules are particularly suitable for application to a class of plants whose hub transcription factor PIL13 expression is below the average for that class of plants or whose hub transcription factor PIL13 is not expressed; thus, the application of the hub transcription factor PIL13 protein or its regulatory molecules can revert this class of plants to a wild-type phenotype or a better phenotype.

[0057] The present invention also provides a method for increasing the expression of hub transcription factor PIL13 in plants, the method comprising: transferring the encoding gene of hub transcription factor PIL13 or an expression construct or vector containing the encoding gene into plants.

[0058] The technical solution of this invention can be applied to molecular design breeding through various pathways.

[0059] Molecular identification

[0060] Based on the inventors' new discovery, this invention provides a gene suitable for identifying photosynthetic traits or plant height traits in plants, namely the hub transcription factor PIL13 gene. This invention also provides specific molecular markers designed for said gene, primers for identifying said molecular markers, and identification strategies.

[0061] Therefore, the present invention provides a method for specifically identifying photosynthetic traits or plant height traits in plants, comprising: analyzing hub transcription factor PIL13 (including protein level, transcript or gene level) in plants (including (but not limited to) their seeds or seedlings); if the expression of hub transcription factor PIL13 is significantly high, then the grass plant has optimized traits; the optimized traits include: promoted photosynthesis and increased plant height; preferably, the promoted photosynthesis includes: increased leaf photosynthetic rate and / or increased expression of photosynthetic genes.

[0062] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0063] The methods for obtaining DNA from the sample to be tested are well known to those skilled in the art, such as the traditional phenol / chloroform / isoamyl alcohol method, or commercially available DNA extraction kits, which are well known to those skilled in the art.

[0064] Polymerase chain reaction (PCR) is a technique well-known to those skilled in the art, and its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of this invention can be performed using conventional PCR techniques.

[0065] Based on the above, the present invention also relates to a kit for identifying photosynthetic traits or plant height traits in plants, said kit containing primers shown in SEQ ID NO:7 and SEQ ID NO:8. In a preferred embodiment, the kit further includes the restriction endonuclease EcoRI. It should be understood that the reagents that can be used for identification according to the present invention are not limited thereto.

[0066] In addition, the kit may also contain instructions for use and / or standard operating procedures for identification. The kit enables rapid, batch detection of photosynthetic traits or plant height traits.

[0067] This invention provides a simple and effective identification method, molecular markers, and preferred primers for identifying photosynthetic traits or plant height traits in plants, thus providing a feasible method for identifying photosynthetic traits or plant height traits and a new tool for plant breeding and screening.

[0068] The ability to identify photosynthetic traits or plant height traits in plants early in the planting process facilitates plant breeding.

[0069] This invention has significant application prospects in molecular design breeding of plant plant type and yield traits, as well as in the improvement of crop varieties using genetic engineering technology.

[0070] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0071] Structure of genes and proteins

[0072] Nucleotide sequence of the PIL13 gene (SEQ ID NO:1):

[0073]

[0074] The amino acid sequence of the PIL13 protein (SEQ ID NO:2):

[0075] MDGNARSAANQTKQIVTDNELVELLWHNGGVVAQPQAAQARVVSSSSGRGQSASVLTGDDTETAAWFPDTLDDALEKDLYTQLWRSVTGDAFPAAAAAGPSSHHAPPPDLPPPAARPPMRSGIGSSWTGDICSAFCGSNHIPETAAQRCRDAGAALPPERPRRSSTHDGAGTSSSGGSGSNFGASGLP SESASAHKRKGREDSDSRSEDAECEATEETKSSSRRYGSKRRTRAAEVHNLSERRRRDRINEKMRALQELIPHCNKTDKASILDEAIEYLKSLQMQVQIMWMTTGMAPMMFPGAHQFMPPMAVGMNSACMPAAQGLSHMSRLPYMNHSMPNHIPLNSSPAMNPMNVANQMQNIQLREASNPFLHPDGW QTVPPQVSGPYASGPQVAQQNQIPKASASTVLPNSGAEQPPTSDGI

[0076] Example

[0077] Example 1: Functional analysis of the PIL13 gene

[0078] 1. Establishment of transgenic lines

[0079] Recombinant overexpression of OsPIL13 (rice-derived PIL13) was carried out using pCAMBIA as the expression vector and Rbcs2 as the promoter. The recombinant vector with the Rbcs2 promoter introduced is called pRbcs2.

[0080] Using GAGCTTGGTGAGCTGCAGAGATGGATGGCAATGCGAGATC (SEQ ID NO:9) and GCCGGAGCCGCCGCCACCAATTCCATCAGAGGTTGGTGG (SEQ ID NO:10) as primers and rice cDNA as a template, OsPIL13 DNA was amplified and inserted downstream of the Rbcs2 promoter of pRbcS2 to obtain pRbcS2-OsPIL13-FLAG.

[0081] Using Agrobacterium-mediated transformation, pRbcS2-OsPIL13-FLAG was introduced into rice (wild-type variety: Nipponbare (Oryza.Sativa L.spp.japonica), WT) to obtain transgenic plants overexpressing OsPIL13.

[0082] The primer sequences used for gene identification and the primer sequences used for vector construction are shown in Table 1.

[0083] Table 1

[0084]

[0085] Note: The underlined part represents the homologous arm required for homologous recombination.

[0086] 2. Detection of OsPIL13 expression in the strain

[0087] After the T2 generation of transgenic rice overexpressing the above-mentioned transgenic rice was planted in the field, rice seedlings 48 days after transplanting were selected, leaf samples were collected using liquid nitrogen, RNA was extracted, and the RNA level of transcription factors was detected and analyzed using RT-qPCR. OsActin1 was used as an internal reference gene during the detection.

[0088] The primers used in the RT-qPCR experiment are shown in Table 2.

[0089] Table 2

[0090]

[0091] RT-qPCR results showed that the relative expression level of the OsPIL13 gene in the pRbcS2-OsPIL13-FLAG line was 5.4 to 6.7 times that of the wild type. Figure 1 A).

[0092] 3. Detection of OsPIL13 protein levels in the strain

[0093] The inventors used Western blotting to verify the expression of transcription factors at the protein level, using an Anti-FLAG antibody.

[0094] The results showed that the OsPIL13-FLAG fusion protein was successfully detected in the corresponding overexpression rice lines. Figure 1 B).

[0095] 4. Phenotypic Analysis

[0096] Transgenic Plants and Wt.T. Culture: Rice was cultivated and experimented with in the field at the crop cultivation and breeding base (address: No. 931, Yexin Branch Road, Songjiang District, Shanghai, latitude and longitude: 30°56′N, 121°8′E). The rice grown in the field was mainly used to measure leaf photosynthesis, extract leaf RNA and protein, measure biomass, yield, and for seed propagation. Rice cultivation in the field mainly consisted of four steps: germination, sowing and seedling raising, transplanting, and harvesting.

[0097] Germination:

[0098] (1) Pack an appropriate amount (generally twice or more the amount to be planted) of rice seeds into a parchment bag, write the material number on the bag, and put it in a 50℃ oven for 24 hours to break dormancy;

[0099] (2) After breaking dormancy, put the parchment bag into a turnover box, add enough tap water to submerge the parchment bag, and place it in a 35℃ incubator for 48 hours, changing the water once every 24 hours during the process.

[0100] (3) Pour out the water from the turnover box, cover the parchment bag with a wet towel, and place it in a 35℃ incubator until the seeds in the parchment bag germinate into 0.5-1cm sprouts, which usually takes 48-72 hours. During the process, rinse the parchment bag in the turnover box thoroughly with tap water every 12 hours.

[0101] sowing:

[0102] (1) Prepare plastic planting tags in advance and write numbers on them. One planting tag corresponds to each pack of seeds;

[0103] (2) When sowing, first insert the planting sign on the corresponding seedbed, then tear open the seed bag, scatter the seeds evenly on the seedbed, and cover them with soil.

[0104] The process of seeds growing in a seedbed is called seedling raising, and the management during this process is as follows:

[0105] (1) After the seed sprouts about 10cm long, pour enough water into the seedbed to cover the soil layer;

[0106] (2) Apply 3.5 kg / mu of urea about 12 days after seedling raising. During the seedling raising process, spray the corresponding pesticides according to the situation of diseases, pests, weeds, etc. Rice seedlings raised for 25-28 days can be used for transplanting.

[0107] Rice transplanting:

[0108] (1) Apply compound fertilizer evenly in the field 2-3 days before transplanting (dosage: 26.7 kg / mu, compound fertilizer ratio: N:P2O5:K2O=15:15:15), and irrigate the field with water, keeping the water depth at 5-10 cm.

[0109] (2) When transplanting rice seedlings, the row and column spacing of each seedling is 20cm. The plots are directly connected in the column direction without any gaps, and the spacing in the row direction is 60cm. The rice used to measure the daytime dataset was planted in one plot with a total of 140 seedlings, with a layout of 20 rows × 7 columns. When the rice lines overexpressing hub transcription factors were used in the field experiment, each line was planted in 3 plots, with 35 seedlings in each plot, with a layout of 5 rows × 7 columns.

[0110] Post-transplanting management is as follows:

[0111] (1) Apply tillering fertilizer 14 days after transplanting (formula and dosage: urea: 4.3 kg / mu, KH2PO4: 1.9 kg / mu, KCl: 0.6 kg / mu);

[0112] (2) Apply appropriate pesticides according to the conditions of diseases, pests, and weeds;

[0113] (3) After transplanting, irrigate the field as needed. Keep the water depth at 5-10cm for 0-14 days, keep the water depth at about 10-20cm for 15-60 days and above, keep the soil slightly moist for 61-90 days, and stop irrigating after 90 days.

[0114] (4) The Nipponbare rice used has a tillering stage approximately 15-45 days after transplanting, a heading stage 46-60 days after transplanting, and a grain-filling stage 61-90 days after transplanting. Seeds can be harvested after the grain-filling stage. Generally, rice plants are of suitable size and grow vigorously 25-60 days after transplanting, making it suitable for various experiments, including: leaf photosynthesis measurement and sampling for RNA and protein-related molecular biology experiments. Biomass and yield can be measured 90 days after transplanting.

[0115] Takeaways:

[0116] (1) Prepare seed bags (kraft paper bags) in advance and write the seed number and harvest time on them;

[0117] (2) When harvesting, cut off the ears and leaves together and put them into seed bags. Put the seeds of one seedling into one seed bag. Do not mix different plants to prevent seeds of different genes and strains from being mixed together by mistake. When you return to the laboratory, remove the leaves and keep only the ears. Place them in an oven at 40°C and dry for 4-7 days. Check and confirm that the seeds are completely dry before storing them in a seed cabinet.

[0118] Phenotypic analysis was performed 90 days after transplanting.

[0119] The corresponding phenotypic images of the transgenic plants show that the transgenic lines have larger plants. The inventors analyzed the aboveground plant height of rice after the grain-filling stage (90 days after transplanting) to study the effect of overexpression of the OsPIL13 transcription factor on the light energy utilization rate of rice.

[0120] In the pRbcS2-OsPIL13-FLAG strain, the plant height of all three strains showed a significant increase. Figure 2 AB).

[0121] In summary, the transgenic experiments demonstrate that expressing OsPIL13 significantly increases the gene expression level, protein content, and plant height of OsPIL13. Furthermore, this significant increase in plant height has been consistently observed in multiple batches of experiments over several years.

[0122] Example 2: Effects of OsPIL13 transcription factor overexpression on photosynthetic gene expression

[0123] The overexpression rice lines were validated, and the gene expression of all overexpression rice lines and wild-type leaves was detected by transcriptome sequencing.

[0124] The inventors first analyzed photosynthetic genes. After data processing, they used the DESeq2 package in R language to perform differential expression analysis. For each line, genes with a log2 fold change > 0 and a corrected p value < 0.05 were defined as significantly upregulated genes, and genes with a log2 fold change < 0 and a corrected p value < 0.05 were defined as significantly downregulated genes.

[0125] In the pRbcS2-OsPIL13-FLAG line, the expression of a large number of photosynthetic genes was significantly upregulated.

[0126] To verify the accuracy of the gene regulatory network, we compared the target genes of pivot transcription factors in the integrated gene regulatory network of rice leaf photosynthesis with the photosynthetic genes whose expression levels were significantly increased in rice lines that overexpressed the corresponding transcription factors.

[0127] Table 3 shows a comparison between the gene regulatory network prediction results and the upregulated photosynthetic genes in OsPIL13 transcription factor overexpression lines.

[0128] Table 3

[0129]

[0130] Because transcriptome sequencing was performed on three overexpressing lines for each transcription factor, to merge the differentially expressed genes from the three transcriptome sequencing results, here we define genes as follows: genes significantly upregulated in all three lines, or significantly upregulated in two lines but not significantly changed in the third line, are considered upregulated genes; genes significantly downregulated in all three lines, or significantly downregulated in two lines but not significantly changed in the third line, are considered downregulated genes; all other cases are considered as having no change in gene expression. Since the target genes were restricted to photosynthetic genes when constructing the gene regulatory network, the two can be directly compared.

[0131] The inventors' analysis showed that, according to the gene regulatory network, 50% of the target genes of the other three transcription factors were significantly overexpressed in the corresponding transcription factor overexpression lines (Table 4).

[0132] Table 4. Differentially expressed photosynthetic genes in OsPIL13 transcription factor overexpression lines

[0133]

[0134]

[0135]

[0136] Note: Transcriptome sequencing was performed on three overexpressing lines for each transcription factor. To merge differentially expressed genes from the three transcriptome sequencing results, each line was first differentially expressed compared to the wild type. Then, for each gene in the three overexpressing rice lines of one transcription factor, it was defined as follows: significantly upregulated in all three lines, or significantly upregulated in two lines but not significantly changed in the third line, was considered an upregulated gene; significantly downregulated in all three lines, or significantly downregulated in two lines but not significantly changed in the third line, was considered a downregulated gene; all other cases were considered as no change in gene expression. In the table, ↑ indicates upregulated genes, ↓ indicates downregulated genes, - indicates no change in gene expression, and (T) indicates the gene's expression level in the integrated rice leaf photosynthetic gene regulatory network. Figure 3 The target genes of this transcription factor are shown in the diagram.

[0137] Example 3: Effects of OsPIL13 transcription factor overexpression on photosynthetic rate in rice leaves

[0138] To investigate the effect of OsPIL13 transcription factor overexpression on leaf photosynthetic rate, the inventors analyzed the changes in leaf photosynthetic rate under saturated photosynthesis in all overexpressing rice lines, with wild-type lines as controls.

[0139] Determination of leaf photosynthetic rate: The photosynthetic rate (A) of rice leaves under saturated light was measured using a portable photosynthesis meter. satThe instrument control parameters before measurement are as follows: CO2 concentration is 400 μmol. -1 The light intensity inside the leaf chamber is 2000 or 1500 μmol m. - 2 s -1 Leaf temperature was determined based on weather forecasts, with the highest temperature recorded for the specified period for each data set. During measurement, the newest fully expanded leaves of the rice plant were selected, and the leaf tip (approximately one-third of its length) was placed in the leaf chamber. The leaves were allowed to acclimatize in the chamber for a period before the photosynthetic rate was recorded. For photosynthetic rate measurements taken during daytime data collection, the acclimatization time was 10 minutes; for measurements of overexpressing hub transcription factors in rice lines, the acclimatization time was 2 minutes. Measurements were taken between 9:00 AM and 1:00 PM daily.

[0140] The results showed that in 2022 (2000 μmol m -2 s -1 ) and 2024 (1500μmol m -2 s -1 The leaf photosynthetic rates of the three rice lines pRbcS2-OsPIL13-FLAG were significantly higher than those of the wild type. Figure 4 ).

[0141] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of a hub transcription factor, PIL13, as a regulatory target in grasses to promote photosynthesis and increase plant height, or to prepare plants with improved photosynthesis and plant height; wherein, The regulation mentioned refers to increasing the expression level or activity of the hub transcription factor PIL13.

2. The application as described in claim 1, characterized in that, The promotion of photosynthesis includes: Increase leaf photosynthetic rate; and / or Increase the expression of photosynthetic genes.

3. The application as described in claim 1, characterized in that, The photosynthetic genes include: Lhca6, Os03g0279950_PsbP, FNR, CYP20-2 or Os02g0744000.

4. The application as described in claim 1, characterized in that, Utilizing regulatory molecules that increase the expression level or activity of hub transcription factor PIL13 to promote photosynthesis and increase plant height; Preferably, the regulatory molecule comprises: the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13, and an expression construct containing a hub transcription factor PIL13 expression cassette; preferably, the expression cassette further comprises a promoter and / or a terminator; preferably, the regulatory molecule is exogenously introduced into the plant; or In the breeding of grass plants, plants with high expression levels or activity of the hub transcription factor PIL13 are selected as preferred plants for cultivation to obtain superior plants with strong photosynthesis and increased plant height; preferably, the traits of the superior plants are passed on to offspring plants.

5. The application as described in claim 1, characterized in that, The hub transcription factor PIL13 is selected from the group consisting of: (a) a protein with the amino acid sequence shown in SEQ ID NO:2; (b) a derivative or active fragment having the function of the protein with a sequence that is ≥85% homology to the amino acid sequence shown in SEQ ID NO:2 (such as ≥88%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98%, or ≥99% homology); and (c) a derivative or active fragment having the function of the protein with the amino acid sequence shown in (a) by substitution, deletion, or addition of one or more amino acid residues.

6. A method for promoting photosynthesis and increasing plant height in grasses, comprising: (a) Using hub transcription factor PIL13 as a regulatory target, increase the expression level or activity of this transcription factor in grasses; or (b) In the breeding of grass plants, plants with high expression levels or activity of hub transcription factor PIL13 are screened as preferred plants for cultivation to obtain superior plants with strong photosynthesis and increased plant height.

7. The method as described in claim 6, characterized in that, In (a), the regulatory molecule used in the method includes: the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13, and an expression construct containing a hub transcription factor PIL13 expression cassette; preferably, the expression cassette further includes a promoter and / or a terminator; preferably, the regulatory molecule is exogenously introduced from the plant.

8. The method as described in claim 6, characterized in that, The promotion of photosynthesis includes: increasing the photosynthetic rate of leaves and / or increasing the expression of photosynthetic genes; preferably, the photosynthetic genes include: Lhca6, Os03g0279950_PsbP, FNR, CYP20-2 or Os02g0744000.

9. Use of hub transcription factor PIL13 for analyzing photosynthetic traits, plant height traits, and / or leaf traits in grasses; preferably, the analysis includes early analysis, such as analysis at the seed or seedling stage of grasses; Preferably, when analyzing the traits of grass plants, the hub transcription factor PIL13 in the plant is analyzed. If the expression of hub transcription factor PIL13 is significantly high, then the grass plant has optimized traits. The optimized traits include: enhanced photosynthesis and increased plant height; preferably, enhanced photosynthesis includes: increased leaf photosynthetic rate and / or increased expression of photosynthetic genes.

10. The use as described in claim 9, characterized in that, The expression level of hub transcription factor PIL13 was detected using primers with the sequences shown in SEQ ID NO:7 and SEQ ID NO:

8.

11. The application or method as described in any one of claims 1 to 10, characterized in that, The grasses mentioned are grasses that have a photosynthetic mechanism and express hub transcription factor PIL13 or its homologs.

12. A cell, tissue, or organ of a grass plant, including increasing the expression level or activity of hub transcription factor PIL13 as a regulatory target; preferably, the regulation is implemented using the encoding nucleic acid of hub transcription factor PIL13, the genomic nucleic acid of hub transcription factor PIL13, or an expression construct containing a hub transcription factor PIL13 expression cassette.

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