Use of a gene regulating leaf angle in rice and method for improving leaf angle in rice

Abstract

This invention discloses the application of a gene regulating rice leaf angle and a method for improving rice leaf angle, relating to the field of biotechnology. The CDS sequence of the gene is shown in SEQ ID NO.2. This invention found that overexpression of this gene can significantly shorten the length of adaxial cells in the leaf pulvinus, thereby greatly reducing the rice leaf angle and resulting in a compact and upright plant structure. High-density planting experiments showed that under low-density conditions, there was no significant difference in single-panicle yield among different lines; however, under high-density planting, the single-panicle yield of the overexpressing lines was significantly higher than that of the wild type, effectively solving the problem of poor light penetration and reduced yield in high-density rice populations with large leaf angles. This gene regulating rice leaf angle can be directly used for molecular breeding of upright rice plants, cultivating high-yielding varieties tolerant to high-density planting, and providing a novel target gene for the brassinosteroid signaling pathway to regulate plant structure, possessing outstanding breeding innovation value and promising industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of a gene that regulates rice leaf angle and a method for improving rice leaf angle. Background Technology

[0002] Improving plant architecture in rice is one of the core directions in high-yield rice breeding. Leaf angle, the angle between a plant's leaf and stem, is a core morphological indicator for regulating plant architecture, and its size directly affects the efficiency of photosynthesis in the canopy. This trait is also an important target for high-yield crop breeding. By improving leaf angle characteristics and optimizing plant architecture, the crop's adaptability to dense planting can be effectively enhanced, thereby fully tapping its yield potential.

[0003] The pulvinus connects the leaf sheath and the leaf blade, directly determining the leaf angle size. The side of the pulvinus closer to the stem is called the adaxial side, and the side farther from the stem is called the abaxial side. The asymmetric proliferation or expansion of cells on the adaxial and abaxial sides forms the leaf angle. The pulvinus achieves precise regulation of the leaf angle by dynamically changing cell morphology, thus affecting the overall plant shape. Pulvinus development can be divided into five consecutive stages, S1-S5, with stage S5 being the key stage. In this stage, the leaf angle size is ultimately determined through the synergistic regulation of environmental factors and hormones.

[0004] Brassinosteroids (BRs) are key hormones regulating leaf angle. Exogenous application of BR promotes increased leaf angle in rice, primarily because BR promotes excessive adaxial growth of the leaf pulvinus. Increasing research links BRs and leaf angle. Brassinolides are a typical type of brassinosteroid. Studies have shown that increased brassinolide synthesis or enhanced signal transduction leads to increased leaf angle in rice, while decreased synthesis or impaired signal transduction results in decreased leaf angle. Brassinolide biosynthesis-related mutants (such as...) d2 , d11 , osdwarf4 , brd2 ) and receptor gene mutants (such as osbri1 / d61 , osbak1 All of them exhibited an upright leaf phenotype, while brassinolide signaling enhancer... OsBSK3Overexpression of [specific gene] increases leaf angle. The RLK-RLCK module, composed of OsBRI1 / OsBAK1 and OsBSK3, can mediate brassinolide signal transduction and regulate the increase of rice leaf angle. An excessively large leaf angle phenotype restricts high-density rice cultivation and yield improvement, and is a trait urgently needing improvement in high-yield rice breeding. Given the rich functional diversity of the RLK family members, this invention aims to discover and identify new RLK family members that can negatively regulate the brassinolide signaling pathway, thereby effectively reducing rice leaf angle and providing novel gene targets for targeted improvement of rice plant architecture. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a gene that regulates rice leaf angle and a method for improving rice leaf angle, thereby solving the problems existing in the prior art. This invention has found that overexpression of this gene can significantly shorten the length of adaxial cells in the leaf pulvinus, thereby greatly reducing the rice leaf angle and making the overall plant compact and upright.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of a gene that regulates rice leaf angle in improving rice leaf angle, and the CDS sequence of the gene is shown in SEQ ID NO.2; The leaf angle of rice was reduced by overexpressing the gene in rice plants.

[0007] The present invention also provides the application of biological materials overexpressing a gene that regulates rice leaf angle in improving rice leaf angle, wherein the CDS sequence of the gene is shown in SEQ ID NO.2; The leaf angle of rice was reduced by overexpressing the gene in rice plants.

[0008] Furthermore, the biological material is a gene expression cassette; the gene expression cassette includes the gene.

[0009] Furthermore, the biological material is a recombinant plasmid; the recombinant plasmid includes the gene expression cassette described above.

[0010] Furthermore, the biological material is a recombinant host cell; the recombinant host cell includes the aforementioned recombinant plasmid.

[0011] Furthermore, the recombinant host cell is recombinant Agrobacterium.

[0012] The present invention also provides a method for improving rice leaf angle, comprising the step of introducing a gene that regulates rice leaf angle into a rice plant to construct transgenic rice that overexpresses the gene; The CDS sequence of the gene is shown in SEQ ID NO.2.

[0013] Furthermore, the gene was introduced into rice plants using Agrobacterium-mediated genetic transformation.

[0014] The present invention also provides a method for breeding rice varieties suitable for high-density planting, comprising the step of introducing a gene that regulates rice leaf angle into rice plants to construct transgenic rice that overexpresses the gene; The CDS sequence of the gene is shown in SEQ ID NO.2.

[0015] Furthermore, the gene was introduced into rice plants using Agrobacterium-mediated genetic transformation.

[0016] The present invention discloses the following technical effects: This invention discovers an RLK family gene that negatively regulates rice leaf angle. OsCrRLK1L9 Stable overexpression lines were obtained through Agrobacterium-mediated transformation. Phenotypic experiments confirmed that overexpression of this gene significantly shortened the length of adaxial cells in the leaf pulvinus, thereby greatly reducing the leaf angle and resulting in a compact and upright plant structure. High-density planting trials showed that under low-density conditions, there was no significant difference in panicle yield among the lines; however, under high-density planting, the panicle yield of the overexpression lines was significantly higher than that of the wild type, effectively solving the problem of poor light penetration and reduced yield in high-density rice populations with large leaf angles.

[0017] The invention discovered OsCrRLK1L9 The gene can be directly used for molecular breeding of rice with an upright plant type, to cultivate high-yield varieties that are tolerant to dense planting, and to provide a novel target gene for the regulation of plant type by the brassinosteroid signaling pathway. It has outstanding breeding innovation value and industrial application prospects. 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 for OsCrRLK1L9 Image showing the results of gene overexpression identification; Figure 2 For wild-type rice ZH11 and OsCrRLK1L9 Plant type observation diagram of the overexpression line, scale bar is 10 cm; Figure 3 For wild-type rice ZH11 and OsCrRLK1L9Leaf angle observation graphs of overexpression lines; where a is a phenotypic observation graph of leaf angle; b is a statistical graph of leaf angle; the scale bar in a is 10 cm; the statistical sample in b is n=10, the error bar is ±SD, and the asterisk represents the significance of the difference (one-way ANOVA, * P <0.05); Figure 4 For wild-type rice ZH11 and OsCrRLK1L9 Pulley length observations of overexpressing lines; where a is a phenotypic observation of pulley; b is a statistical graph of pulley length; the scale bar in a is 1 mm; the statistical sample in b is n=10, the error bar is ±SD, and the asterisk represents the significance of the difference (one-way ANOVA). *** P <0.001), ns represents no significant difference; Figure 5 This is a statistical graph showing the yield per ear under different planting densities; where a represents the yield per ear at low density; b represents the yield per ear at high density; asterisks indicate significant differences (one-way ANOVA). * P <0.05, ** P <0.01), ns indicates that the difference is not significant. 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] The present invention relates to OsCrRLK1L9 The genomic sequence of the gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2; the amino acid sequence of the OsCrRLK1L9 protein is shown in SEQ ID NO.3.

[0026]

[0027]

[0028] SEQ ID NO.3: MRLLALAVASIVLANLHLLGVHGRDLLLSCGSNATVDAGGRRWIGDMAPGLNFTLSSPGIAASQAGSSNGNEIFGLVYHSARFFSTASWYNFSVLPGNYCLRLHFFPYTFGNFSGNDSLFDVTANDFKLVSKFNVSEEIVWRSTVSNSAINAVVKEYFLLVGSRGLQVEFDPSPGSFAFVNAIEVMLTPDNLFNDTVNKVGSAGNGQLPLGLSNRGLETMYRLNVGGHALNSSSDQYLHRPWYTDEAFMFSANAAQIVSNTSSVSYLSNNDSSISPIDVYETARIMSNNMVVDKRFNVSWRFYVHPNFDYLVRLHFCELFYDKPNQRVFKIYINNKTAAEDYDVYVRAGGINKAYHEDYFDNLPQQVDSLWLQLGPDSLTSASGTDPLLNGLEIFKLSRNGNLAYVLGHIDMGNQRGISKDRNRKILWEEVGIGSASFVTLTSVVLFAWCYVRRKRKADEKEAPPGWHPLVLHEAMKSTTDARAAGKSPLTRNSSSIGHRMGRRFSISEIRAATKNFDEALLIGTGGFGKVYKGEVDEGTTVAIKRANPLCGQGLKEFETEIEMLSKLRHRHLVAMIGYCEEQKEMILVYEYMAKGTLRSHLYGSDLPPLTWKQRVDACIGAARGLHYLHTGADRGIIHRDVKTTNILLDENFVAKIADFGLSKTGPTLDQTHVSTAVKGSFGYLDPEYFRRQQLTQKSDVYSFGVVLFEVACGRPVIDPTLPKDQINLAEWAMRWQRQRSLDAIVDPRLDGDFSSESLKKFGEIAEKCLADDGRSRPSMGEVLWHLEYVLQLHEAYKRNNVDCESFGSSELGFADMSFSLPHIREGEEEHHSKPSSIREDPDT.

[0029] Example 1 Constructing a vector carrying 5×Flag tag OsCrRLK1L9 overexpression vector, and transforming the vector into wild-type rice variety ZH11: Amplification was performed using primer pair F / R OsCrRLK1L9 After the gene coding region, the amplified fragment was ligated into the 5×Flag-MCS overexpression vector (purchased from Thermo Fisher Scientific) using homologous recombination (reaction conditions: 37℃, 30 minutes) to obtain the recombinant plasmid.

[0030] Amplification OsCrRLK1L9 The reaction procedure for the gene coding region was as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 53℃ annealing for 30 seconds, 72℃ extension for 3 minutes, for 35 cycles.

[0031] Upstream primer F: gatgacgatgataaaaagcttATGAGGCTACTTGCGCTGGCAG (SEQ ID NO.4); Downstream primer R: caattcacacttgtaggatccAGTGTCTGGATCTTCTCTTA (SEQ ID NO.5).

[0032] Mature ZH11 seeds were selected to induce callus tissue. The recombinant plasmid was introduced into the callus tissue via Agrobacterium EHA105-mediated genetic transformation. The callus tissue was then induced to differentiate into seedlings on a culture medium. Transgenic rice plants were screened using hygromycin. Total protein was extracted from 7-day-old transgenic rice seedlings and analyzed by Western blot using Flag antibody. Ponceau S staining was used as a protein loading control.

[0033] The results showed that the two independent transgenic lines (OE2 and OE3) were able to detect the OsCrRLK1L9 protein carrying the Flag tag, while the wild-type ZH11 line was not. Figure 1 This indicates that the present invention yields two independent overexpressions. OsCrRLK1L9 Transformed strains of genes.

[0034] Example 2 Will OsCrRLK1L9 Overexpression lines (OE2 and OE3) and wild-type rice ZH11 were sown normally and transplanted into rice cultivation pots. The rice was cultured until the grain-filling stage. See the rice plant architecture observation diagram below. Figure 2 ,visible OsCrRLK1L9 The two overexpressing lines, OE2 and OE3, had relatively compact plant types overall.

[0035] OsCrRLK1L9 After the overexpression lines and wild-type rice ZH11 were cultured to the grain-filling stage, the following measurements and statistics were performed: 1. Measurement and statistics of rice leaf angle Rice leaf angles were measured using a protractor, with 1-2 main tillers measured per plant, and 10 samples collected from each line. Rice leaf angles were photographed using a camera, and plotted using Graghpad software. Results are shown below. Figure 3 The results showed that the average leaf angle of wild-type rice ZH11 was 23.9°, that of OE2 was 10.6°, and that of OE3 was 12.1°. It is evident that... OsCrRLK1L9 The leaf angle of the overexpressing lines was significantly smaller than that of ZH11, indicating that... OsCrRLK1L9 Genes negatively regulate rice leaf angle size, i.e., overexpression. OsCrRLK1L9 Genes can effectively reduce the leaf angle of rice.

[0036] 2. Measurement and statistics of rice leaf pillow length Tillers containing leaf pulvinus were photographed, with 10 samples collected from each line. ImageJ software was used to calculate the axillary and axillary lengths of the leaf pulvinus for each line, and Graghpad software was used to create plots. Results are shown below. Figure 4 The results showed that the average length of the paraxial leaf pulvinus in wild-type rice ZH11 was 1.8 mm, in OE2 it was 1.14 mm, and in OE3 it was 1.02 mm. This indicates that... OsCrRLK1L9 The adaxial pulvinus length of the overexpressing lines was significantly smaller than that of ZH11; however, the abaxial pulvinus length did not differ significantly, indicating that... OsCrRLK1L9 The reduced leaf angle in overexpression-expressing rice lines is caused by a decrease in the length of the adaxial side of the leaf pulvinus.

[0037] Example 3 OsCrRLK1L9 Density tolerance test of overexpression lines: Will OsCrRLK1L9 Seeds of the overexpression lines (OE2 and OE3) and wild-type rice ZH11 were germinated and hydroponically cultured until they reached 30 days of size, then transplanted at different planting densities. Low density was set at 25 rice plants per square meter, and high density at 49 rice plants per square meter. After natural maturity, the weight of a single panicle was measured for each line at different densities. Results are shown below. Figure 5 The results showed that under low-density planting conditions, there was no significant difference in the yield per ear among the different lines, while under high-density planting conditions, compared with ZH11, OsCrRLK1L9 The overexpression lines had higher yields per ear, indicating their potential for high-density planting.

[0038] 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 a gene regulating rice leaf angle in improving rice leaf angle, characterized in that, The CDS sequence of the gene is shown in SEQ ID NO.2; The leaf angle of rice was reduced by overexpressing the gene in rice plants.

2. Application of biomaterials overexpressing genes regulating rice leaf angle in improving rice leaf angle, characterized in that, The CDS sequence of the gene is shown in SEQ ID NO.2; The leaf angle of rice was reduced by overexpressing the gene in rice plants.

3. The application according to claim 2, characterized in that, The biological material is a gene expression cassette; the gene expression cassette includes the gene.

4. The application according to claim 2, characterized in that, The biological material is a recombinant plasmid; the recombinant plasmid includes the gene expression cassette described in claim 3.

5. The application according to claim 2, characterized in that, The biomaterial is a recombinant host cell; the recombinant host cell includes the recombinant plasmid described in claim 4.

6. The application according to claim 5, characterized in that, The recombinant host cell is recombinant Agrobacterium.

7. A method for improving the leaf angle of rice, characterized in that, The method includes the step of introducing a gene that regulates rice leaf angle into a rice plant to construct transgenic rice that overexpresses the gene; The CDS sequence of the gene is shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that, The gene was introduced into rice plants using Agrobacterium-mediated genetic transformation.

9. A method for breeding rice varieties suitable for high-density planting, characterized in that, The method includes the step of introducing a gene that regulates rice leaf angle into a rice plant to construct transgenic rice that overexpresses the gene; The CDS sequence of the gene is shown in SEQ ID NO.

2.

10. The method according to claim 9, characterized in that, The gene was introduced into rice plants using Agrobacterium-mediated genetic transformation.

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