Rice osbadh2 alleles and their use in improving rice aroma and salt tolerance

Abstract

The present application relates to the field of genetic engineering, and discloses a rice OsBADH2 allele and application thereof in improving rice aroma and salt-tolerant traits. A novel excellent allele of the rice OsBADH2 gene is successfully identified. The gene has unique double excellent functions: on the one hand, it can effectively promote the accumulation of rice aroma substances and significantly improve the eating quality of rice; on the other hand, it can significantly enhance the salt-tolerant ability of rice, and ensure the normal growth and fruiting of plants in saline-alkali environment. Three sets of differential breeding technical solutions are provided, and the three sets of solutions are suitable for different breeding needs: traditional crossing is suitable for conventional breeding units, and is simple to operate and has no biological safety controversy; vector transformation is directional and efficient, and is suitable for rapid preparation of transgenic materials; PE leading editing precisely modifies endogenous genes, has no foreign gene residues, meets the supervision requirements of gene editing crops, and has a wider application scenario.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding and genetic engineering, specifically relating to a novel allele in the OsBADH2 promoter region of rice and its application in improving rice aroma and salt tolerance traits. It also discloses three supporting breeding techniques: traditional hybridization breeding, recombinant vector-mediated genetic transformation, and PE-guided editing site-directed single-base mutation breeding. Background Technology

[0002] Rice is one of the world's most important food crops, and its aroma, quality, and salt tolerance are core objectives of current breeding efforts. However, the high-yield orientation and intensive, high-fertilizer-water cultivation methods of modern agriculture have led to insufficient accumulation of flavor compounds, diluted aroma, and deteriorated eating quality in major rice varieties, making it difficult to meet the growing market demand for high-quality rice. On the other hand, saline-alkali land resources, as important reserve arable land, may actually promote the enrichment of flavor compounds in rice due to their specific adverse environment, resulting in rice with superior eating quality. Therefore, they represent a unique land resource for developing a high-quality rice industry.

[0003] However, existing fragrant rice breeding technologies face numerous bottlenecks: current improvement strategies for the OsBADH2 gene mostly focus on knockout mutations in the coding region. While these strategies can effectively enhance rice aroma (e.g., Research Advances of Fragrance Substances in Rice, Chin J Rice Sci, 2025, 39(2): 171-186), they often lead to a significant decrease in the plant's salt and alkali tolerance, thus failing to simultaneously improve quality and enhance stress resistance. Furthermore, naturally occurring rice germplasm resources possessing both fragrance and strong salt and alkali tolerance are extremely scarce. Traditional hybridization breeding is time-consuming and inefficient, making it difficult to meet the rapid breeding needs of high-quality fragrant rice in saline-alkali land. The industry has consistently lacked an efficient editing strategy that can simultaneously improve rice aroma and salt and alkali tolerance by performing site-directed mutations on only a few bases in the promoter while preserving the complete coding region. Summary of the Invention

[0004] In response to the aforementioned industry status quo and technological pain points: First, this invention successfully identified a novel superior allele of the rice OsBADH2 gene. This gene possesses a unique dual superior function: on the one hand, it can effectively promote the accumulation of aroma substances in rice, significantly improving the eating quality of rice; on the other hand, it can significantly enhance the salt and alkali tolerance of rice, ensuring the normal growth and grain filling of plants in saline-alkali environments. This invention fills the gap in gene resources and editing technologies that can simultaneously regulate rice aroma and salt and alkali tolerance, providing key gene targets and technical support for targeted molecular breeding of high-quality fragrant rice in saline-alkali land. It has significant theoretical and industrial application value for the efficient development and utilization of saline-alkali land and the high-quality upgrading of the rice industry.

[0005] Secondly, this invention provides three differentiated breeding technology solutions, each adapted to different breeding needs: traditional hybridization is suitable for conventional breeding units, is simple to operate, and has no biosafety controversies; vector transformation is directional and efficient, suitable for the rapid creation of transgenic materials; PE leader editing precisely modifies endogenous genes, leaving no exogenous gene residues, meets the regulatory requirements for gene-edited crops, and has a wider range of applications.

[0006] The specific technical solution of the present invention includes: In a first aspect, the present invention provides a rice OsBADH2 allele, which includes the nucleotide sequence shown in SEQ ID NO.1; the coding region, i.e., the exon region sequence of the allele is completely identical to the wild-type OsBADH2 gene, and the mutation sites are two single-point mutations in the promoter: the promoter -2352bp site C is mutated to T, which destroys the G-box functional element; the promoter -1901bp site C is mutated to T, which generates the LTR low-temperature response element in situ; the allele can significantly increase the content of 2-acetyl-1-pyrrolline (2-AP) in rice, and at the same time improve the salt and alkali tolerance of rice.

[0007] The aforementioned new OsBADH2 allele in rice is a naturally occurring superior variant gene derived from the superior rice restorer line Yuhui 2103, and its core characteristics are as follows: (1) Sequence characteristics: The entire exon region of the coding region is completely identical to the wild-type OsBADH2 gene, with no base variations; (2) Mutation characteristics: The mutation is located in the promoter region. The C→T mutation at the -2352bp site destroys the G-box photoresponse element, and the C→T mutation at the -1901bp site forms the LTR low temperature response element. (3) Functional characteristics: It significantly increases the 2-AP content of rice grains, giving rice a fragrance; at the same time, it enhances the rice seedlings' tolerance to salt, alkali and salt-alkali combined stress, and maintains the plant's ion homeostasis and osmotic balance. (4) Genetic characteristics: It is derived from the excellent rice restorer line Yuhui 2103, and has stable genetics and high safety.

[0008] Secondly, the present invention provides a recombinant vector whose nucleotide sequence contains the aforementioned rice OsBADH2 allele.

[0009] Thirdly, this invention provides the application of the rice OsBADH2 allele or the aforementioned recombinant vector in improving the aroma and salt tolerance traits of rice.

[0010] Fourthly, this invention provides the application of the rice OsBADH2 allele or the aforementioned recombinant vector in creating new rice germplasm with high 2-AP content and salt tolerance.

[0011] Fifthly, the present invention provides a transgenic rice cell, rice plant or rice germplasm, which has been introduced with the aforementioned rice OsBADH2 allele or integrated with the aforementioned recombinant vector.

[0012] Sixthly, this invention provides a traditional hybridization breeding method for improving the aroma and salt tolerance traits of rice, comprising the following steps: introducing the rice OsBADH2 allele into the rice variety to be improved, and selecting improved rice with increased 2-AP content and significantly enhanced salt tolerance. 2-AP is a characteristic aroma substance of rice; salt tolerance includes the ability of rice seedlings to tolerate salt stress, alkali stress, and combined salt and alkali stress.

[0013] Furthermore, the method includes: using Yuhui 2103 carrying the aforementioned new OsBADH2 allele as the donor parent, and using the main cultivated rice variety with poor aroma and salt tolerance as the recurrent parent, conducting artificial hybridization, self-pollination, and backcrossing selection; combining molecular marker screening to select single plants carrying the target allele, ultimately obtaining a new rice line with excellent agronomic traits and both aroma and salt tolerance. This scheme relies on natural germplasm hybridization, without artificial gene modification, and is suitable for conventional field breeding scenarios.

[0014] In a seventh aspect, the present invention provides a PE leader editing vector targeting the rice OsBADH2 promoter, comprising a PE leader editing protein expression cassette and two pegRNA transcription expression cassettes; wherein the OsBADH2 comprises the nucleotide sequence shown in SEQ ID NO.1; wherein: The first pegRNA targets the OsBADH2 promoter at the -2352bp site and contains the nucleotide sequence shown in SEQ ID NO.2. It is used to achieve a site-specific C→T single-base substitution at this site, thereby disrupting the wild-type G-box functional element. The second pegRNA targets the OsBADH2 promoter at a 1901 bp site and contains the nucleotide sequence shown in SEQ ID NO.3. It is used to achieve a site-directed C→T single-base substitution at this site to form a complete LTR cryogenic response element.

[0015] After editing rice endogenous OsBADH2 using this vector, the coding region of the endogenous gene remains unchanged from the wild type, and only the promoter region forms the genotype with the two C→T single base variations mentioned above.

[0016] Furthermore, the vector backbone is selected from any one of pRGEB32, pCAMBIA1300, pYLCRISPR, and Cas9Pubi-H; the PE leader editing fusion protein is driven by the Ubiquitin promoter, the peggRNA is driven by the rice OsU6 promoter, and the selection marker is the hygromycin resistance gene HPT.

[0017] Eighthly, the present invention provides a method for improving the aroma and salt tolerance traits of rice using a recombinant vector, comprising: constructing the aforementioned recombinant vector, transforming the recombinant vector into rice to be improved using Agrobacterium-mediated transformation, and obtaining rice material integrating the OsBADH2 allele, wherein the material has increased 2-AP content and significantly enhanced salt tolerance.

[0018] Furthermore, the method includes: (1) Construct a recombinant vector containing the new OsBADH2 allele shown in SEQ ID NO.1. The vector backbone can be a commonly used plant expression vector such as pCAMBIA1300, and the hygromycin resistance gene is carried as a selection marker. (2) The recombinant vector was introduced into Agrobacterium and the rice callus was transformed using Agrobacterium-mediated transformation. (3) Transformed plants were obtained through resistance screening, differentiation and rooting. Positive lines were screened after molecular identification. The obtained plants stably expressed new alleles, and the aroma and salt tolerance were improved simultaneously.

[0019] Ninthly, the present invention provides a method for improving the aroma and salt tolerance traits of rice using a PE pilot editing system, comprising the following steps: 1) Construct the aforementioned PE-based pre-editing platform; 2) The PE leader editing vector was transformed into the target rice callus using Agrobacterium-mediated transformation, and T0 generation gene-edited rice was obtained after resistance screening; 3) Sequencing to identify the OsBADH2 promoter genotype and screening homozygous single plants: C→T homozygous mutations were achieved at two sites in the promoter, -2352bp and -1901bp, and the entire exon sequence of endogenous OsBADH2 was consistent with that of wild type. 4) Self-pollination and separation to remove exogenous elements from the PE editing vector yielded a new gene-edited rice line with no exogenous transgenic fragments and only site-directed mutations in the endogenous promoter; the resulting new line had significantly increased 2-AP content in the grains and enhanced tolerance to salt and alkali stress.

[0020] This approach eliminates the need for introducing exogenous alleles, directly and precisely modifying the endogenous OsBADH2 promoter in rice to artificially replicate the natural variant of Yuhui 2103. Furthermore, the pegRNA nucleotide sequence targeting the -2352bp site of the OsBADH2 promoter is shown in SEQ ID NO.2, and the pegRNA nucleotide sequence targeting the -1901bp site is shown in SEQ ID NO.3.

[0021] Furthermore, the method includes: (1) Design of two-site pegRNA A. Targeting the OsBADH2 promoter at the -2352bp site, using the pegRNA shown in SEQ ID NO.2, a C→T single base substitution was achieved, disrupting the G-box element; B. Targeting the OsBADH2 promoter -1901bp site, using the pegRNA shown in SEQ ID NO.3, a C→T single base substitution is achieved to form an LTR element; C. Editing principle: PE-guided editing relies on the repair sequence of pegRNA to complete single base replacement, without the need for additional homologous repair fragments, and without DNA double-strand breaks, fragment insertions or deletions.

[0022] (2) Construction of dual-site PE leader editing vector The vector backbone was pYLCRISPR / Cas9Pubi-H, pRGEB32, or pCAMBIA1300; the Ubiquitin promoter drove the PE leader to edit the fusion protein, and rice OsU6a and OsU6b drove the two pegRNAs SEQ ID NO.2 and SEQ ID NO.3, respectively; the vector carried the hygromycin resistance gene HPT as a selection marker, and the vector construction was completed.

[0023] (3) Agrobacterium-mediated genetic transformation of rice Embryogenic callus was induced in mature embryos of rice varieties such as Nipponbare, Zhonghua 11, and Huanghuazhan. The T0 generation edited seedlings were obtained by infection with Agrobacterium tumefaciens EHA105 / GV3101 and screening with hygromycin.

[0024] (4) Genotype screening and exogenous fragment removal T0 generation plants were sequenced and identified, and single plants with homozygous C→T mutations of -2352bp and -1901bp and no variation in the coding region were screened. T1 generation plants were self-crossed and segregated, and all exogenous elements of the PE editing system were removed to obtain homozygous edited lines with single-base mutations in the endogenous promoter.

[0025] (5) Identification of physical characteristics The 2-AP content, salt / alkali stress tolerance, and physiological indicators of the edited plant were consistent with those of Yuhui 2103, achieving simultaneous improvement in aroma and salt and alkali tolerance.

[0026] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention clarifies that the core mutation of the superior allele of OsBADH2 (SEQ ID NO.1) is two C→T single base mutations in the promoter, which is different from the traditional exon inactivation mutation, and enriches the gene resources for the synergistic improvement of rice aroma and salt tolerance.

[0027] (2) This invention provides three differentiated breeding technology solutions: first, traditional hybridization breeding, which introduces naturally superior alleles into the main cultivated varieties; second, Agrobacterium genetic transformation mediated by recombinant vectors, which directionally introduces new alleles; and third, PE lead editing single-base mutation technology, which artificially replicates naturally mutated genotypes and avoids the defects of long-cycle hybridization breeding. The three technology solutions are suitable for different breeding needs: traditional hybridization is suitable for conventional breeding units, is simple to operate, and has no biosafety disputes; vector transformation is directional and efficient, suitable for the rapid creation of transgenic materials; PE lead editing precisely modifies endogenous genes, leaving no exogenous gene residues, meets the regulatory requirements for gene-edited crops, and has a wider range of applications.

[0028] (3) The alleles of the present invention are derived from the superior rice restorer line Yuhui 2103. They are naturally stable and heritable variations without artificial gene editing or modification. There is no risk of transgenic safety. They can be directly applied to various breeding methods such as conventional breeding and molecular marker-assisted breeding, and are suitable for large-scale promotion and application.

[0029] (4) The PE lead editing scheme has significant advantages over the CRISPR / Cas9 large fragment editing scheme: it does not require complex homologous recombination operations, and only uses two pegRNAs, SEQ ID NO.2 and SEQ ID NO.3, to complete single base substitution. It has low technical difficulty, high editing efficiency, and low off-target risk; it does not rely on Yuhui 2103 as a donor, and the improvement can be completed in one generation of transformation, which greatly shortens the breeding cycle.

[0030] (5) The final gene-edited strain has only a single base variation in the endogenous promoter, and its genotype is completely consistent with the natural superior germplasm. It has high safety and is conducive to variety approval and large-scale promotion.

[0031] (6) This invention can help develop and utilize saline-alkali land, cultivate high-quality fragrant rice domestically, expand the rice planting area, enhance the economic value of rice, and ensure the safe production of food in saline-alkali land. Attached Figure Description

[0032] Figure 1The OsBADH2 promoter sequence was compared between the Yuhui 2103 and control materials. The figure clearly shows the specific variation characteristics of G-box functional element disruption and LTR sequence insertion in the OsBADH2 promoter of the Yuhui 2103 material, providing sequence evidence for the molecular variation mechanism of the new allele.

[0033] Figure 2 Results of 2-AP content detection in grains of Yuhui 2103 carrying the new OsBADH2 allele. Compared with the control material, the content of the key aroma compound 2-AP in grains of Yuhui 2103 carrying this new allele was significantly increased, confirming that this new allele can effectively improve the aroma quality of rice grains.

[0034] Figure 3 Statistical results of the survival rate of Yuhui 2103 seedlings carrying the new OsBADH2 allele under salt and alkali stress conditions. A and B represent salt stress treatment experiments, and C and D represent alkali stress treatment experiments. Under salt and alkali stress treatments, the survival rate of Yuhui 2103 seedlings carrying the new allele was significantly higher than that of the control material, indicating that the new allele can significantly enhance the salt and alkali tolerance of rice seedlings.

[0035] Figure 4 The results of physiological index measurements of Yuhui 2103 plants carrying the new OsBADH2 allele and control materials under salt and alkali stress were compared. AF represents salt stress treatment, and GL represents alkali stress treatment. The differences in various physiological indexes between the two types of materials under salt and alkali stress were systematically compared to clarify the regulatory role of the new allele in the physiological characteristics of rice under stress.

[0036] Figure 5 Under salt and alkali stress, the Na content in the Yuhui 2103 plant carrying the new OsBADH2 allele and the control material was significantly different. + K + Comparison of ion content determination results. AC represents salt stress treatment, and DF represents alkali stress treatment. By detecting the accumulation of sodium and potassium ions in plants under stress conditions, the mechanism by which a new allele regulates the ion homeostasis of rice under salt and alkali stress was revealed.

[0037] Figure 6 The results of comparing the betaine content and BADH enzyme activity in the leaves of Yuhui 2103 carrying the new OsBADH2 allele with those of the control material under salt and alkali stress were shown. A and B represent salt stress treatment experiments, and C and D represent alkali stress treatment experiments. This study verified that the new allele can enhance the tolerance and adaptability of rice to salt and alkali stress by regulating betaine synthesis and BADH enzyme activity.

[0038] Figure 7A: Schematic diagram of the PE leader editing dual-site target and recombinant vector structure; B: Marking of the positions of the two single-base editing target sites of the OsBADH2 promoter; C: Schematic diagram of the PE dual-site editing vector elements. Detailed Implementation

[0039] The technical solution of the present invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but do not limit the scope of protection of the present invention.

[0040] The genetic materials involved in this invention are Yuhui 2103 and Yuhui 7109: both are excellent indica rice restorer lines preserved in the applicant's laboratory. Among them, Yuhui 2103 is a fragrant rice restorer line with strong restorative ability, excellent rice quality, and aroma, and its mutation type has been identified as a new promoter mutation in our laboratory; Yuhui 7109 is a sister line of Yuhui 2103, with excellent rice quality and strong combining ability, and is used as a control material for Yuhui 2103.

[0041] General Implementation Examples Firstly, a rice OsBADH2 allele comprising the nucleotide sequence shown in SEQ ID NO.1.

[0042] Secondly, a recombinant vector containing the aforementioned rice OsBADH2 allele in its nucleotide sequence.

[0043] Thirdly, the application of the rice OsBADH2 allele or the aforementioned recombinant vector in improving the aroma and salt tolerance traits of rice.

[0044] Fourthly, the application of the rice OsBADH2 allele or the aforementioned recombinant vector in creating new rice germplasm with high 2-AP content and salt tolerance.

[0045] Fifthly, a transgenic rice cell, rice plant, or rice germplasm, which has been introduced with the aforementioned rice OsBADH2 allele or integrated with the aforementioned recombinant vector.

[0046] Sixthly, a traditional hybridization breeding method for improving the aroma and salt tolerance traits of rice includes the following steps: introducing the rice OsBADH2 allele into the rice variety to be improved, and breeding improved rice with increased 2-AP content and significantly enhanced salt tolerance.

[0047] In some specific implementation cases, the method includes: using Yuhui 2103 carrying the above-mentioned new OsBADH2 allele as the donor parent, and using the main cultivated rice varieties with poor aroma and salt tolerance as the recurrent parents, to carry out artificial hybridization, self-pollination, and backcrossing selection; combining molecular marker screening to select single plants carrying the target allele, and finally obtaining a new rice line with excellent agronomic traits and both aroma and salt tolerance.

[0048] Sixthly, a PE leader editing vector targeting the rice OsBADH2 promoter, comprising a PE leader editing protein expression cassette and two pegRNA transcription expression cassettes; wherein the OsBADH2 includes the nucleotide sequence shown in SEQ ID NO.1; wherein: The first pegRNA targets the OsBADH2 promoter at a position of -2352bp and contains the nucleotide sequence shown in SEQ ID NO.2. It is used to achieve a site-specific C→T single-base substitution at this site, thereby disrupting the wild-type G-box functional element. The second pegRNA targets the OsBADH2 promoter at a 1901 bp site and contains the nucleotide sequence shown in SEQ ID NO.3. It is used to achieve a site-directed C→T single-base substitution at this site to form a complete LTR cryogenic response element.

[0049] In some specific implementation cases, the vector backbone is selected from any one of pRGEB32, pCAMBIA1300, pYLCRISPR and Cas9Pubi-H; the PE leader editing fusion protein is driven by the Ubiquitin promoter, the peggRNA is driven by the rice OsU6 promoter, and the screening marker is the hygromycin resistance gene HPT.

[0050] Seventhly, a method for improving the aroma and salt tolerance traits of rice using a recombinant vector includes: constructing the aforementioned recombinant vector, transforming the recombinant vector into rice to be improved using Agrobacterium-mediated transformation, and obtaining rice material integrating the OsBADH2 allele, which has increased 2-AP content and significantly enhanced salt tolerance.

[0051] In some specific implementation cases, the method includes: (1) Construct a recombinant vector containing the new OsBADH2 allele shown in SEQ ID NO.1. The vector backbone can be a commonly used plant expression vector such as pCAMBIA1300, and the hygromycin resistance gene is carried as a selection marker. (2) The recombinant vector was introduced into Agrobacterium and the rice callus was transformed using Agrobacterium-mediated transformation. (3) Transformed plants were obtained through resistance screening, differentiation and rooting. Positive lines were screened after molecular identification. The obtained plants stably expressed new alleles, and the aroma and salt tolerance were improved simultaneously.

[0052] Eighthly, a method for improving the aroma and salt tolerance traits of rice using a PE pilot editing system, comprising the following steps: 1) Construct the aforementioned PE pilot editing platform; 2) The PE leader editing vector was transformed into the target rice callus using Agrobacterium-mediated transformation, and T0 generation gene-edited rice was obtained after resistance screening; 3) Sequencing to identify the OsBADH2 promoter genotype and screening homozygous single plants: C→T homozygous mutations were achieved at two sites in the promoter, -2352bp and -1901bp, and the entire exon sequence of endogenous OsBADH2 was consistent with that of wild type. 4) Self-pollination and separation to remove exogenous elements from the PE editing vector yielded a new gene-edited rice line with no exogenous transgenic fragments and only site-directed mutations in the endogenous promoter; the resulting new line had significantly increased 2-AP content in the grains and enhanced tolerance to salt and alkali stress.

[0053] Furthermore, the pegRNA nucleotide sequence targeting the -2352bp site of the OsBADH2 promoter is shown in SEQ ID NO.2, and the pegRNA nucleotide sequence targeting the -1901bp site is shown in SEQ ID NO.3.

[0054] In some specific implementation cases, the method includes: (1) Design of two-site pegRNA A. Targeting the OsBADH2 promoter at the -2352bp site, using the pegRNA shown in SEQ ID NO.2, a C→T single base substitution was achieved, disrupting the G-box element; B. Targeting the OsBADH2 promoter -1901bp site, using the pegRNA shown in SEQ ID NO.3, a C→T single base substitution is achieved to form an LTR element; C. Editing principle: PE-guided editing relies on the repair sequence of pegRNA to complete single base replacement, without the need for additional homologous repair fragments, and without DNA double-strand breaks, fragment insertions or deletions.

[0055] (2) Construction of dual-site PE leader editing vector The vector backbone was pYLCRISPR / Cas9Pubi-H, pRGEB32, or pCAMBIA1300; the Ubiquitin promoter drove the PE leader to edit the fusion protein, and rice OsU6a and OsU6b drove the two pegRNAs SEQ ID NO.2 and SEQ ID NO.3, respectively; the vector carried the hygromycin resistance gene HPT as a selection marker, and the vector construction was completed.

[0056] (3) Agrobacterium-mediated genetic transformation of rice Embryogenic callus was induced in mature embryos of rice varieties such as Nipponbare, Zhonghua 11, and Huanghuazhan. The T0 generation edited seedlings were obtained by infection with Agrobacterium tumefaciens EHA105 / GV3101 and screening with hygromycin.

[0057] (4) Genotype screening and exogenous fragment removal T0 generation plants were sequenced and identified, and single plants with homozygous C→T mutations of -2352bp and -1901bp and no variation in the coding region were screened. T1 generation plants were self-crossed and segregated, and all exogenous elements of the PE editing system were removed to obtain homozygous edited lines with single-base mutations in the endogenous promoter.

[0058] (5) Identification of physical characteristics The 2-AP content, salt / alkali stress tolerance, and physiological indicators of the edited plant were consistent with those of Yuhui 2103, achieving simultaneous improvement in aroma and salt and alkali tolerance. Specific Implementation Example 1: Introduction of new alleles through conventional hybridization combined with marker-assisted selection (1) First, select widely planted rice varieties with excellent agronomic traits but lacking aroma and with weak salt and alkali tolerance (such as Yuhui 7109) as recurrent parents; at the same time, select rice materials carrying new alleles in the OsBADH2 promoter region (such as Yuhui 2103, whose promoter region functional variation sites are as follows) Figure 1 (As shown) as the donor parent.

[0060] (2) Artificial hybridization was carried out between the donor parent (Yuhui 2103) and the recurrent parent to be improved (Yuhui 7109) to obtain the F1 generation hybrid. After the F1 plants matured, the F2 generation segregating population was harvested by self-pollination.

[0061] (3) During the F2 generation seedling stage, about 0.2g of tender leaves were collected from each rice plant and quick-frozen in liquid nitrogen. Genomic DNA was extracted using the CTAB method. Specific SNP molecular markers were developed based on the two mutation sites of the new alleles in the OsBADH2 promoter region. Genotyping of individual plants in the F2 population was performed using PCR amplification and electrophoresis or sequencing methods.

[0062] Individual plants carrying the new donor allele and exhibiting agronomic traits similar to the recurrent parents were selected as the chosen plants. These plants were then continuously self-pollinated to obtain F3 generations. The molecular marker detection was repeated on the F3 plants to select homozygous lines carrying the new OsBADH2 allele, exhibiting significant aroma, improved salt and alkali tolerance, and stable and consistent agronomic traits.

[0063] (4) If necessary, in order to further restore the background of the recurrent parents, the heterozygous single plants obtained by screening can be backcrossed with the recurrent parents continuously (3-5 generations of backcrossing). In each generation, the new OsBADH2 allele introduced is tracked using the above molecular markers, and the background recovery rate is finally obtained. A new improved rice line carrying homozygous target alleles, with a rich aroma and significantly enhanced salt and alkali tolerance.

[0064] (5) The improved rice varieties have significantly increased 2-AP content in their grains and have a richer rice aroma. Figure 2 Under stress conditions of 150 mM NaCl solution and 20 mM Na2CO3 solution, the seedling survival rate, proline and betaine content, and antioxidant enzyme activity were significantly higher than those of the original recurrent parent, showing a significantly enhanced salt and alkali tolerance. Figure 3-6 ).

[0065] Example 2: Agrobacterium-mediated genetic transformation of recombinant vectors (1) First, select the rice variety to be improved, such as Zhonghua 11, a widely planted but lacking fragrance and with weak salt tolerance. Obtain mature seeds of this variety and perform surface disinfection treatment. Specifically, soak the seeds in 70% ethanol for 1 minute, then disinfect them with 2.5% sodium hypochlorite solution by shaking for 15 minutes, and finally rinse them 5 times with sterile distilled water to thoroughly remove residual disinfectant. The disinfected seeds are inoculated on induction medium, which is MS basal medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg / L 6-benzylaminopurine (6-BA), with the pH adjusted to 5.8. Culture in the dark at 25℃±1℃ for 4 weeks to induce callus formation. Select vigorous, dense, and pale yellow embryogenic callus as the recipient material for gene transformation.

[0066] (2) This invention constructs a plant expression vector containing a new allele of rice OsBADH2. The new OsBADH2 allele has a completely conserved coding sequence compared to the wild-type OsBADH2 gene (MSU / RGAP number: LOC_Os08g32870), but two key natural C→T mutations occur in the gene promoter regulatory region, triggering cis-acting element remodeling. The specific mutation characteristics and functional mechanisms are as follows: First, a C→T single-base mutation occurs at the -2352 bp position in the promoter region of this allele, changing the core sequence of the wild-type G-box photoresponsive element from TCCATATGGCA to TCTATATTGGCA. This results in the loss of function of the endogenous G-box photoresponsive element in the promoter, altering the normal expression regulation pattern of the OsBADH2 gene. Second, a C→T single-base mutation occurs at the -1901 bp position in the promoter region of this allele, generating an LTR low-temperature response element with the core motif TTTCGG. These two specific mutations in promoter elements can significantly regulate the expression level of the OsBADH2 gene and reduce the conversion efficiency of γ-aminobutyraldehyde to γ-aminobutyric acid in rice. Under the influence of substrate compensation, a large amount of γ-aminobutyraldehyde substrates are redirected to the synthesis pathway of betaine and the aroma core substance 2-AP, significantly increasing the accumulation of 2-AP in rice grains, thus enabling rice to form a rich and stable aroma quality. At the same time, the effective increase in betaine content can significantly enhance the osmotic regulation capacity of rice, synergistically improve the tolerance of rice to salt, alkali and salt-alkali combined stress, and ultimately achieve synergistic improvement of rice aroma quality and salt-alkali tolerance.

[0067] The complete coding sequence of the novel allele (including its own promoter and terminator regions, with the promoter being the natural sequence 3000 bp upstream of the OsBADH2 gene) was digested with restriction endonucleases HindIII and EcoRI, and then ligated to the corresponding restriction sites of the plant binary expression vector pCAMBIA1301 to construct the recombinant plasmid pCAMBIA1301-OsBADH2. This vector also carries the hygromycin phosphotransferase gene (HPT) as a selection marker for subsequent transformation screening. The recombinant plasmid was transformed into competent cells of Agrobacterium tumefaciens strain EHA105 using the heat shock method. Positive clones were screened on YEB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and the correctness of the vector construction was verified by colony PCR and sequencing.

[0068] (3) The new alleles were introduced into rice callus using Agrobacterium-mediated genetic transformation. The specific steps were as follows: A verified Agrobacterium single colony was inoculated into 5 mL of YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured with shaking at 28℃ and 200 rpm until the logarithmic growth phase (OD600 was approximately 0.7). The bacterial suspension was centrifuged at 4℃ and 5000 rpm for 5 minutes to collect the bacterial cells, and the cells were resuspended in an equal volume of Agrobacterium infection solution (AA medium containing 100 μmol / L acetylsyl acetophenone, pH 5.2) to adjust the OD600 to 0.5. The embryogenic callus obtained in step (1) was immersed in the above Agrobacterium suspension and vacuum permeated for 10 minutes under a vacuum of 0.08 MPa, followed by slow shaking and co-culture on a shaker at 28℃ and 50 rpm for 20 minutes. Remove the callus tissue, blot off excess bacterial solution on the surface with sterile filter paper, and transfer it to co-culture medium (MS medium containing 100 μmol / L acetylsuccinone, pH 5.8) and co-culture for 3 days in the dark at 25℃±1℃.

[0069] (4) After co-culture, the callus tissue was transferred to a selection medium for screening of resistant callus. The selection medium was MS solid medium containing 2.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 50 mg / L hygromycin and 250 mg / L cephalosporin, with the pH adjusted to 5.8. The callus was cultured in the dark at 25℃±1℃ for 3 weeks, and subcultured every 2 weeks. During this period, browned or dead callus tissue was discarded, and vigorous, bright yellow resistant callus tissue was retained. The selected resistant callus tissues were transferred to differentiation medium, which was MS medium containing 2.0 mg / L 6-BA, 0.5 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L kinetin (KT), 50 mg / L hygromycin, and 250 mg / L cephalosporin, at pH 5.8. The medium was cultured at 25℃±1℃, with a light intensity of 2000–3000 lux and a photoperiod of 16 hours light / 8 hours dark to induce shoot differentiation. After 4 weeks, the differentiated green shoots or seedlings were transferred to rooting medium (1 / 2 MS medium containing 0.5 mg / L NAA and 50 mg / L hygromycin, pH 5.8) and cultured under the same light conditions for 3 weeks to promote root development and form complete regenerated plants.

[0070] (5) Molecular identification and phenotypic analysis of the obtained regenerated plants. First, genomic DNA was extracted from the leaves of the regenerated plants, and genotyping was performed using a four-primer amplification inhibited mutation PCR system (Tetra-primer ARMS-PCR) targeting the specific SNP site of the new allele. This PCR system contains two outer primers and one inner primer, one of which has a 3' end complementary to the mutation site, enabling specific amplification of the mutant allele fragment. The PCR reaction conditions were: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 35 cycles; and 72℃ final extension for 5 minutes. The amplification products were analyzed by agarose gel electrophoresis, and the genotype of the plants was determined based on the band size. Homozygous or heterozygous positive plants carrying the new allele were screened. At the same time, the relative expression level of the OsBADH2 gene in the leaves of the positive plants was detected by real-time quantitative PCR (qRT-PCR), with the rice Actin gene as an internal control. Furthermore, the content of 2-AP in mature rice was determined by gas chromatography-mass spectrometry (GC-MS). The specific steps were as follows: 1 gram of polished rice was ground into powder, 5 mL of dichloromethane was added, and ultrasonic extraction was performed for 30 minutes. After centrifugation, the supernatant was collected for GC-MS analysis. The chromatographic column was an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The temperature program was 50℃ for 2 minutes, increased to 150℃ at 5℃ / min, and then increased to 250℃ at 10℃ / min for 5 minutes. Finally, the salt and alkali stress tolerance of positive plants was identified: seedlings that had grown to the three-leaf stage were transferred to Yoshida nutrient solution containing 150 mM NaCl and 20 mM Na2CO3 (pH 8.5) for stress treatment. After 14 days of treatment, the survival rate, plant height, root length, and fresh weight of the plants were recorded.

[0071] Example 3: Site-directed mutagenesis of the OsBADH2 promoter using the PE-guided single-base editing system to cultivate aromatic salt-tolerant rice. In this embodiment, wild-type Zhonghua 11 was used as the experimental material. The original bases at the -2352bp and -1901bp sites of its OsBADH2 promoter are both C (same as above). Figure 1 As shown in Figure 7109, the PE lead editing system was used to achieve precise C→T single base substitution at two target sites, destroying the G-box element and generating the LTR low-temperature response element in situ, thus creating a new target rice germplasm.

[0072] (1) Construction of PE pilot editing carrier Two specific pegRNAs were designed, with sequences SEQ ID NO.2 and SEQ ID NO.3, respectively, targeting the -2352bp and -1901bp sites of the promoter. Both template sequences carried C→T editing information. A recombinant vector containing an nCas9-M-MLV RT fusion protein expression cassette and a dual pegRNA expression cassette was constructed. Figure 7 The vector backbone was selected from the pCAMBIA series. The expression of the fusion protein was driven by the maize Ubiquitin promoter and the pegRNA was driven by the rice U3 promoter. The hygromycin resistance gene HPT was carried as a selection marker. After molecular cloning and sequencing verification, the recombinant plasmid pPE-OsBADH2-PE was obtained.

[0073] (2) Rice callus induction and Agrobacterium-mediated transformation Healthy seeds of *Hydrocotyle spp. 11* were selected and disinfected sequentially with 70% ethanol and 2.5% sodium hypochlorite. The resulting inoculum was inoculated onto N6 induction medium and cultured in the dark at 26°C for 4 weeks to obtain embryogenic callus. After hyperosmolar pretreatment, the callus was transformed into recombinant plasmids using *Agrobacterium* EHA105. Following bacterial infection, the callus was co-cultured for 3 days, then transferred to a selection medium supplemented with hygromycin and cephalosporin. Resistance selection was continued for 6 weeks to obtain positive resistant callus.

[0074] (3) Callus differentiation, plant regeneration and hardening and transplanting The resistant callus was transferred to MS differentiation medium and cultured at 26℃ under 16 h light / 8 h dark conditions to induce green shoot differentiation. When the seedlings grew to 3-5 cm, they were transferred to 1 / 2 MS rooting medium for cultivation. After the roots were strong, the seedlings were hardened off for 3 days and then transplanted into greenhouse nutrient soil to obtain T0 generation regenerated plants.

[0075] (4) Genotyping of T0 generation plants Genomic DNA was extracted from leaves of T0 generation plants. Specific primers were designed for PCR amplification targeting two editing sites. The products were sequenced by Sanger sequencing and compared with wild-type sequences. Positive edited plants with simultaneous C→T substitutions at -2352bp and -1901bp sites were screened.

[0076] (5) Genetic purification of editing sites and screening of strains without exogenous fragments Seeds of the T1 generation were harvested from the positive T0 generation plants through self-pollination. Using hygromycin resistance combined with PCR sequencing, a line with homozygous C→T mutation at two sites and complete removal of the exogenous backbone sequence of the vector was selected and named OsBADH2-PE1. The OsBADH2 coding region of this line is consistent with that of the wild type, with only the promoter showing the target single-base variation.

[0077] (6) Identification of aroma characteristics of seeds Mature grains of OsBADH2-PE1 and wild-type Zhonghua 11 were harvested, and the content of the characteristic aroma compound 2-AP was detected using headspace solid-phase microextraction combined with GC-MS. The results showed that the 2-AP content of the edited line was significantly increased compared with the wild type, and its aroma quality reached the level of mainstream high-quality fragrant rice.

[0078] (7) Identification of salt and alkali tolerance during the seedling stage Edited plants and wild-type seedlings were cultivated to the three-leaf-one-heart stage. Three hydroponic treatments were set up: normal nutrient solution, mild salinity, and severe salinity. After 7 days of stress, morphological and multiple physiological and biochemical indicators were measured. The results showed that under salinity stress, OsBADH2-PE1 plants exhibited less wilting and yellowing, and their chlorophyll, proline content, and antioxidant enzyme activity were significantly higher than those of the wild type, while their malondialdehyde content was lower. Under severe salinity stress, the survival rate and salt tolerance of the edited plants were significantly improved.

[0079] (8) Evaluation of field agronomic traits OsBADH2-PE1 and wild-type Zhonghua 11 were planted under conventional field conditions, and yield-related traits such as growth period, number of effective panicles, number of grains per panicle, seed setting rate, and thousand-grain weight were investigated. The results confirmed that there were no significant differences in agronomic indicators between the two lines, and that single-base mutations in the promoter did not affect normal growth and yield of rice.

[0080] In summary, this embodiment achieves precise C→T substitution at two sites in the OsBADH2 promoter through PE-guided editing, simultaneously enhancing the aroma and salt tolerance of rice without altering the gene coding region, and exhibiting excellent germplasm and agronomic traits, making it a suitable core breeding material for high-quality salt-tolerant fragrant rice.

Claims

1. A rice OsBADH2 allele, characterized in that: It includes a nucleotide sequence as shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that: Its nucleotide sequence contains the rice OsBADH2 allele as described in claim 1.

3. The application of the rice OsBADH2 allele according to claim 1 or the recombinant vector according to claim 2 in improving the aroma and salt tolerance traits of rice.

4. The application of the rice OsBADH2 allele according to claim 1 or the recombinant vector according to claim 2 in the creation of new rice germplasm with high 2-AP content and salt tolerance.

5. A transgenic rice cell, rice plant, or rice germplasm, characterized in that: The rice OsBADH2 allele of claim 1 is introduced, or the recombinant vector of claim 2 is integrated.

6. A hybridization breeding method for improving the aroma and salt tolerance traits of rice, characterized in that: include: The rice OsBADH2 allele described in claim 1 was introduced into the rice variety to be improved, and improved rice with increased 2-AP content and significantly enhanced salt and alkali tolerance was bred.

7. A PE leader editing vector targeting the rice OsBADH2 promoter, characterized in that: It includes a PE leader editing protein expression cassette and two pegRNA transcription expression cassettes; The OsBADH2 comprises the nucleotide sequence shown in SEQ ID NO.1; The first pegRNA targets the OsBADH2 promoter at a position of -2352 bp and contains the nucleotide sequence shown in SEQ ID NO.2; The second pegRNA targets the OsBADH2 promoter at a 1901 bp site and contains the nucleotide sequence shown in SEQ ID NO.

3.

8. The PE pilot editing carrier according to claim 7, characterized in that: The vector backbone is selected from any one of pRGEB32, pCAMBIA1300, pYLCRISPR and Cas9Pubi-H.

9. A method for improving the aroma and salt tolerance traits of rice using a recombinant carrier, characterized in that: The recombinant vector as described in claim 2 was constructed, and the recombinant vector was transformed into rice to be improved using Agrobacterium-mediated transformation to obtain rice with OsBADH2 allele integrated.

10. A method for improving the aroma and salt tolerance traits of rice using a PE pilot editing system, characterized in that: include: 1) Construct the PE pilot editing carrier as described in claim 7; 2) The PE leader editing vector was transformed into the target rice callus using Agrobacterium-mediated transformation, and T0 generation gene-edited rice was obtained after resistance screening; 3) Sequencing to identify the OsBADH2 promoter genotype and screening homozygous single plants: C→T homozygous mutations were achieved at two sites in the promoter, -2352bp and -1901bp, and the entire exon sequence of endogenous OsBADH2 was consistent with that of wild type. 4) Self-pollination and separation to remove exogenous elements from the PE editing vector yields gene-edited rice with no exogenous transgenic fragments and only site-directed mutations in the endogenous promoter.

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