New rice Gn1a allele Gn1aG156D and application thereof in breeding

Abstract

The invention relates to the technical field of molecular breeding, in particular to a novel rice Gn1a allele Gn1a G156D and application thereof in breeding. The invention provides a new allele Gn1a G156D of the rice Gn1a gene and a cytokinin oxidase / dehydrogenase OsCKX2 mutant coded by the allele, the allele can improve the ear type (including ear length, primary branch number and secondary branch number) and increase the yield, and can be used for improving germplasm resources of plants and cultivating new varieties of high-yield plants; the method has important application value in the field of plant breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more particularly to new rice varieties. Gn1a Allele Gn1a G156D And its application in breeding. Background Technology

[0002] Rice ( Oryza sativa L. Rice is an indispensable staple food for nearly half the world's population and the third most cultivated crop globally. With the continued growth of the global population and the reduction of arable land posing challenges to global food security, increasing yield remains a key objective of agricultural research. Rice yield is influenced by many factors, primarily determined by the number of panicles per plant, the number of grains per panicle, and grain weight. The main components of a rice panicle include the number of spikelets per panicle, panicle length, and the number of primary and secondary branches. Therefore, improving panicle structure represents a crucial strategy for increasing rice yield.

[0003] Plant hormones are key factors determining panicle type and yield in rice, among which cytokinins (CKs) exert their influence by precisely regulating the activity of panicle meristems. CK signaling in rice is sensed through membrane-localized kinase receptors OsHKs / OHKs. Inactivation of OsHK4 / OHK4, OsHK5 / OHK3, and OsHK6 / OHK5 leads to a reduction in panicle length, branch number, and grain number. Type A and Type B response regulators (RRs) are downstream response factors to cytokinin signaling. Overexpression or loss of function of some A-RRs or B-RRs results in similar changes in panicles with receptor mutations.

[0004] In addition to CK signal transduction, maintaining CK homeostasis is also crucial for regulating rice panicle structure and determining grain yield. LONELY GUY ( LOG It encodes a CK activating enzyme that directly converts inactive CK nucleosides into free active CK. log The mutant stops generating inflorescence meristems, resulting in a reduction in branches and spikelets. GRAIN YIELD 3 ( GY3 This is a major QTL for rice yield, encoding LOGL5, and involved in a two-step pathway of CK synthesis. (Inhibition) GY3 This leads to the production of more active CK in the panicle, thereby increasing rice yield.

[0005] CK homeostasis is also regulated by cytokinin oxidase / dehydrogenase (CKX), whose activity catalyzes the irreversible degradation of biologically active CKs. The enzyme encoding OsCKX2... GRAIN NUMBER 1a ( Gn1a ) is the main QTL for the number of grains per panicle of rice. Gn1aLoss of function or reduced expression of CK leads to accumulation of CK in the inflorescence meristem, thereby increasing spike length and grain number, ultimately promoting yield. OsCKX4 or OsCKX9 Overexpression of this leads to smaller ears, fewer branches, and fewer kernels. Recent studies have shown that... Gn1a It is a key regulator in rice panicle development and yield formation. Multiple upstream genetic components converge on this enzyme hub to coordinate inflorescence morphogenesis, such as the DENSE AND ERECT PANICLE1 (DEP1)-GRAIN NUMBER ASSOCIATED (GNA) module, chromatin interaction factors, etc.

[0006] Given Gn1a Its significant contribution to rice yield has led to the widespread use of its natural alleles in excellent high-yielding varieties such as Habataki, Shuhui498, Yihui3551, and 93-1. These varieties all belong to the indica rice family. To our knowledge, Gn1a Successful application of allelic variation in japonica rice breeding remains limited. On one hand, this difference may stem from varying selective pressures during the domestication of japonica rice. On the other hand, interspecific hybridization sterility remains a major reproductive obstacle, making it difficult to successfully integrate alleles into the rice variety. Gn1a The high-yielding allele was introduced from indica rice into japonica rice. Recent studies have shown that knocking out the high-yielding allele in japonica rice using CRIPR / Cas9... Gn1a It can also increase yield, which means that the use of inactive [products] in molecular breeding of japonica rice [is possible]. Gn1a It has application potential. Summary of the Invention

[0007] In this invention, we identified a superior japonica rice variety... Gn1a The three alleles serve as the CK receptor gene. OHK4 The suppressors. These alleles partially saved [their lives]. ohk4 The morphology of the mutants included plant height, number of initial tillers, ear length, number of branches, and number of grains. Further analysis showed that the inhibition of OsCKX2 activity was significantly reduced in the mutants, leading to CK accumulation in the ear. Through hybridization, the inactive... Gn1a Introducing the improved lines into a wild-type background resulted in increased tiller number, grains per spike, and yield per plot. Based on this, the present invention proposes the following technical solution.

[0008] First, the present invention provides a cytokinin oxidase / dehydrogenase OsCKX2 mutant, which is based on wild-type cytokinin oxidase / dehydrogenase OsCKX2, in which the 156th amino acid glycine (G) is replaced by aspartic acid (D), and the amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0009] Furthermore, the present invention provides a nucleic acid encoding the mutant.

[0010] Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2; the nucleic acid is Gn1a Alleles Gn1a G156D .

[0011] Gn1a Alleles Gn1a G156D In the wild type Gn1a Based on the gene, the 467th base in the coding region was mutated from G to A (position 623 of SEQ ID NO.1), resulting in Gn1a In the encoding OsCKX2, amino acid glycine at position 156 is replaced by aspartic acid, and the wild-type Gn1a The gene sequence is shown in SEQ ID NO.1.

[0012] The mutation method G156D described in this invention is a commonly used amino acid mutation site representation in the art, where G represents the amino acid before mutation, 156 represents the mutation site, and D represents the amino acid after mutation. One way to achieve this mutation is in rice. Gn1a A mutation occurs at position 467 of the start codon in the gene coding region, changing from G to A.

[0013] Furthermore, the present invention provides biological materials containing the mutant or the nucleic acid.

[0014] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable cells.

[0015] Furthermore, the present invention provides the use of the said mutant, the said nucleic acid, or the said biological material in at least one of the following aspects: (1) Reduce the activity of cytokinin oxidase / dehydrogenase OsCKX2 and increase the content of cytokinin CK in the spike of the plant; (2) Increase plant height; (3) Increase the number of plant tillers; (4) Increase the length of plant spikelets; (5) Increase the number of primary and / or secondary branches of the plant; (6) Increase the number of grains per spike in plants.

[0016] Furthermore, the present invention provides the application of the mutant, the nucleic acid, or the biological material in plant breeding or plant germplasm resource improvement.

[0017] Preferably, the plant is a monocotyledonous plant or a dicotyledonous plant, and more preferably rice.

[0018] Furthermore, the present invention provides a method for preparing a transgenic plant, comprising introducing the mutant, the nucleic acid, or the biological material into the plant.

[0019] Preferably, the introduction is performed using genetic engineering or hybridization techniques; Preferably, the genetic engineering method includes: inserting a sample containing the nucleic acid ( Gn1a Allele Gn1a G156D The expression vectors of Ti were introduced into plant cells using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, or electroporation. Preferably, the hybridization method includes: mixing plant seeds with a mixture containing the nucleic acid ( Gn1a Allele Gn1a G156D Hybridization of plant seeds.

[0020] The above preparation method is particularly suitable for japonica rice breeding.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides rice Gn1a New alleles of the gene Gn1a G156D And its encoded cytokinin oxidase / dehydrogenase OsCKX2 mutant, which can improve spike type (including spike length, number of primary branches and secondary branches) and increase yield, can be used for plant germplasm resource improvement and breeding of high-yielding plant varieties, and has important application value in the field of plant breeding. Attached Figure Description

[0022] Figure 1 The wild-type Huai rice 5 and spikelet mutant provided in Example 1 of this invention pal1 and pal1 Suppressor pal1s205 The phenotypes were analyzed as follows: A represents the plant morphology at maturity, B represents the panicle type in the closed state, C represents the panicle type in the unfolded state, and D and I represent the statistical analysis of plant height, number of tillers, panicle length, number of primary branches, number of secondary branches, and number of grains per panicle.

[0023] Figure 2 Provided for Embodiment 2 of the present invention pal1s205 Reversal mutant in Gn1a Analysis of gene mutation sites; where A is... pal1s205 Schematic diagram of base mutations and amino acid changes. B shows the conservation analysis of the mutated amino acid G156 in homologous proteins of different species. C shows the activity analysis of the mutated protein. D shows the wild-type Huai rice 5. pal1 mutants and pal1s205 Comparison of CK content in young spikelets of mutants.

[0024] Figure 3 The wild type provided in Embodiment 3 of the present invention pal1 mutants and Gn1a Phenotypic comparison of knockout line plants; where A represents... Gn1a A schematic diagram of the target sites for the knockout line. B represents the plant morphology at maturity, C represents the ear type in the closed state, D represents the ear type in the unfolded state, and E and J represent the statistical analysis of plant height, number of tillers, ear length, number of primary branches, number of secondary branches, and number of grains per ear, respectively.

[0025] Figure 4 The present invention, in embodiment 4, provides a background of Huai Dao 5 carrying... Gn1a G156D homozygous mutants of the new allele gn1a G156D Phenotypic analysis; where A represents plant morphology at maturity, B represents statistical analysis of plant height, C represents statistical analysis of tiller number, D represents ear type in the expanded state, E represents ear type in the closed state, and F and J represent statistical analysis of ear length, number of primary branches, number of secondary branches, number of grains per ear, and plot yield, respectively. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0027] Example 1: Reversal Mutant pal1s205 Acquisition and phenotypic analysis In previous studies, through 60 Co-γ radiation-induced mutagenesis of the japonica rice variety Huai Dao 5 yielded a short-spike mutant. pal1 ( panicle length 1 ), its mutated gene PAL1 Encoding the CK receptor OHK4 / OsHK4. Compared with Huaidao 5, pal1 The mutant ears were shorter, with significantly fewer branches and grains. In this study, EMS mutagenesis... pal1A suppressor mutant was identified from the mutant library. pal1s205 (This material is stored in Room 515, Major Engineering Building, Institute of Crop Science, Chinese Academy of Agricultural Sciences. It is available to the public from the applicant and is used solely for verifying this invention.) pal1 Compared to mutants, pal1s205 The panicle length, number of primary branches, number of secondary branches, and number of grains per panicle all increased significantly, and nearly returned to the level of Huai Dao 5.

[0028] Reversal mutant pal1s205 EMS mutagenesis pal1 Long spike mutants (phenotypic reversal mutants) screened from mutant libraries Figure 1 (AC). Compared to the wild type, pal1 The mutant has a decreased plant height and an increased number of tillers. Figure 1 (A, DE). Statistical analysis also shows that rice pal1 The mutant ear length was reduced by 30.80% compared to the wild type, the number of primary and secondary branches was reduced by 20.00% and 38.51% respectively, and the number of grains per ear was reduced by 24.00% compared to the wild type. Figure 1 (FI). pal1 Compared to the mutant, the revertant mutant pal1s205 Plant height increases, tiller number decreases ( Figure 1 (A, DE). Statistical analysis also showed that the reversion mutant... pal1s205 ear length pal1 The mutant increased by 42.99%, and the number of primary and secondary branches increased by [percentage missing]. pal1 The mutant strain increased by 13.98% and 55.87%, respectively, and the number of grains per ear was higher than that of the mutant strain. pal1 The mutant count increased by 27.37% ( Figure 1 FI).

[0029] Example 2: Reversal Mutant pal1s205 Gene positioning Restore mutant pal1s205 and pal1 The mutants were crossed to obtain the F1 generation, and the F1 generation was self-crossed to obtain the F2 segregating population. Genetic analysis and gene mapping were performed on the F2 population. Analysis of the F2 generation lines that showed phenotypic segregation indicated that the mutant plants and normal plants had a segregation ratio of 3:1, suggesting that the mutant trait is controlled by a pair of dominant genes.

[0030] Analysis of reversal mutants using the Mutmap method pal1s205 Candidate genes. From pal1 and pal1s205From the F3 progeny produced by hybridization, 30 individuals exhibiting wild-type and 30 individuals exhibiting mutant phenotypes were selected. Genomic DNA was extracted from leaves of these individuals and uniformly mixed to form two DNA libraries. Whole-genome sequencing was then performed on the DNA from the mixed libraries. Candidate genes were screened according to the criteria of SNP index1 ≥ 0.9 and index2 ≤ 0.5, identifying one candidate SNP. Gene annotation showed... pal1s35 The SNP identified is located at the end of the fourth exon of LOC_Os01g10110, where the 467th base G in the coding region is mutated to A, resulting in the substitution of the 156th amino acid, glycine (G), with aspartic acid (D). Figure 2 (A). The function comment indicates that LOC_Os01g10110 is the previously reported... Gn1a ( Grain Number 1a The gene encodes OsCKX2, a cytokinin oxidase / dehydrogenase that catalyzes the degradation of active cytokinin (CK). Gn1a Rice yield can be controlled by regulating cytokinin, panicle length, and spikelet number.

[0031] The amino acid sequence of wild-type OsCKX2 was further aligned using BLAST in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Homologous proteins of OsCKX2 in different plant species were downloaded, and the conservation of mutant amino acids in different species was analyzed using MEGA11 and GENEDOC. The results showed that the amino group at position 156 of OsCKX2 in rice is highly conserved. Figure 2 The presence of residue B indicates that this residue is important for the function of the OsCKX2 protein. A review of literature and relevant databases currently... Gn1a The G467A mutation site has not been reported and there is no natural variation, therefore Gn1a G156D It is a new Gn1a allele.

[0032] To further construct the pCold-MBP-OsCKX2 plasmid, the OsCKX2 coding region fragment was amplified from the japonica rice variety Nipponbare, with a PCR product size of 1698 bp. Then, it was cloned into the NdeI and EcoRI sites of the pCold-MBP vector using the In-Fusion HD cloning kit (Clontech). To produce the recombinant protein, the constructed expression vector was introduced into *E. coli* BL21(DE3)pLysS cells. The bacterial cells were cultured in Luria-Bertani (LB) medium at 37°C to 0.6–0.8 (OD200). 600Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and the mixture was continuously cultured at 16°C for 10 hours. The cells were collected by centrifugation at 4000 rpm for 10 minutes at 4°C, and then lysed using an ultrasonic homogenizer at 4°C. The lysate was centrifuged at 12000 rpm for 30 minutes at 4°C, and amylase resin (NEB) was added to the supernatant. The mixture was then gently shaken at 4°C for 1 hour, centrifuged at 2000 rpm for 1 minute, the supernatant was discarded, and elution buffer was added. Finally, the purified recombinant protein was desalted using a BeyoDesalt™ G-25 Spin desalting column (Beyotime).

[0033] The degradation activity of CKX2 against cytokinins was determined using a modified endpoint method. The reaction system consisted of 75 mM Tris / HCl buffer (pH 8.5), 500 μM 2,6-dichlorophenol indophenol (Sigma-Aldrich) (as electron acceptor), 250 μM 2iP (Sigma-Aldrich) (2iP dissolved in DMSO as a reaction substrate), and MBP-OsCKX2 recombinant protein (the total volume of the reaction mixture in a 1.5 mL tube was 0.6 mL). After adding the recombinant protein, the reaction mixture was incubated at 37°C for 1 hour. The enzymatic reaction was stopped by adding 0.3 mL of 40% (m / v) trichloroacetic acid (TCA), and the sample was then centrifuged at 12,000 rpm for 5 minutes to remove protein precipitate. Then, 0.2 mL of 4-aminophenol [2% (m / v) solution in 6% (m / v) trichloroacetic acid] was added to the supernatant, and the absorption spectrum at wavelengths of 300 to 700 nm was immediately (within 3 minutes) scanned to determine the concentration (ε) of the Schiff base product for a specific substrate. 352 = 15.2mM -1 cm -1 In the control experiment, 15 μL of DMSO was used as a blank control instead of the substrate. The results showed that, compared to the wild-type protein (OsCKX2), the mutant protein OsCKX2... G156D Activity decreased by 64.76% ( Figure 2 (C).

[0034] We further tested the wild-type Huai Rice No. 5 (WT). pal1 mutants and pa1ls205 The CK content in the young spikelets of the mutant showed that... pal1 In comparison, pa1ls205 The concentrations of tZ and iP were significantly increased in the mutant. Figure 2(D).

[0035] Example 3: Knockout using the CRISPR / Cas9 system Gn1a To confirm pa1ls205 Phenotypic reversion in mutants is caused by Gn1a Caused by mutation, a gene editing system was constructed using CRISPR / Cas9. Gn1a exist pal1 Loss-of-function mutants in the context of ( Figure 3 (A). The construction method is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a format Gn1a The target subcloned into the sgRNA intermediate vector, and then cloned into the target vector pYLCRISPR / Cas9Pubi-H. The construct was introduced into rice callus tissue via Agrobacterium-mediated transformation (primers used are shown in Table 1).

[0036] Table 1. Primer sequences associated with Gn1a gene knockout

[0037] and pal1 Compared to two homozygous mutant lines with gene knockout ( pal1 gn1a-ko1 , pal1 gn1a-ko2 All of them exhibited the characteristics of increased plant height and decreased tiller number. Figure 3 B, EF). Simultaneously, the length of the spikelet, the number of primary branches, and the number of secondary branches in the knockout line all significantly increased (B, EF). Figure 3 The CD and GI values ​​also increased significantly, and the number of grains per ear also increased significantly. Figure 3 J), which is pa1ls205 The mutants exhibited similar characteristics. Except for having more tillers than WT, the knockout lines showed no significant difference in other phenotypic numbers compared to WT, indicating that... pa1ls205 Phenotype is determined by Gn1a This is caused by the loss of function.

[0038] Example 4 Gn1a G156D Breeding applications To further clarify Gn1a G156D The contribution of alleles to rice yield, we will include... Gn1a G156D of pa1ls205 The mutant was crossed with wild-type Huaidao 5, and backcrossed using Huaidao 5 as a recurrent parent, ultimately yielding a mutant containing the Huaidao 5 gene. Gn1a G156D Allele lines and named gn1a G156D .

[0039] Compared with Huai Dao No. 5, gn1aG156D The plant height and the number of tillers both increased. Figure 4 (AC). Meanwhile, statistical data shows that compared to Huai Dao No. 5, gn1a G156D The plant spike length increased by 5.51%, the number of primary branches and secondary branches increased by 18.94% and 18.69% respectively, and the number of grains per spike increased by 30.19%, ultimately leading to a yield increase of 20.61%-21.76% in the plot. Figure 4 DJ).

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cytokinin oxidase / dehydrogenase OsCKX2 mutant, characterized in that, Based on the wild-type cytokinin oxidase / dehydrogenase OsCKX2, the 156th amino acid glycine (G) is replaced by aspartic acid (D), and the amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. The nucleic acid encoding the mutant of claim 1.

3. The nucleic acid according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.2; the nucleic acid is Gn1a Allele Gn1a G156D .

4. Biological material containing the mutant of claim 1 or the nucleic acid of claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-regenerative cells.

6. The use of the mutant of claim 1, the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5 in at least one of the following aspects: (1) Reduce the activity of cytokinin oxidase / dehydrogenase OsCKX2 and increase the content of cytokinin CK in the spike of the plant; (2) Increase plant height; (3) Increase the number of plant tillers; (4) Increase the length of plant spikelets; (5) Increase the number of primary and / or secondary branches of the plant; (6) Increase the number of grains per ear of plant.

7. The application of the mutant of claim 1, the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5 in plant breeding or plant germplasm resource improvement.

8. The application according to claim 6 or 7, characterized in that, The plant is a monocotyledonous or dicotyledonous plant, preferably rice.

9. A method for preparing a transgenic plant, characterized in that, This includes introducing into plants the mutant of claim 1, the nucleic acid of claim 2 or 3, or the biological material of claim 4 or 5.

10. The preparation method according to claim 9, characterized in that, The importation is performed using genetic engineering or hybridization techniques; Preferably, the genetic engineering method includes: introducing an expression vector carrying the nucleic acid of claim 2 or 3 into plant cells using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, or electroporation. Preferably, the hybridization method includes: hybridizing plant seeds with plant seeds containing the nucleic acid described in claim 2 or 3.

Images