Application of multidrug resistance efflux transporter gene OsMRET1 in regulation and control of chalkiness character of rice

Abstract

The invention discloses an application of a multidrug-resistant efflux transporter gene OsMRET1 in regulation and control of chalkiness traits of rice. A nucleotide sequence of a coding region of the gene is shown as SEQ ID No.1, and a coded amino acid sequence is shown as SEQ ID No.2. The invention further discloses an application of the multidrug-resistant efflux transporter gene OsMRET1 in regulation and control of chalkiness traits of rice. It is found for the first time that the gene OsMRET1 can regulate and control formation of chalkiness, an OsMRET1 gene knockout mutant is created through a gene editing technology, an OsMRET1 gene overexpression strain is obtained through a transgenic technology, it is verified that the OsMRET1 gene is a new gene for regulating and controlling rice chalkiness, and overexpression of the gene can reduce rice chalkiness and improve rice chalkiness. And a new gene resource and a technical route are provided for rice quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding and gene editing technology, specifically relating to multidrug resistance efflux transport genes. OsMRET1 Application in regulating the chalky trait of rice. Background Technology

[0002] Rice is one of China's most important food crops. In recent years, with the improvement of living standards, people's requirements for rice quality have also been continuously increasing. The demand for high-quality rice will continue to grow (Peng et al., 2023; Zhao et al., 2020). Therefore, breeding high-quality new rice varieties is one of the most important goals pursued by rice breeders. Chalkiness, as one of the important factors affecting rice quality, has always received much attention. Rice chalkiness refers to the opaque white part in the rice endosperm, mainly caused by loose arrangement of starch granules or uneven distribution of protein bodies, which seriously affects the processing quality and appearance quality of rice.

[0003] Chalk whitening is a quantitative trait associated with the endosperm, and its genetic characteristics are controlled by the genotype of triploid endosperm, diploid maternal parent, and cytoplasmic factors. The formation of chalk whitening involves complex molecular and physiological mechanisms. The chalk whitening phenotype is significantly influenced by the production environment; under different environmental conditions, the expression levels of genes regulating chalk whitening and the corresponding protein activities can vary greatly, potentially leading to significant differences in chalk whitening phenotypes for the same variety in different years and locations.

[0004] Although research on genes regulating chalkiness has been reported, efficient and stable techniques for effectively improving chalkiness traits still lack. This is because the number of discovered chalkiness-regulating genes in rice is insufficient, and their molecular regulatory mechanisms are poorly understood. Therefore, identifying new gene resources capable of precisely regulating chalkiness formation and utilizing new breeding methods or molecular techniques based on these genes is of great significance for promoting rice quality breeding in my country. Summary of the Invention

[0005] To address the problem of slow progress in the genetic improvement of chalkiness in rice breeding due to the lack of key genes controlling chalkiness, this invention provides a multidrug resistance efflux transport gene. OsMRET1 Application in regulating the chalky trait in rice, gene OsMRET1 It can regulate the formation of chalkiness and create it through gene editing technology. OsMRET1 Gene knockout mutants are obtained through transgenic technology. OsMRET1 Gene overexpression lines were verified. OsMRET1 The gene is a novel gene that regulates chalkiness in rice. Overexpression of this gene can reduce chalkiness in rice, providing new genetic resources and technical routes for improving rice quality.

[0006] To achieve the above objectives, the present invention provides a multidrug resistance efflux transport mechanism. OsMRET1 Genes, confirmed based on the Rice Genome Database (RGAP) OsMRET1 Sequence characteristics of (LOC_Os04g34530, chromosome 4). The nucleotide sequence of its coding region is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2.

[0007] The second aspect of the present invention provides the above-mentioned rice. OsMRET1 Any of the following applications of genes: (1) Regulating the chalky trait in rice; (2) Used for rice variety improvement; (3) Used to prepare genetically modified rice.

[0008] Specifically, the chalkiness trait of rice includes chalkiness degree and chalky grain rate.

[0009] A third aspect of this invention provides a method for improving the chalkiness and chalky grain rate of rice, comprising: using genetic engineering techniques to knock out [a specific gene] in rice. OsMRET1 Gene.

[0010] Specifically, with OsMRET1 Using genes as targets, CRISPR-based sgRNA primer sequences were designed. DNA fragments containing the primer sequences encoding the sgRNA were ligated into vectors carrying CRISPR / Cas9, transformed into rice, and thus obtained... OsMRET1 Genetically modified rice with missing gene function.

[0011] Specifically, OsMRET1 The target site is located in exon 4, and the sequence is shown in SEQ ID No. 3 and SEQ ID No. 4.

[0012] Specifically, the sgRNA primers include: OsMRET1 The primer sequences for gRT1 of the gene are shown in SEQ ID No. 5, the primer sequence for OsU3T1 is shown in SEQ ID No. 6, the primer sequence for gRT2 is shown in SEQ ID No. 7, and the primer sequence for OsU6aT2 is shown in SEQ ID No. 8.

[0013] A fourth aspect of this invention provides a method for reducing chalkiness and chalky grain rate in rice, comprising: using genetic engineering techniques to overexpress a gene in rice... OsMRET1 Gene.

[0014] The fifth aspect of this invention provides the application of the transgenic rice obtained by the above-described method in plant breeding.

[0015] Through the above technical solution, the present invention achieves the following beneficial effects: This invention is the first to discover genes OsMRET1 It can regulate the formation of chalkiness and create it through gene editing technology. OsMRET1 Gene knockout mutants are obtained through transgenic technology. OsMRET1 Gene overexpression lines were verified. OsMRET1 The gene is a novel gene that regulates chalkiness in rice. Overexpression of this gene can reduce chalkiness in rice, providing new genetic resources and technical routes for improving rice quality. Attached Figure Description

[0016] Figure 1 yes OsMRET1 Gene expression analysis; Figure 2 yes OsMRET1 Mutant gene identification; where A represents gene structure and B represents... OsMRET1-cr1 The gene editing method of the mutant, C is OsMRET1-cr2 The gene editing method of the mutant, D is OsMRET1 The protein sequence of the mutant; Figure 3 yes OsMRET1 Field phenotypic analysis of mutants; where A is OsMRET1 The mutant strain type, B is OsMRET1 Tillering data of mutants, C is OsMRET1 Particle width data of mutants, D is OsMRET1 Particle length data of mutants; Figure 4 yes OsMRET1 Chalky phenotype and analysis of gene mutants; where A is OsMRET1 Seed appearance of the mutant, B is OsMRET1 The chalkiness data of the mutant, C is OsMRET1 Data on the chalky grain rate of the mutant; Figure 5 yes OsMRET1 Gene expression level detection and analysis; Figure 6 yes OsMRET1 Investigation and statistical analysis of agronomic traits in rice plants with gene overexpression; where A is... OsMRET1 Tillering data of overexpression material, B is OsMRET1 Particle length data of overexpression materials, C is OsMRET1 Particle width data of overexpression materials, D is OsMRET1 The thousand-particle weight data of the overexpression material, E is OsMRET1 Plant height data of overexpression materials; Figure 7 yes OsMRET1 Gene overexpression and chalky phenotype analysis; where A is OsMRET1 The chalky grain rate data of the overexpression material, B is OsMRET1chalkiness data of overexpression materials. Detailed Implementation

[0017] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Example 1: Target gene analysis and sgRNA design 1.1 Gene Sequence Acquisition: OsMRET1 The gene is located on chromosome 4 of the rice genome, and its gene number is [gene number missing]. Os04g0422600 (RAP-DB naming rules), or LOC_Os04g34530 (MSU naming rules). OsMRET1 The coding region of the gene has a nucleotide sequence length of 1074 bp, as shown in SEQ ID No. 1. OsMRET1 The gene encodes an amino acid sequence of 357 amino acids, as shown in SEQ ID No. 2, and the protein size is approximately 38.9 kDa.

[0019] SEQ ID No. 1: SEQ ID No. 2: MMGSESGRAVAAMVSLQLLFSALQVFIKLALNDGMDARVLVAYRFMFAATFLCPIAFLRERKKRPPLTMKVVLQLFLCGLFGFSINQNLYVLAIKLTSATFITAISNLTPATTFLLAILTRLETLKLKKPAGQAKLLGTLVGMGGAMLLTFYKGPKIMVLDQLPHPKFAHLTENPQSHP ISTGNQIIGSFLGIISCFTYATWLVIQAKVSKVYPCHYSIAAMVCLFGALQSTVMALCVHRDMEHWRLGLNIRLYSSAYAGLIASGSAFPLLSWCLRKKGPLFISVFSPLMLIFVALMSSIILNEALHLGSVLGSVLIVGGLYMVLWGKAKEAADLSEDENQGKESIPVTTGGENEMK.

[0020] 1.2 Gene expression pattern analysis: The gene expression pattern was predicted using the gene expression pattern prediction website (https: / / ricexpro.dna.affrc.go.jp / ). The prediction results showed that: OsMRET1 Genes specifically expressed in seeds ( Figure 1 ).

[0021] 1.3 Target Selection: Targets were screened using the CRISPR target prediction website (http: / / crispr.dbcls.jp / ) to ensure they were located in conserved functional domains and had a low risk of off-target effects. Based on the website's prediction results, suitable target sequences were selected. OsMRET1 The gene's target site 1 is located in exon 4, with a sequence SEQ ID No. 3, such as: AGTCGCACCCAATATCGACGGG. OsMRET1 The gene target 2 is located in exon 4, with the sequence SEQ ID No. 4, such as: ATGTTGCTTACATTCTACAAGG.

[0022] 1.4 Primer Design: sgRNA primers were synthesized using the CRISPR primer design platform (http: / / skl.scau.edu.cn / primerdesign / vector / ). OsMRET1 The gRT1 primer sequence for the gene, SEQ ID No. 5, is as follows: GTCGCACCCAATATCGACGGGgttttagagctagaaat.OsMRET1 The OsU3T1 primer sequence for the gene, SEQ ID No. 6, is as follows: CCCGTCGATATTGGGTGCGTgccacggatcatctgc. OsMRET1 The gRT2 primer sequence for the gene is SEQ ID No. 7, such as: ATGTTGCTTACATTCTACAAGGgttttagagctagaaat. OsMRET1 The primer sequence for the OsU6aT2 gene, SEQ ID No. 8, is as follows: CCTTGTAGAATGTAAGCAATgccacggatcatctgc.

[0023] Example 2: Construction of knockout vector and rice transformation 2.1 Carrier Construction: First, a first round of PCR amplification was performed, using the U3 vector as a template, and... OsMRET PCR amplification was performed using primers -gRT1 and B1' to obtain the DAN fragment a, which was then used... ​ DNA fragment b was obtained by PCR amplification using primers -OsU3T1 and B2. ​ DNA fragment c was obtained by PCR amplification using primers -gRT2 and B2'. ​ DNA fragment d was obtained by PCR amplification using primers -OsU6aT2 and BL. The PCR products were then recovered by agarose gel electrophoresis. Primer sequence B1' (SEQ ID No. 9) is as follows: TTCAGAGAGGGG; primer sequence B2' (SEQ ID No. 10) is as follows: AGCTGggtctcGtcagGGTCCATCCACTCCAAGCTC; primer sequence B2' (SEQ ID No. 11) is as follows: TTCAGAGGGGGCACTGGAATCGGCAGCAAAGG; primer sequence BL' (SEQ ID No. 12) is as follows: AGCTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC.

[0024] Next, a second round of PCR amplification was performed. Using the products from the first round as a template, DNA fragments a and b were mixed and used as a template for PCR amplification using primers B1' and B2 to obtain DNA fragment A. Similarly, DNA fragments c and d were mixed and used as a template for PCR amplification using primers B2' and BL to obtain DNA fragment B. The PCR products A and B were then mixed and recovered after agarose gel electrophoresis to obtain DNA fragment AB.

[0025] The Golden Gate cloning method was used to insert DNA fragment AB into the pYLCRISPR / Cas9-MH vector. First, the circular pYLCRISPR / Cas9-MH plasmid vector was digested with BstB1, and the linearized pYLCRISPR / Cas9-MH vector was recovered by agarose gel electrophoresis. Then, DNA fragment A was ligated into the linearized vector using T4 DNA ligase. The reaction products were transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for shaking culture and Sanger sequencing verification. The sequencing primers MH-F (SEQ ID No. 13) were used, such as CGGTGTCATCTATGTTACTAG, and the sequencing primers MH-R (SEQ ID No. 14) were used, such as CCGACATAGATGCAATAACTTC. Finally, it was confirmed that DNA fragments A and B were correctly constructed into the pYLCRISPR / Cas9-MH vector.

[0026] 2.2 Rice Conversion: Using the japonica rice variety Nipponbare as the recipient, the constructed pYLCRISPR / Cas9-MH vector was transformed into rice callus tissue via Agrobacterium-mediated transformation (strain EHA105). Positive transgenic plants were screened using hygromycin (50 mg / L), ultimately yielding 20 T0 generation transgenic plants. Rice transformation was performed by Wuhan Boyuan Company. Preliminary PCR detection of the pYLCRISPR / Cas9-MH vector revealed the following sequences: Cas9-F sequence (SEQ ID No. 15: GTCGCCTACCACGAGAAGTA), and Cas9-R sequence (SEQ ID No. 16: GTGAGGTCCTGGTGGTGCTC). This confirmed successful vector transfer into the transgenic plants.

[0027] Example 3 ​ Mutant identification and phenotypic analysis 3.1 Gene Editing Detection: Genomic DNA was extracted from T0 generation transgenic plants, and PCR amplification and sequencing were performed using primers near the target site. ​ The sequencing primers used included the aF sequence (SEQ ID No. 17) as follows: CAACCACCTTCCTTCTTGCT, and the aR sequence (SEQ ID No. 18) as follows: TAGTGGCACGGGTAAACCTT. PCR sequencing results showed... ​ The target site contains an 11 bp base deletion ( ​ A, B), causing the 245th bit to terminate prematurely ( ​ D). ​ The target site contains a 1 bp base deletion ( ​A, C), causing the 186th bit to terminate prematurely ( ​ D). The T0 generation transgenic mutant was self-crossed to obtain the T1 generation, which was then further validated by sequencing. ​ Gene knockout results in homozygous mutant lines.

[0028] 3.2 ​ Phenotypic determination of gene knockout mutants: Wild-type Nipponbare was cultivated under summer field conditions in Yangzhou. ​ Mutant strain, under normal water and fertilizer management. Observation of agronomic traits revealed that, compared with the wild type, ​ The mutants showed no significant differences in grain shape, plant height, tiller number, etc. ​ ).

[0029] Further studies on wild-type and ​ When the seeds of the mutant were ground into polished rice, the chalky phenotype was observed, revealing that compared to the wild-type Nipponbare, it exhibited a different appearance. ​ The mutant showed a highly significant increase in chalkiness and chalky grain rate, increasing by approximately 73% and 80%, respectively. ​ ).

[0030] Example 4 ​ Construction of overexpression vectors and rice transformation: according to ​ Based on the coding region sequence, primers for the overexpression vector were designed. OE-F primer sequences (SEQ ID No. 19) are as follows: ttcgagctcagatctggtaccATGATGGGGAGCGAGTCGG; OE-R primer sequences (SEQ ID No. 20) are as follows: acgggggactctagtggatccTCACTTCATTTCATTCTCACCGC. PCR amplification was then performed. ​ The coding region sequence was obtained by glue recovery. ​ The DNA fragment was obtained. The pCAMBIA1300 vector was digested with Kpn1 and BamH1 enzymes, and the linearized pCAMBIA1300 vector was obtained by gel purification. Homologous recombinase was used to... ​ The gene fragment was homologously recombinated into the overexpression vector pCAMBIA1300. The reaction product was transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for culture by shaking and Sanger sequencing for verification, ultimately confirming the positive results. ​ It was correctly constructed onto the pCAMBIA1300 vector.

[0031] Using the japonica rice variety Nipponbare as the recipient, the pCAMBIA1300 vector constructed above was transformed into rice callus tissue via Agrobacterium-mediated transformation. Positive transgenic plants were screened using hygromycin, and 20 T0 generation transgenic plants were finally obtained. Rice transformation was completed by Wuhan Boyuan Company.

[0032] Example 5 ​ Identification and phenotypic analysis of overexpression plants 5.1 ​ Gene overexpression level detection: RNA was extracted from leaves of wild-type and transgenic plants and reverse transcribed into cDNA. The qRT-PCR primers (qRT-PCR-F sequence SEQ ID No. 21, e.g., AGAAACGACCGCCTCTAACC) and qRT-PCR-R sequence (SEQ ID No. 22, e.g., GGATGGGGAAGTTGGTCGAG) were used. qRT-PCR results showed that the overexpressing plants… ​ Expression levels were significantly higher compared to wild type ( ​ ).

[0033] 5.2 ​ Phenotypic determination of plants with gene overexpression: Wild-type Nipponbare and overexpression plants were planted in open fields in Yangzhou during the summer, with normal water and fertilizer management. Agronomical traits were observed, and compared to the wild type, ​ There were no significant differences in thousand-grain weight, grain type, plant height, and tiller number among the gene-overexpressing mutants. ​ ).

[0034] Further analysis of the seeds of wild-type and overexpression plants into polished rice revealed that, compared with the wild-type Nipponbare, the chalkiness and chalky grain rate of the overexpression plants were significantly reduced, decreasing by approximately 50% and 33%, respectively. ​ ).

[0035] The above research shows ​ It is a novel gene that regulates chalkiness. Overexpression of this gene can significantly reduce the occurrence of chalkiness in rice, which plays an important role in improving rice quality.

[0036] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0037] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0038] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A multidrug-resistant efflux transport mechanism OsMRET1 Genes, characterized by, The nucleotide sequence of its coding region is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No.

2.

2. The rice according to claim 1 OsMRET1 Any of the following applications of genes: (1) Regulating the chalky trait in rice; (2) Used for rice variety improvement; (3) Used to prepare genetically modified rice.

3. The application according to claim 2, characterized in that, The chalkiness trait of rice includes chalkiness degree and chalky grain rate.

4. A method for improving the chalkiness and chalky grain rate of rice, characterized in that, include: Using genetic engineering techniques to knock out the gene in rice OsMRET1 Gene.

5. The method according to claim 4, characterized in that, by OsMRET1 Using genes as targets, CRISPR-based sgRNA primer sequences were designed. DNA fragments containing the primer sequences encoding the sgRNA were ligated into vectors carrying CRISPR / Cas9, transformed into rice, and thus obtained... OsMRET1 Genetically modified rice with missing gene function.

6. The method according to claim 5, characterized in that, OsMRET1 The target site is located in exon 4, and the sequence is shown in SEQ ID No. 3 and SEQ ID No.

4.

7. The method according to claim 5, characterized in that, sgRNA primers include: OsMRET1 The primer sequences for gRT1 of the gene are shown in SEQ ID No. 5, the primer sequence for OsU3T1 is shown in SEQ ID No. 6, the primer sequence for gRT2 is shown in SEQ ID No. 7, and the primer sequence for OsU6aT2 is shown in SEQ ID No.

8.

8. A method for reducing the chalkiness and chalky grain rate of rice, characterized in that, include: Using genetic engineering techniques, overexpressing rice OsMRET1 Gene.

9. The use of the transgenic rice obtained by the method of any one of claims 4 to 8 in plant breeding.

Images