Application of phb9 gene in regulating rice leaf morphology

Abstract

The application discloses application of a PHB9 gene in regulating rice leaf morphology and belongs to the technical field of genetic engineering.The CDS sequence of the PHB9 gene is shown as SEQ ID NO.2.It is found through experiments that overexpression of the PHB9 gene increases leaf length and leaf width, and the PHB9 gene has a significant effect on regulating rice leaf morphology.Further, changes in vascular systems, mitochondria and chloroplast structures in the internal structure of the leaf are analyzed, and it is shown that the vascular systems, mitochondria and chloroplast structures change when the leaf morphology is regulated, and the morphological changes in the internal structure of the leaf affect the changes in the leaf phenotype, and have a significant influence on improving the photosynthetic rate of the leaf.It is also found that overexpression of the PHB9 gene reduces the sensitivity of rice seeds to ABA, promotes seed germination and root growth.The application provides a new gene for breeding of leaf morphology building, root development and seed germination.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the PHB9 gene in regulating rice leaf morphology. Background Technology

[0002] Rice plant architecture and leaf type are core agronomic traits that determine photosynthetic efficiency, nutrient allocation, and final yield. According to the classic source-sink theory, crop yield formation depends on a coordinated balance between the carbon assimilation capacity of the photosynthetic source (leaves) and the storage capacity of the grain sink (panicle). However, current research still has significant shortcomings in understanding the regulatory network of rice plant and leaf type and the source-sink interaction mechanism: Insufficient exploration of gene resources: Although key genes such as IPA1 / WFP (ideal plant type), D53 (tillering angle), and NAL1 (leaf width) have been cloned, the genetic network regulating plant height, tillering angle, leaf angle, leaf curling degree, and ear configuration has not been fully elucidated. In particular, pleiotropic genes that synergistically regulate the development of source organs (leaves) and the filling of sink organs (grains) are still scarce.

[0003] The source-sink synergistic regulation mechanism is unclear: most studies only focus on improving a single trait (such as simply increasing the number of tillers or enlarging the ear type), neglecting the dynamic balance of source-sink flow. For example, excessively increasing tillers may lead to canopy shading and decreased photosynthetic efficiency; simply increasing the sink capacity without corresponding source supply support will lead to poor grain filling and increased chalkiness.

[0004] Molecular design breeding targets are limited: current breeding methods mainly rely on QTL aggregation and lack precise design based on molecular mechanisms. In particular, key node genes that connect to plant architecture and plasticity regulators that dynamically optimize source-liquid allocation in response to the environment urgently need to be explored in depth.

[0005] Therefore, based on the above issues, it is of great significance to discover new genes that regulate the development of rice plant architecture and leaf type. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the PHB9 gene in regulating rice leaf morphology, so as to solve the problems existing in the prior art. Overexpression of the PHB9 gene can increase the width and length of rice leaves and promote leaf morphogenesis.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; The CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0008] This invention also provides the use of the protein encoded by the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; The CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0009] The present invention also provides the use of recombinant vectors containing the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; The recombinant vector is constructed by connecting the PHB9 gene with an expression vector, and the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0010] The present invention also provides the use of recombinant bacteria containing the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; The method for constructing the recombinant bacteria includes: ligating the PHB9 gene to an expression vector to construct a recombinant vector, and then transferring the recombinant vector into a host bacterium to obtain the recombinant bacteria; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0011] Preferably, the regulation of rice leaf morphology is performed by overexpressing the PHB9 gene to increase leaf width and leaf length, and knocking out the PHB9 gene to decrease leaf width and leaf length.

[0012] Preferably, the regulation of rice leaf photosynthetic efficiency is achieved by overexpressing the PHB9 gene to increase stomatal conductance, intercellular CO2 concentration, and / or transpiration rate of rice leaves, thereby improving the photosynthetic efficiency of rice leaves.

[0013] Preferably, overexpressing the PHB9 gene reduces the sensitivity of rice seeds to ABA, thereby promoting seed germination.

[0014] The present invention also provides a method for regulating rice leaf morphology, comprising (1) overexpressing the PHB9 gene in rice to increase leaf width and leaf length, or (2) knocking out the PHB9 gene in rice to decrease leaf width and leaf length; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0015] The present invention also provides a method for improving the photosynthetic efficiency of rice leaves, comprising overexpressing the PHB9 gene in rice to increase the stomatal conductance, intercellular CO2 concentration and / or transpiration rate of rice leaves, thereby improving the photosynthetic efficiency of rice leaves; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0016] The present invention also provides a method for reducing the sensitivity of rice seeds to ABA, comprising overexpressing the PHB9 gene in rice to reduce the sensitivity of rice seeds to ABA; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

[0017] The present invention discloses the following technical effects: This invention, through overexpression or knockout of the PHB9 gene in rice, found that compared to wild-type rice NGZ, PHB9 gene overexpression lines showed significantly increased leaf width and length, while PHB9 gene knockout lines showed significantly decreased leaf width and length, indicating that the PHB9 gene plays a significant role in regulating leaf morphology. The net photosynthetic rate and chlorophyll content of leaves in PHB-overexpression lines did not change significantly, but stomatal conductance, intercellular CO2 concentration, and transpiration rate all increased significantly, indicating that PHB9 gene overexpression can improve the photosynthetic efficiency of rice leaves. Furthermore, the thylakoid stacking in the chloroplasts of leaves in PHB-overexpression lines was more loose, indicating that PHB9 can also affect photosynthetic efficiency by influencing chloroplast morphology in rice leaves. This invention also found that PHB9 gene overexpression reduces rice's sensitivity to ABA and promotes seed germination and root development. This invention provides new genes for rice leaf shape, seed germination, and root development. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Phylogenetic analysis diagram of PHB9; Figure 2 The purpose of this study was to analyze the expression pattern of PHB9 and identify transgenic plants. Among them, (A) PHB9 gene expression map; (B) qRT-PCR analysis of PHB9 gene expression in different parts of Osmanthus fragrans during the flowering period; (C) comparative analysis of sequencing results of PHB9 gene-edited lines; (D) detection of PHB9 gene overexpression lines. Figure 3 Phenotypic observation and statistics of leaves of PHB9 transgenic plants; (A) Leaf morphology of wild-type NGZ (left), mutant phb9 (middle), and PHB9 overexpression line (right) during the grain-filling stage, bar=5cm; (B) Statistics on the length of flag leaves of plants during the grain-filling stage; (C) Statistics on the width of flag leaves of plants during the grain-filling stage, n=20; (D) Freehand sections of flag leaves of wild-type NGZ (top), phb9 mutant (middle), and PHB9 overexpression line (bottom) during the flowering stage, with arrows indicating large veins, bar=1mm; (E) Statistics on the number of large veins on one side; n=20; Figure 4 Paraffin sections of leaves from wild-type and PHB9 lines were prepared. (A) Midrib cross-section of wild-type NGZ leaf (top), large vascular bundle cross-section of wild-type NGZ leaf (middle), and bottom cross-section of wild-type NGZ leaf; (B) Midrib cross-section of phb9 mutant leaf (top), large vascular bundle cross-section of phb9 mutant leaf (middle), and bottom cross-section of phb9 mutant leaf; (C) Midrib cross-section of PHB9 overexpression line leaf (top), large vascular bundle cross-section of PHB9 overexpression line leaf (middle), and bottom cross-section of PHB9 overexpression line leaf; VBS, vascular bundle sheath; PC, parenchyma cells; PH, phloem; M, mesophyll cells; X, xylem; BC, vesicular cells; LV, large vascular bundle; SV, small vascular bundle, bar=200μm; Figure 5 The photosynthetic characteristics of wild-type and PHB9 strains are shown; among them, (A) net photosynthetic rate; (B) stomatal conductance; (C) intercellular CO2 concentration; (D) transpiration rate; (E) chlorophyll content; Chl a, chlorophyll a; Chl b, chlorophyll b; Chl, total chlorophyll; n=20; Figure 6Transmission electron microscopy observations of leaves from wild-type and PHB9 lines; (A) Chloroplast morphology of wild-type NGZ leaves (top), internal structure of chloroplasts of wild-type NGZ leaves (middle), mitochondrial morphology of wild-type NGZ leaves (bottom); (B) Chloroplast morphology of phb9 mutant leaves (top), internal structure of chloroplasts of phb9 mutant leaves (middle), mitochondrial morphology of phb9 mutant leaves (bottom); (C) Chloroplast morphology of PHB9 overexpression lines (top), internal structure of chloroplasts of PHB9 overexpression lines (middle), mitochondrial morphology of PHB9 overexpression lines (bottom); Cp, chloroplast; Thy, thylakoid; OG, glutathione granules; SG, starch granules; Mt, mitochondria; bar=500nm; Figure 7 The plant type and panicle agronomic traits of wild-type and PHB9 transgenic plants were observed and statistically analyzed. Among them, (A) plant morphology of wild-type NGZ (left), phb9 mutant (middle), and PHB9 overexpression line (right) at the grain-filling stage, bar=20cm; (B) plant height statistics at the grain-filling stage; (C) effective tiller number statistics; n=20; (D) panicle morphology of wild-type NGZ (left), mutant phb9 (middle), and PHB9 overexpression line (right), bar=5cm; (E) grain morphology of mature wild-type NGZ (top), phb9 mutant (middle), and PHB9 overexpression line (bottom), bar=2cm; (F) panicle length statistics, n=20; (G) number of filled grains statistics; (H) grain length statistics; (I) grain width statistics; (J) thousand-grain weight statistics; n=200; Figure 8 The results of root observation during germination of wild-type and PHB9 transgenic lines are shown; (A) Root phenotypes during germination of wild-type NGZ (left), phb9 mutant (middle), and PHB9 overexpression line (right); (B) Statistical analysis of root length during germination of each line; n=30; Figure 9Phenotypic results of wild-type NGZ, phb9 mutant lines and PHB9 overexpression lines under ABA treatment; (A) germination of seeds of wild-type NGZ, phb9 mutant lines and overexpression lines after 5 days of treatment with 0, 2 and 5 µM ABA; (B) germination rate of seeds of each line after 5 days of treatment with 0, 2 and 5 µM ABA, n=3; (C) growth of seeds of wild-type NGZ, phb9 mutant lines and PHB9 overexpression lines after seed germination on 1 / 2 MS medium with 0, 2 and 5 µM ABA added for 5 days, bar=4 cm; (D) plant height of wild-type NGZ, phb9 mutant lines and PHB9 overexpression lines after treatment with 0, 2 and 5 µM ABA, n=10; (E) root length of wild-type NGZ, phb9 mutant lines and PHB9 overexpression lines after treatment with 0, 2 and 5 µM ABA, n=10. Figure 10 Transcriptome data of roots from PHB9 overexpressing lines during germination; (A) Statistical analysis of the number of significantly different genes between wild-type NGZ and PHB9 overexpressing lines; (B) Volcano plot of differential expression in transcriptome; (C) KEGG pathway classification statistics of differential transcripts in PHB9 overexpressing lines; (D) GO functional classification statistics of differential transcripts in PHB9 overexpressing lines. Figure 11 The following are heatmaps of differentially expressed genes in PHB9 overexpression lines: (A) Heatmap of differentially expressed genes related to brassinosteroids, ethylene, jasmonic acid, salicylic acid, cytokinins and gibberellins; (B) Heatmap of differentially expressed genes related to abscisic acid; (C) Heatmap of genes related to salt stress; (D) Heatmap of differentially expressed genes related to root development; (E) Heatmap of genes related to high temperature and drought stress. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 1. Conservation analysis of the PHB9 protein sequence Based on phylogenetic analysis, this invention selected typical model organisms of dicotyledonous and monocotyledonous plant groups, such as Arabidopsis thaliana, soybean (Glycine max), wheat (Triticum aestivum), and maize (Zea mays), as well as members of the PHB family from major food crops, for homology comparison to assess the sequence conservation and functional differentiation patterns of this gene family during plant evolution. The phylogenetic tree shows that PHB proteins can be divided into two major subfamilies: Type I and Type II. Figure 1 This is consistent with the classification framework reported in previous studies. Cluster analysis further indicates that PHB9 belongs to the Type II subfamily.

[0026] 2. Analysis of PHB9 expression pattern To analyze the expression characteristics of the PHB9 gene in different rice tissues, expression profiles were first predicted using the RiceVarMap website. The results showed that ( Figure 2 (A) The PHB9 gene is highly expressed in the roots, plumules, mature inflorescences, and callus tissue during the germination period.

[0027] Subsequently, using wild-type NGZ at flowering stage as material, root, stem, leaf, root-stem transition zone, and floral organ tissues were collected. Total RNA was extracted using the TRIzol method and reverse transcribed into cDNA. The expression pattern was quantitatively analyzed using qRT-PCR technology (with OsActin1 as an internal reference gene). Figure 2 (B). The qRT-PCR primers are: Internal reference primer sequence: OsActin1-F: GCATCTCTCAGCCATTGCA (SEQ ID NO.4); OsActin1-R: ACCACAGGTAGCATAGGTA (SEQ ID NO. 5).

[0028] PHB9 primer sequence: PHB9-F: GAAGCCCTTCGTCTTCGACA (SEQ ID NO. 6); PHB9-R: CCTTGTCGTCGTACTCGAGG (SEQ ID NO. 7).

[0029] The qRT-PCR reaction system is as follows: 2 μL cDNA (diluted 5 times), 5 μL 2×ChamQ Universal SYBR qPCRMaster Mix, 0.25 μL Primer-F, 0.25 μL Primer-R, and 2.5 μL ddH2O.

[0030] The reaction program was: 95℃, 3 min; (90℃, 15 s; 60℃, 30 s; 72℃, 30 s), 40 cycles.

[0031] The results showed that PHB9 was constitutively expressed in all tested tissues.

[0032] 3. Identification of PHB9 gene-edited lines and overexpression lines 3.1 Identification of PHB9 gene-edited lines To further investigate the effects of PHB9 on rice leaf morphology, a CRISPR / Cas9 gene knockout target was designed in the PHB9 coding region. The constructed knockout vector was transformed into callus tissue of the background material Nanguizhan (NGZ) to obtain a positive transgenic knockout mutant. The specific steps are as follows: (1) Target design To further investigate the effects of PHB9 on rice leaf morphology, a CRISPR / Cas9 gene knockout target was designed in the PHB9 coding region. The knockout target sequence is as follows: OsPHB9-OsU6aT1F: gccgGCGCTCTCTCGTCTTCCGG (SEQ ID NO. 8); OsPHB9-OsU6aT1R:aaacCCGGAAGACGAGAGAGCGC (SEQ ID NO.9); OsPHB9-OsU3T2F: ggcaGAGCCACGGCACGAGGAAGT (SEQ ID NO. 10); OsPHB9-OsU3T2R:aaacACTTCCTCGTGCCGTGGCTC (SEQ ID NO. 11).

[0033] (2) Amplification of the target gene First, a high-fidelity enzyme was used to perform PCR to amplify the target gene. The PCR reaction system consisted of: 2 μL 10× buffer, 2 μL 2mM dNTPs, 2 μL DNA template, 1 μL DMSO, 0.8 μL 10pmol / L upstream / downstream primers, 0.5 U KOD DNA polymerase (KOD-401, TOYOBO), and water added to a final volume of 20 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50–64℃ annealing for 30 s, 68℃ extension for 1 min / kb, 40 cycles; and 68℃ incubation for 8 min.

[0034] (3) Electrophoresis and recycling Add an appropriate amount of 10× loading buffer to each reaction tube and perform electrophoresis on an agarose gel containing 1%–3% (10 μL EB, 1–3 g agarose / 100 mL 0.5× TBE buffer). Electrophoresis is then performed in 0.5× TBE buffer at 5–10 V / cm. After electrophoresis, images are taken using a gel imaging system.

[0035] DNA fragments were recovered from PCR amplification using agarose gel (GK2042, Jereh Biotechnology).

[0036] (4) Plasmid digestion 40 μL restriction enzyme digestion system: plasmid (30 μL), 4 μL 10× digestion buffer, 4 μL 10× BSA (add or omit according to the instructions), 6 U restriction endonuclease (NEB), add water to make up to 40 μL, and incubate in a 37℃ water bath for about 1 h.

[0037] (5) Plasmid construction steps After the PCR fragment was recovered by agarose gel, it was mixed with the enzyme-digested empty vector and ligated into the EasyGenoDNA recombination system (# VI201-02, Tiangen Biotech). The 10 μL recombination system consisted of 5 μL 2×EasyGeno Assembly Mix, 2.5 μL enzyme-digested vector DNA, and 2.5 μL amplified fragment DNA.

[0038] The reaction mixture was added to a 250 μL EP tube, treated in a 50°C water bath for 30 minutes, transformed into E. coli, plated, and incubated at 37°C for 16 hours. After that, bacteria were picked and sent for sequencing. If the sequencing was correct, it was used for subsequent plasmid extraction.

[0039] (6) Small-scale extraction of coli plasmid DNA using conventional alkaline method Inoculate a single colony into 3 mL of LB broth containing 3 μL of antibiotic and incubate overnight at 37°C with shaking at 200 rpm. Take 1.5 mL of the culture (3 mL of low-copy plasmid), centrifuge at 12,000 rpm for 30 seconds. Aspirate the supernatant and resuspend the cells in 100 μL of Solution I (50 mmol / L glucose, 10 mmol / L EDTA, 25 mmol / L Tris-HCl, pH 8.0). Add 200 μL of freshly prepared Solution II (0.2 mol / L NaOH, 1% SDS) and immediately mix gently up and down. Add 150 μL of Solution III (5 mol / L KAc, pH 4.8), mix quickly up and down, and incubate at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes. Transfer the supernatant to another centrifuge tube, add twice the volume of ethanol, mix well, and centrifuge at 12,000 rpm for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes; remove supernatant, wash DNA precipitate with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, remove supernatant; vacuum dry precipitate; dissolve in 60 μL of double-distilled water containing 10 g / mL RNase A.

[0040] The extracted plasmids were stored at -20℃ for a long period of time.

[0041] (7) Genetic transformation of rice S1: Induction Select rice grains without mold spots and with normal sprouts. Disinfect with 75% alcohol for 1 min, rinse with sterile water for 1 min each time; disinfect with 15% sodium hypochlorite for 20 min, rinse with sterile water 3 times for 1 min each time; inoculate the disinfected rice grains into induction medium (purchased from Boyuan Yanxuan #RFB01) and culture at 26℃ under light for 20 days.

[0042] S2: Agrobacterium infection Agrobacterium was picked into the infection solution to prepare OD. 600 For the callus resuspension at 0.2 g / L, pick up the callus tissue into an Erlenmeyer flask, add the Agrobacterium resuspension, infect for 10-15 min and discard the bacterial solution. Inoculate the callus tissue into co-culture medium (purchased from Boyuan Yanxuan #RFC01) and co-culture at 20℃ for 48-72 h.

[0043] S3: Callus Screening Inoculate the callus tissue from S2 into the selection medium (Boyuan Yanxuan #RFA02) and incubate in the dark at 26℃ for 20-30 days; inoculate the positive callus tissue into the secondary selection medium, and be sure to select single-clonal callus tissue during the callus tissue selection process, and incubate in the dark at 26℃ for 7-10 days. S4: Differentiation and Rooting Positive callus tissue was inoculated into differentiation medium (Boyuan Yanxuan #RFB01, #RJN00) and cultured at 25-27℃ under light for 15-20 days. After the 2-5cm shoots differentiated, they were inoculated into rooting medium (Boyuan Yanxuan #RFB01, #RFR06) and cultured at 30℃ under light for 7-10 days. S5: Positive vaccine detection Rice genomic DNA was extracted using the CTAB method and then detected by PCR, using the same method as for Agrobacterium tumefaciens detection.

[0044] After sequencing, such as Figure 2 In the C region, compared to the wild-type NGZ, the PHB9 mutant lacks two bases in the cds region, causing premature termination of protein sequence translation.

[0045] 3.2 Identification of overexpression lines Simultaneously, this invention utilizes the plasmid vector pCAMBIA-1306-5xflag, which contains a strong maize Ubi promoter, to construct an overexpression vector for the PHB9 gene. The methods for cloning the gene and introducing the vector are similar to those for knockout vectors. The overexpression vector was transformed into rice by Boyuan Biotechnology Co., Ltd., and then transferred into NGZ to obtain overexpressing plants. Subsequently, specific primers for the marker gene were used for screening, and the expression level of the PHB9 gene in these plants was measured. The specific primers are as follows: OsPHB9-OE-F: AAAACTGCAGATGGCCGGCGGTCCCGCGGC (SEQ ID NO. 12); OsPHB-OE-R: TTTTGAGCTCATTCAGCAAGGCAACATCAG (SEQ ID NO. 13).

[0046] like Figure 2 As shown in Figure D, the results indicate that two PHB9 overexpression-positive plants (OE-1 and OE-2) were selected and can be used for subsequent research.

[0047] Example 2: Leaf phenotype study of PHB9 gene-edited lines To elucidate the effects of PHB9 gene loss of function and overexpression on rice leaf morphology, this invention systematically observed the length of the third leaf from the top and the maximum leaf width during the grain-filling stage in wild-type (NGZ) and PHB9 gene knockout mutant (phb9 mutant) and overexpression mutant lines (PHB9 overexpression lines). Figure 3 As shown in the AC diagram, compared with the wild-type NGZ, the phb9 mutant had a significantly reduced leaf length of about 7.5% (P<0.001) and a significantly reduced leaf width of about 7% (P<0.001); the PHB9 overexpression lines had a significantly increased leaf length of about 17.5% (P<0.001) and a significantly increased leaf width of about 8% (P<0.001) compared with the wild-type NGZ, demonstrating its significant role in regulating rice leaf morphology.

[0048] Meanwhile, in order to explore the potential mechanism of leaf morphological changes in different PHB9 lines, this invention used the freehand sectioning method to perform anatomical analysis on the mature flag leaves of each line. The results showed that PHB9 significantly affected the development of the leaf vascular system. Figure 3 (D).

[0049] Figure 3 As shown in DE, the microscopic observation results revealed ( Figure 3 In the phb9 mutant line (DE), the number of large veins on one side of the leaves was not significantly different from that of the wild-type NGZ (both were 4). The decrease in leaf width was not related to the number of large veins, but was due to the shortening of the distance between two large veins. However, the number of large veins on one side of the leaves of the PHB9 overexpression line increased to 6 compared to the 4 in the wild-type NGZ. The increase in the number of large veins was significantly positively correlated with the PHB9 expression level (P<0.001).

[0050] Example 3: Cellular mechanism of PHB9 regulating vascular development in rice leaves Based on the above anatomical observations, in order to further elucidate the cellular mechanism by which PHB9 regulates the vascular development of rice leaves, this invention uses paraffin sectioning technology to analyze the cellular structure of the midrib, large vascular bundles and small vascular bundles of the leaves of various transgenic lines.

[0051] like Figure 4 As shown, the results indicate that the phb9 mutant lines exhibited phenotypes with smaller midribs, smaller bubble-like cells and mesophyll cells, and abnormal xylem development compared to the wild-type NGZ, but the number of small vascular bundles remained unchanged compared to the wild-type. Meanwhile, the PHB9 overexpression lines showed phenotypes with larger midrib areas, larger xylem, bubble-like cells, and mesophyll cells, while the number and size of small vascular bundles remained unchanged compared to the wild-type. This suggests that PHB9 affects rice leaf development by regulating vascular bundle morphogenesis and mesophyll cell differentiation.

[0052] Example 4: Effects of PHB9 on photosynthetic efficiency of rice leaves Changes in leaf morphology are often accompanied by changes in photosynthetic efficiency. To systematically evaluate the impact of PHB9-induced changes in leaf morphology on leaf photosynthetic function, this invention further measured the chlorophyll content and photosynthetic parameters of each genotype using ultraviolet spectrophotometry and a LI-6400XT photosynthesis system.

[0053] like Figure 5 As shown, the results indicate that, compared with wild-type NGZ, the net photosynthetic rate and chlorophyll content of leaves in the PHB9 overexpression lines did not change significantly, but stomatal conductance, intercellular CO2 concentration and transpiration rate all increased significantly.

[0054] Example 5: PHB9 affects the development of chloroplasts and mitochondria in rice leaves. Chloroplast structural integrity is a core factor determining plant photosynthetic efficiency. To elucidate the cellular basis of the aforementioned changes in photosynthetic parameters, this invention conducted ultrastructural observations of leaf tissues from wild-type and transgenic lines using transmission electron microscopy, focusing on the morphological characteristics of the chloroplast membrane system (thylakoid stacking and grana lamellar arrangement).

[0055] like Figure 6 As shown, the results indicate that, compared with wild-type NGZ, the thylakoid stacking of chloroplasts in the leaves of PHB9-overexpressing lines was more loose, starch granules were significantly larger, and mitochondrial morphology remained largely unchanged. This suggests that PHB9 may affect photosynthetic efficiency by influencing the morphology of chloroplasts and mitochondria in rice leaves.

[0056] Example 6: PHB9 affects rice root development Experimental results showed that PHB9 had no significant effect on root development during rice germination. Figure 8 There was no significant difference in root length between the phb9 mutant lines, overexpression lines, and wild-type NGZ within 96 hours of germination.

[0057] Example 7: The effect of PHB9 on the sensitivity of rice to ABA To investigate the biological function of PHB9 in rice seed germination, wild-type NGZ, phb9 mutant lines, and overexpression lines were used as materials. Exogenous ABA was applied at doses of 0 μM, 2 μM, and 5 μM (30 seeds per treatment, 3 biological replicates), and the differences in germination response were systematically evaluated. Figure 9The results showed that (1) there was no significant difference in seed germination rate among different genotypes of PHB9 under no ABA treatment (96.67% ± 3.3%); (2) under 2 μM ABA treatment, the seed germination rate of PHB9-OE (96.67% ± 3.3%) was significantly higher than that of wild-type NGZ (90% ± 3.3%, P<0.01), while the germination rate of phb9 mutant was not significantly different from that of NGZ (P>0.05); (3) under 5 μM ABA treatment, the germination rate of wild-type NGZ and phb9 mutant decreased sharply to 26.67% ± 3.3%, while PHB9-OE maintained a high germination rate (66.67% ± 3.3%). The results indicate that PHB9 overexpression significantly reduces the sensitivity of rice seeds to ABA, while loss of function of this gene enhances the ABA-mediated germination inhibition effect.

[0058] Meanwhile, to further investigate the relationship between PHB9 expression and exogenous ABA, seeds of each strain were germinated on 1 / 2 MS medium for 2 days, then transferred to 1 / 2 MS medium containing different concentrations of ABA for 5 days to assess seedling growth. Figure 9 (CE). The results showed that: (1) In the absence of ABA treatment, the differences in seedling height and root length among the PHB9 lines changed from no significant difference at 96 h of germination. The seedling height and root length of the phb9 mutant lines were significantly lower than those of NGZ (the seedling height of the phb9 mutant lines was 138 mm, and that of the NGZ lines was 160 mm; the root length of the phb9 mutant lines was 28 mm, and that of the NGZ lines was 33 mm); the seedling height and root length of the PHB9-OE mutant lines were significantly higher than those of NGZ (the seedling height of the phb9 mutant lines was 187 mm, and that of the NGZ lines was 160 mm; the root length of the phb9 mutant lines was 41 mm, and that of the NGZ lines was 33 mm). (2) When treated with 2 μMABA, the seedling height and root length of each line decreased sharply, and the difference between each transgenic line and the wild type was significantly reduced. The seedling height of the phb9 mutant line was significantly lower than that of the NGZ line (40 mm for phb9 mutant line and 25 mm for NGZ line), while the root length showed no significant difference. The root length of the PHB9-OE mutant line was significantly higher than that of the NGZ line (23 mm for PHB9-OE mutant line and 15 mm for NGZ line), while the seedling height showed no significant difference. (3) When treated with 5 μM ABA, there was almost no significant difference between the transgenic lines and NGZ. Only the seedling height of the PHB9-OE mutant line was significantly higher than that of the NGZ line (15 mm for PHB9-OE mutant line and 10 mm for NGZ line); indicating that PHB9 positively regulates root development during rice germination and negatively regulates the sensitivity of rice seeds to ABA.

[0059] Example 8 Transcriptome Analysis This invention used roots of wild-type rice and PHB9 overexpressing lines at 3 days of germination as materials for transcriptome RNA-seq analysis. Each treatment had three replicates, and each replicate consisted of a mixture of 10 individual seedlings. To compare gene expression differences among different samples, |Fold Change|≥2 and FDR<0.01 were used as the screening criteria for differentially expressed genes (DEGs), resulting in a large number of differentially expressed genes. Figure 10 As shown, compared with wild-type NGZ, the PHB9 overexpression line yielded a total of 3194 DEGs, of which 1113 were upregulated and 2081 were downregulated. The enrichment of DEGs was mainly concentrated in cellular anatomical entities, metabolic processes, cellular processes, biological regulation, protein binding, catalytic activity, and transporter activity. The KEGG enrichment pathways were concentrated in the MAPK signaling pathway, phenylpropanoid biosynthesis, carbon metabolism, amino acid biosynthesis, starch and sucrose metabolism, glycolysis / gluconeogenesis, and plant hormone signal transduction.

[0060] To further understand the regulatory network of PHB9 genes, this invention, based on the phenotype of PHB9 gene mutant lines and combined with data on differentially expressed genes in the transcriptome, found that most genes jointly regulated by the three mechanisms are related to root development, plant hormone signal transduction, and abiotic stress response. Figure 11Genes regulating root growth and development showed synergistic upregulation, while genes related to plant hormone biosynthesis, metabolic pathways, and stress responses (including genes directly regulating rice resistance to salt and drought stress) showed a systematic downregulation trend. Overexpression of PHB9 significantly upregulated the expression levels of root development-related genes such as OsCSLD1, OsRSL5, OsRSL9, and OsRHL1, thereby inducing synergistic growth of root length and plant height, ultimately accelerating the overall growth and development of the plant. Simultaneously, the expression profiles of genes related to high temperature, salt stress, and drought resistance showed systematic downregulation, and these genes were synchronously suppressed with key nodes in the stress resistance regulatory network mediated by plant hormones (such as abscisic acid, jasmonic acid, and gibberellin). This suggests that PHB8 may play a negative regulatory role in rice's abiotic stress response.

[0061] The PHB9 gene (LOC_Os04g38900) and its protein sequence involved in this invention are as follows: (1) PHB9 protein sequence (SEQ ID NO.1): >LOC_Os04g38900.1 MAGGPAAVSFLTNIAKAAAGLGAAASLLSASLYTVDGGERAVIFDRFRGVLPETVGEGTHFLVPWLQKPFVFDIRTRPHNFSSNSGTKGPCRWVYPHPSGLLSPPPTSVPFPTIFTSLGLEYDDKVLPSIGNEVLKAVVAQFNADQLLT ERPHVSALVRDALIRRAREFNIILDDVAITHLSYGIEFSQAVEKKQVAQQEAERSKFLVAKAEQERRAAIVRAEGESESARLISEATAAAGTGLIELRRIEAAREIAAELARSPNVAYVPAGDNGRMLLGLNAAGGVSLVTDVALLN*.

[0062] (2) PHB9 CDS sequence (SEQ ID NO.2): >LOC_Os04g38900.1 ATGGCCGGCGGTCCCGCGGCGGTGTCGTTCCTGACCAACATCGCCAAGGCGGCGGCGGGGCTCGGCGCCGCGGCCTCCCTCCTGTCGGCGTCGCTCTACACCGTCGACGGCGGCGAGCGCGCCGTCATCTTCGACCGGTTCCGCGGGGTGCTCCCGGAGACCGTCGGCGAGGGGACCCACTTCCTCGTGCCGTGGCTCCAGAAGCCCTTCGTCTTCGACATCCGCACGCGCCCCCACAACTTCTCCTCCAACTCCGGCACCAAAGGACCCTGCAGATGGGTTTACCCTCACCCTTCCGGCCTCCTTTCCCCGCCCCCGACGTCCGTGCCCTTCCCCACCATCTTCACCTCCCTCGGCCTCGAGTACGACGACAAGGTGCTCCCCTCCATCGGCAACGAGGTGCTCAAGGCCGTCGTCGCGCAGTTCAACGCCGACCAGCTCCTCACCGAGCGGCCCCACGTCTCAGCCCTCGTCCGCGACGCCCTCATCCGCCGCGCCCGCGAGTTCAACATCATCCTCGACGACGTCGCCATCACCCACCTCTCCTACGGCATCGAGTTCTCGCAGGCCGTCGAGAAGAAGCAGGTGGCGCAGCAGGAGGCCGAGCGCTCCAAGTTCCTCGTGGCCAAGGCTGAGCAGGAGAGGCGCGCGGCCATCGTGCGCGCGGAGGGAGAGAGTGAGTCCGCGCGGCTCATTTCTGAGGCCACCGCCGCCGCTGGGACAGGGCTGATTGAGCTGAGGAGGATCGAGGCGGCCAGGGAGATTGCTGCCGAGCTGGCCCGTTCTCCTAATGTTGCATACGTTCCTGCTGGGGACAATGGCCGGATGCTGCTCGGCCTCAACGCTGCCGGGGGTGTATCTTTGGTTACTGATGTTGCCTTGCTGAATTAG。

[0063] (3) Full-length sequence of PHB9 gene (SEQ ID NO.3): >LOC_Os04g38900

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; in, The CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

2. The application of the protein encoded by the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; in, The CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

3. Application of recombinant vectors containing the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; in, The recombinant vector is constructed by ligating the PHB9 gene with an expression vector, and the CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

4. Application of recombinant bacteria containing the PHB9 gene in any of the following: (1) Application in regulating rice leaf morphology; (2) Application in regulating the photosynthetic efficiency of rice leaves; (3) Application in regulating the sensitivity of rice seeds to ABA; (4) Application in promoting rice seed germination; in, The method for constructing the recombinant bacteria includes: ligating the PHB9 gene to an expression vector to construct a recombinant vector, and then transferring the recombinant vector into a host bacterium to obtain the recombinant bacteria; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

5. The application as described in any one of claims 1-4, characterized in that, The regulation of rice leaf morphology is achieved by overexpressing the PHB9 gene to increase leaf width and length, and knocking out the PHB9 gene to decrease leaf width and length.

6. The application as described in any one of claims 1-4, characterized in that, The regulation of rice leaf photosynthetic efficiency is achieved by overexpressing the PHB9 gene to increase stomatal conductance, intercellular CO2 concentration, and / or transpiration rate of rice leaves, thereby improving the photosynthetic efficiency of rice leaves.

7. The application as described in any one of claims 1-4, characterized in that, Overexpression of the PHB9 gene reduces the sensitivity of rice seeds to ABA, thereby promoting seed germination.

8. A method for regulating the morphology of rice leaves, characterized in that, This includes (1) overexpressing the PHB9 gene in rice to increase leaf width and leaf length, or (2) knocking out the PHB9 gene in rice to decrease leaf width and leaf length; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

9. A method for improving the photosynthetic efficiency of rice leaves, characterized in that, This includes overexpressing the PHB9 gene in rice to increase stomatal conductance, intercellular CO2 concentration, and / or transpiration rate in rice leaves, thereby improving the photosynthetic efficiency of rice leaves; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.

2.

10. A method for reducing the sensitivity of rice seeds to ABA, characterized in that, This includes overexpressing the PHB9 gene in rice to reduce the sensitivity of rice seeds to ABA; wherein the CDS sequence of the PHB9 gene is shown in SEQ ID NO.2.

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