OsGID1L2 gene for regulating and controlling rice plant height and application of OsGID1L2 gene

Abstract

The invention discloses an OsGID1L2 gene for regulating and controlling the plant height of rice and application of the OsGID1L2 gene. A nucleotide sequence of a whole genome of the OsGID1L2 gene, a nucleotide sequence of a coding region of the OsGID1L2 gene and an amino acid sequence of protein of the OsGID1L2 gene are respectively as shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. According to the invention, an overexpression vector containing a complete CDS sequence of the OsGID1L2 gene or a CRISPR / Cas9 gene editing vector of the OsGID1L2 gene is constructed, and the constructed vector is introduced into a rice receptor material, so that an OsGID1L2 gene overexpressed transgenic rice plant or an OsGID1L2 gene knockout rice plant is obtained, and directional regulation and control of the rice plant height are realized. The method can be widely applied to rice plant height reduction and lodging-resistant breeding, and rice varieties suitable for different planting environments (such as high-fertilizer fields, lodging-prone areas and close planting cultivation) are cultivated by directionally regulating and controlling the rice plant height; meanwhile, the regulation and control mechanism of the gene relates to a gibberellin signal channel, can be further expanded and applied to lodging resistance improvement of rice, and has important theoretical value and industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to an OsGID1L2 gene that regulates rice plant height and its application. Background Technology

[0002] Rice is one of the world's most important food crops. Plant height is a key agronomic trait affecting rice yield and lodging resistance, and rationally controlling rice plant height is one of the core goals of high-yield and stable-yield rice breeding. The "Green Revolution" of the last century significantly improved rice lodging resistance and planting density through the application of semi-dwarf genes, resulting in a substantial increase in yield. However, existing semi-dwarf varieties still face problems such as insufficient precision in plant height control and difficulties in the synergistic improvement of agronomic traits.

[0003] The regulation of plant height is complex, with the gibberellin (GA) signaling pathway being one of the core pathways regulating rice plant height. The gibberellin receptor GID1 (Gibberellin Insensitive Dwarf1) family is a key component of GA signaling; its encoded protein specifically binds to gibberellin and mediates downstream GA signaling through interaction with DELLA proteins, thereby regulating plant cell elongation and division, ultimately affecting plant height formation. Currently, several GID1 family members have been identified in rice, but the functions of different members are specific. Among them, the OsGID1L2 gene, as a homologous similar gene of the GID1 family, has not yet been clearly reported to reveal its specific function and application value in rice plant height regulation.

[0004] Many plant height genes that have been discovered and identified currently exhibit poor overall agronomic traits in individuals, making them difficult to utilize in production. The main problem in current plant height breeding is the limited number of applicable plant height genes. Existing technologies primarily focus on known genes such as sd1, sd-g, and SBI, which have been widely applied but suffer from drawbacks such as limited genetic background and limited improvement effects. With the development of molecular breeding techniques, discovering new plant height-regulating genes and developing new regulatory methods are of great significance for enriching the genetic resources for rice plant height improvement and cultivating high-quality, high-yield rice varieties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as the scarcity of rice plant height regulating gene resources, the underutilization of the OsGID1L2 gene function, and insufficient precision in plant height regulation, this invention provides an OsGID1L2 gene for regulating rice plant height and its applications. The aim is to provide a new application of the rice OsGID1L2 gene in regulating rice plant height, clarify the plant height regulatory function of this gene, and provide a method for regulating rice plant height based on this gene, thus providing new gene resources and technical approaches for rice plant architecture improvement and high-yield breeding.

[0006] The rice OsGID1L2 gene and its applications provided by this invention can be widely used in breeding rice varieties to reduce plant height and improve lodging resistance. By directionally regulating rice plant height, rice varieties suitable for different planting environments (such as high-fertility fields, lodging-prone areas, and dense planting) can be cultivated. Furthermore, the regulatory mechanism of this gene involves the gibberellin signaling pathway, which can be further extended to improving rice lodging resistance, possessing significant theoretical value and promising prospects for industrial application. In addition, the OsGID1L2 gene also has homologous genes in other gramineous crops (such as maize and wheat), and its regulatory methods can provide a reference for improving plant height in other crops, further expanding its application scope.

[0007] The technical solution adopted in this invention is as follows: The OsGID1L2 gene, and its complete genome nucleotide sequence are shown in SEQ ID NO. 3, specifically: The nucleotide sequence of the coding region of the OsGID1L2 gene is shown in SEQ ID NO. 4, specifically: The amino acid sequence of the protein encoded by the OsGID1L2 gene is shown in SEQ ID NO. 5, specifically: msqtelpestspsesspamasttpapyvvedcgpnlqlfsdgtvirfedynilpppvlppalatvqwkdvvydagrglklrvyrppaatvageklpvlvyfhgggyvigsfemdnfhacclrlahelpavvlsadyrlapehrlpaahddaatamswvrdqavasgdaadpwlaesadfg rvfvsgdsagagivhhvalrlgsgqiavdparvagcallfpyfggeertrseaenppgpfltlpfsdqgwrlalprgatrdhplanpfgpenpamdavalppllvvvaqldllrdrdvdyaarlramgkqvemvefegqhhgffaveplgdagselvrvvrrfvygnggdaaaaaaaaask.

[0008] Preferably, the OsGID1L2 gene belongs to the gibberellin receptor GID1 family, with an open reading frame length of 1080 bp, encoding a gibberellin receptor protein of 360 amino acids.

[0009] Preferably, the OsGID1L2 gene is located on chromosome 3 of rice and contains one exon.

[0010] Application of the OsGID1L2 gene in regulating rice plant height.

[0011] Preferably, the application involves regulating the expression level of the OsGID1L2 gene in rice to achieve targeted regulation of rice plant height.

[0012] Preferably, the regulation includes overexpressing the OsGID1L2 gene to increase rice plant height and knocking out the OsGID1L2 gene to dwarf rice plant height.

[0013] Preferably, the method for overexpressing the OsGID1L2 gene includes the following steps: (1) Construct an overexpression vector containing the complete CDS sequence of the OsGID1L2 gene; (2) The vector constructed in step (1) was introduced into rice recipient material and transgenic rice plants with high expression of OsGID1L2 gene were screened.

[0014] Preferably, the method for knocking out the OsGID1L2 gene includes the following steps: (1) Constructing a CRISPR / Cas9 gene editing vector for the OsGID1L2 gene; (2) The vector constructed in step (1) was introduced into rice recipient material and rice plants with OsGID1L2 gene knockout were screened.

[0015] A method for regulating rice plant height includes the following steps: (1) Regulates the expression level of the OsGID1L2 gene in rice; (2) Select rice plants with the expected plant height.

[0016] A recombinant vector for regulating rice plant height, wherein the recombinant vector is an overexpression vector containing the complete CDS sequence of the rice OsGID1L2 gene, or a CRISPR / Cas9 gene editing vector containing OsGID1L2 gene-specific sgRNA.

[0017] The beneficial effects of this invention are as follows: 1. The plant height regulation function of the rice OsGID1L2 gene was clarified for the first time, and new applications of this gene were explored, enriching the gene resources for rice plant height regulation, filling the gap in the existing technology where the function of the OsGID1L2 gene was not fully utilized, and providing a new target gene for rice plant type improvement.

[0018] 2. Precise and targeted regulation of rice plant height has been achieved: By overexpressing or knocking out the OsGID1L2 gene, tall or dwarf rice plants can be obtained respectively, with stable regulation effect, which solves the problem of "tall plants are not resistant to lodging" that is easy to occur in the existing technology for plant height regulation.

[0019] 3. The regulation method based on the OsGID1L2 gene is simple to operate and highly efficient: Using genetic engineering technology (Agrobacterium-mediated transformation, gene editing, etc.), rice plants of the target height can be obtained quickly. Compared with traditional hybridization breeding, it significantly shortens the breeding cycle and reduces breeding costs, making it suitable for large-scale application in rice breeding practices.

[0020] 4. Wide range of applications: The OsGID1L2 gene is highly conserved in different rice varieties. Its regulation methods can be applied to different types of rice varieties such as japonica rice and indica rice. It can also work synergistically with other plant architecture improvement genes to further optimize rice plant architecture and improve rice yield and stress resistance.

[0021] 5. Strong technological innovation: It utilizes the signal transduction characteristics of the gibberellin receptor gene to regulate plant height. Compared with existing single-function plant height regulating genes, its regulatory mechanism is more explicit. Moreover, it can indirectly affect the stress resistance of rice (such as lodging) by regulating the gibberellin signaling pathway, achieving the dual effect of "plant height improvement + stress resistance enhancement". Attached Figure Description

[0022] Figure 1 The diagram shows the identification and cloning results of the OsGID1L2 gene. In the diagram: A shows the segregation of plant height between the parents and the population materials; B shows the statistical results of plant height of the genetic population lines; C shows the gene localization results using the interval mapping method, located on rice chromosome 3; D is a schematic diagram of the structure of the OsGID1L2 gene, which has only one exon; E shows a comparison of the changes in the protein domains of Y11 and R998 after frameshift mutation.

[0023] Figure 2 This is a predicted structure diagram of the OsGID1L2 gene.

[0024] Figure 3 This is a model structure diagram of OsGID1L2 in overexpressed rice lines.

[0025] Figure 4 The figure shows the gene expression and plant height of GID1L2-OE rice plants. In the figure: A is the expression level of OsGID1L2 gene in GID1L2-OE plants, and B is the plant height phenotype of GID1L2-OE rice plants.

[0026] Figure 5 This diagram illustrates the base editing of the OsGID1L2 gene in rice knockout lines.

[0027] Figure 6 The figure shows the gene expression and plant height of GID1L2-CR rice plants. In the figure, A represents the OsGID1L2 gene expression level of GID1L2-CR plants; B represents the plant type and plant height phenotype of GID1L2-CR plants.

[0028] Figure 7 Phenotypic diagram of plant height of FB10-gid1l2 rice plants. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but this does not limit the scope of protection and application of the present invention.

[0030] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the following examples are all commercially available products; and the experimental methods used are all conventional molecular biology experimental methods unless otherwise specified.

[0031] In the following specific embodiments provided by this invention, the following experimental subjects were selected: the superior indica rice variety R998 (semi-dwarf plant height, carrying the OsGID1L2 dwarf allele, and the recipient material for overexpression experiments), the Guangxi Zhuang Autonomous Region common wild rice Y11 introgression line NIL200 (tall plant height, carrying the OsGID1L2 tall allele, and the recipient material for gene knockout experiments), and the high-quality hybrid rice parent intermediate material FB10 (tall plant height but not resistant to lodging, and the material for plant height improvement applications). R998 was bred and provided by the Rice Research Institute of Guangdong Academy of Agricultural Sciences, while NIL200 and FB10 were independently bred by the inventors' team.

[0032] Example 1: Identification and cloning of the OsGID1L2 gene regulating rice plant height 1. Identification of the OsGID1L2 gene A BC3F9 genetic population was constructed using the common wild rice Y11 from Guangxi Zhuang Autonomous Region and its recurrent parent R998. High-density genetic maps were established through simplified resequencing of the genomes of both parents and 281 introgression lines. Gene localization was performed using the mapQTL interval mapping method. A novel QTL locus (OsGID1L2) affecting plant height was discovered in the 31.95-33.45 Mb region on chromosome 3 of rice. This locus contains an annotation gene that is predicted to encode the gibberellin receptor-associated protein GID1L2 (accession number: L). OC_Os03g57640, 1358 bp in length, contains one exon and an open reading frame (ORF) of 1080 bp, encoding a 360-amino acid gibberellin receptor protein. PlantCARE prediction indicates that its promoter region contains a GA-responsive element (GARE), an abscisic acid-responsive element (ABRE), an auxin-responsive element (TGA-element), and a cis-regulatory element involved in the methyl jasmonic acid response (TGACG-motif), enabling it to integrate multiple signals to regulate its own expression. Statistics on plant height in the genetic population and gene localization results using interval mapping are shown below. Figure 1 AC. The predicted OsGID1L2 gene structure diagram is shown below. Figure 2 .

[0033] 2. Cloning of the OsGID1L2 gene The amino acid sequence of the protein encoded by the OsGID1L2 gene is: msqtelpestspsesspamasttpapyvvedcgpnlqlfsdgtvirfedynilpppvlppalatvqwkdvvydagrglklrvyrppaatvageklpvlvyfhgggyvigsfemdnfhacclrlahelpavvlsadyrlapehrlpaahddaatamswvrdqavasgdaadpwlaesadfgrvfvsgdsagagivhhvalrlgsgqiavdparvagcallfpyfggeertrseaenppgpfltlpfsdqgwrlalprgatrdhplanpfgpenpamdavalppllvvvaqldllrdrdvdyaarlramgkqvemvefegqhhgffaveplgdagselvrvvrrfvygnggdaaaaaaaask (as SEQ ID) As shown in NO.5), the predicted OsGID1L2 gene structure and protein domain of Y11 and R998 are shown in [reference needed]. Figure 1 DE.

[0034] Table 1 Amplification Primer Sequences

[0035] Example 2: Validation of the function of OsGID1L2 in increasing rice plant height through gene overexpression 1. Obtaining the full-length CDS fragment of the rice OsGID1L2 gene Using cDNA from wild rice Y11 as a template, and based on the sequence of the OsGID1L2 gene in the Rice Genome Database (RGAP) (accession number: LOC_Os03g57640), a specific primer pair containing the restriction enzyme sites required for overexpression vector construction was designed for OsGID1L2. F2 / R2 (primer sequences are shown in Table 1, OsGID1L2) F: SEQ ID NO. 6, OsGID1L2 R: SEQ ID NO.7), was subjected to PCR amplification, and the nucleotide sequence of the amplified gene fragment (i.e., the CDS sequence of OsGID1L2) was obtained:

[0036] 2. Construction of OsGID1L2 gene overexpression vector pBWA(V)HS-GID1L2 Primer pair OsGID1L2 will be used The product obtained from F / R amplification was inserted into the basic vector pBWA(V)HS containing the 35S promoter. Positive clones were screened using the marker gene on the basic vector to obtain the recombinant expression vector pBWA(V)HS-GID1L2.

[0037] 3. Obtaining transgenic plants overexpressing the OsGID1L2 gene The constructed pBWA(V)HS-GID1L2 vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation or heat shock. Positive Agrobacterium strains that could be used to infect rice tissues were screened using the recombinant expression vector and the characteristics of Agrobacterium itself.

[0038] Rice R998 was infected with a recombinant Agrobacterium strain containing the recombinant plasmid pBWA(V)HS-GID1L2. Figure 1 A) Callus tissue was cultured in the dark on selection medium containing 50 mg / L hygromycin to obtain positive transgenic callus. The positive callus was differentiated, rooted, and transplanted to obtain T0 generation plants. T1 generation plants were obtained through routine molecular testing and rice cultivation methods.

[0039] The model structure of gene OsGID1L2 in overexpressed rice lines is as follows: Figure 3 The CDS sequence of the gene OsGID1L2 is shown below: msqtelpestspsesspamasttpapyvvedcgpnlqlfsdgtvirfedynilpppvlppalatvqwkdvvydagrglklrvyrppaatvageklpvlvyfhgggyvigsfemdnfhacclrlahelpavvlsadyrlapehrlpaahddaatamswvrdqavasgdaadpwlaesadfgrv fvsgdsagagivhhvalrlgsgqiavdparvagcallfpyfggeertrseaenppgpfltlpfsdqgwrlalprgatrdhplanpfgpenpamdavalppllvvvaqldllrdrdvdyaarlramgkqvemvefegqhhgffaveplgdagselvrvvrrfvygnggdaaaaaaaask (as SEQ ID NO.5 shown).

[0040] 4. Identification of plants overexpressing gene OsGID1L2 (1) Molecular identification: T1 generation overexpressing rice leaves were taken 2 weeks after transplanting. Total RNA was extracted using conventional RNA extraction methods and reverse transcribed into cDNA using a reverse transcription kit (purchased from Invitrogen). The cDNA was then extracted using primer OsGID1L2. RT F1 / R1 (primer sequences are shown in Table 1, SEQ ID NO. 8 / SEQ ID NO. 9) were used for qRT. PCR was used to detect the expression level of the OsGID1L2 gene; primers Actin were used. The PCR product of the F / R primer sequence (see Table 1, SEQ ID NO. 10 / SEQ ID NO. 11) was used as an internal control; using wild-type rice R998 as a control, plants with significantly higher OsGID1L2 gene expression levels than wild-type plants were screened, which were designated as positive overexpression rice plants (named GID1L2-OE). The OsGID1L2 gene expression level of GID1L2-OE plants is shown in the table below. Figure 4 A.

[0041] (2) Phenotypic identification: Positive overexpressing plants OsGID1L2-OE and wild-type R998 plants were planted simultaneously and managed normally. After the plants matured, the plant height was measured and data were collected. OsGID1L2 gene overexpressing plants (GID1L2-OE) 1. GID1L2-OE 2) See plant type Figure 4As shown in Figure B, the plant height statistics of OsGID1L2 gene overexpression plants are shown in Table 2. The results show that the plant height of GID1L2-OE plants is significantly shorter than that of wild-type plants. 1 and GID1L2-OE The results showed that the OsGID1L2 gene overexpression significantly promoted the increase of rice plant height, with increases of 70.3% and 93.1% respectively.

[0042] Table 2. Plant height statistics of plants overexpressing the OsGID1L2 gene.

[0043] Example 3: Gene knockout verification of the function of OsGID1L2 in reducing rice plant height 1. OsGID1L2 gene editing target selection and sgRNA design Based on the CDS sequence of the OsGID1L2 gene, two specific sgRNAs, sgRNA1 (sequence shown in Table 1, SEQ ID NO. 12) and sgRNA2 (sequence shown in Table 1, SEQ ID NO. 13), were designed using CRISPR-P 2.0 software, with the target site located in the coding region of the OsGID1L2 gene.

[0044] 2. Construction of OsGID1L2 gene editing vector The synthesized sgRNA1 and sgRNA2 primers were annealed to form double strands and inserted into the CRISPR / Cas9 basic vector pEGCas9Pubi-H to construct the OsGID1L2 gene editing vector (named pEGCas9Pubi-H-GID1L2). The vector construction was confirmed to be correct by colony PCR and sequencing.

[0045] 3. Agrobacterium-mediated transformation and genetic transformation of rice The gene editing vector pEGCas9Pubi-H-GID1L2 was transformed into Agrobacterium tumefaciens EHA105, and then transformed into the tall introgression line NIL200 in the Y11 and R998 genetic populations of common wild rice in Guangxi Zhuang Autonomous Region via Agrobacterium-mediated transformation. Figure 1 In A), positive regenerated plants were obtained. T1 generation plants were obtained through conventional molecular detection and rice cultivation methods.

[0046] 4. Identification of OsGID1L2 knockout plants (1) Molecular identification: Leaves of T1 generation OsGID1L2 knockout rice plants 2 weeks after transplanting were taken, and genomic DNA was extracted. Specific primer pairs GID1L2-F3 / R3 were designed according to the target region (primer sequences are shown in Table 1, SEQ ID NO. 14 / SEQ ID NO. 15) for PCR amplification. The amplified products were sequenced, and plants with base deletion, insertion, or substitution in the OsGID1L2 gene target region, resulting in loss of gene function, were screened. These were identified as positive knockout rice plants (named GID1L2-CR). The base editing of the OsGID1L2 gene in the rice knockout line GID1L2-CR is as follows: Figure 5 As shown.

[0047] Leaves from T1 generation overexpressing rice plants two weeks after transplanting were collected. Total RNA was extracted using standard RNA extraction methods and then reverse transcribed into cDNA using a reverse transcription kit (Invitrogen). The cDNA was then processed using primer OsGID1L2. RT F2 / R2 (primer sequences are shown in Table 1, SEQ ID NO. 16 / SEQ ID NO. 17) were used for qRT. PCR was used to detect the expression level of the OsGID1L2 gene; primers Actin were used. The PCR products of the F / R primers (see Table 1, SEQ ID NO. 10 / SEQ ID NO. 11) were used as internal controls; using wild-type rice NIL200 as a control, plants with lower OsGID1L2 gene expression levels than wild-type plants were screened, which were designated as positive gene knockout rice plants (named GID1L2-CR). The OsGID1L2 gene expression level of GID1L2-CR plants is shown in the table below. Figure 6 A.

[0048] (2) Phenotypic identification: GID1L2-CR plants and wild-type R998 plants were planted simultaneously and managed normally. After the plants matured, the plant height was measured and data were collected. OsGID1L2 gene knockout plants (GID1L2-CR) 1. GID1L2-CR 2) See plant type and height. Figure 6 As shown in Figure B, the plant height statistics of OsGID1L2 gene knockout plants are shown in Table 3. The results show that the plant height of GID1L2-CR plants is significantly greater than that of wild-type R998. 1 and GID1L2-CR The dwarfing effect was significantly reduced by 33% and 29.8% respectively, further proving that the OsGID1L2 gene is a key gene regulating rice plant height.

[0049] Table 3. Plant height statistics of GID1L2-CR plants

[0050] Example 4: Application of OsGID1L2 gene in rice plant height improvement breeding To verify the effectiveness of the OsGID1L2 gene in rice height improvement breeding, we used the same method as in Example 3, employing Agrobacterium-mediated genetic transformation, to introduce the gene editing model into FB10, an intermediate material of the high-quality hybrid rice maintainer line prone to lodging, bred by our research team. T1 generation plants were molecularly identified to remove the transgenic tag, and homozygous mutant plants (named FB10-gid1l2) were selected based on the target mutation sequence. The base editing of the OsGID1L2 gene in the rice knockout line FB10-gid1l2 is shown below. Figure 5 As shown.

[0051] GID1L2-CR plants and wild-type FB10 plants were planted simultaneously and managed normally. After maturity, plant height was measured and data were collected. Plant type and height of the OsGID1L2 gene knockout plants of FB10 (FB10-gid1l2-1, FB10-gid1l2-2) are shown below. Figure 7 As shown in Table 4, the plant height statistics of OsGID1L2 gene knockout plants are presented. The results show that the plant height of FB10-gid1l2 plants is significantly greater than that of wild-type FB10. 1 and FB10-gid1l2 The dwarfing effect was significantly reduced by 33% and 29.8% respectively, which will improve its lodging resistance and further prove that the OsGID1L2 gene can be effectively applied to rice plant height improvement breeding.

[0052] Table 4. Plant height statistics of OsGID1L2 gene knockout plants

[0053] In this invention, all experiments in all embodiments were repeated more than three times, with a sample size of no less than 10 plants in each experiment. The experimental results showed good repeatability and stability, proving that the plant height regulation function of the OsGID1L2 gene and its application method are reliable and can be stably applied to rice breeding practices.

Images