New rice Gn1a allele Gn1aG556D 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 G556D and application of the novel rice Gn1a allele Gn1a G556D in breeding. The invention provides a new allele Gn1a G556D of the rice Gn1a gene and a cytokinin oxidase / dehydrogenase OsCKX2 mutant encoded by the same, and the allele can improve the panicle type structure and increase the yield, can be used for improving germplasm resources of plants and cultivating new varieties of high-yield plants, and has important application value in the field of plant breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, and particularly relates to a new Gn1a allele Gn1a G556D and its application in breeding. BACKGROUND

[0002] Rice (Oryza sativa L.) is the staple food for nearly half of the world's population and the third largest cultivated crop in the world. With the continued growth of the global population and the reduction of arable land, global food security is being challenged, and improving yield remains a key goal of agricultural research. Rice yield is affected by many factors, mainly determined by panicle number per plant, grain number per panicle and grain weight. The main factors affecting grain number per panicle include panicle length, primary branch and secondary branch number and spikelet number per panicle, so panicle structure improvement is one of the important ways to improve rice yield. Oryza sativa L. In recent years, several genes that change panicle structure and ultimately affect yield have been characterized, such as

[0003] GRAIN ( NUMBER1a )、 Gn1a ( DENSE AND ERECTPANICLE1 )、 DEP1 ( Frizzy panicle )、 FZP 、 ABERRANT PANICLE ORGANIZATION1 ( APO1 )、 APO2 / RICE FLORICAULA ( RFL )、 ABERRANT SPIKELET AND PANICLE1 ( ASP1 )、 CLASS I KNOTTED1-LIKE HOMEOBOX ( KNOX1 ) 、 PANICLE LENGTH1 ( PAL1 )。 Gn1a is the first major QTL identified to be associated with grain number per panicle, which encodes a cytokinin oxidase / dehydrogenase (OsCKX2) that is an enzyme capable of catalyzing the degradation of active cytokinins (CKs). The plant hormone cytokinin plays a decisive role in the activity of meristems, Gn1a mutation or reduced expression will lead to the accumulation of cytokinin in the inflorescence meristem and increase the panicle length, branch number and grain number per panicle, ultimately leading to an increase in rice grain yield. Some other genes can also affect CK levels and panicle structure in vivo by directly or indirectly regulating the expression of Gn1a DROUGHT AND SALT TOLERANCE ( DST )、 Frizzy panicle ( FZP )、 ERECTA1 ( OsER1 )。​LARGER PANICLE ( LP )and VIN3-LIKE 2 ( OsVIL2 ).Although Gn1a The allele has been successfully used in indica rice breeding and has increased grain yield, but its application in japonica rice breeding is still less. However, due to the reduced fertility or even sterility of hybrid offspring between indica and japonica rice, it is difficult to use traditional breeding methods to remove the allele in indica rice. Gn1a It becomes very difficult to use alleles for japonica rice breeding.

[0004] Therefore, further exploration Gn1a The alleles of japonica rice, especially the superior alleles in japonica rice, will provide a theoretical basis and genetic resources for high-yield breeding of japonica rice. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, first, the present invention provides a cytokinin oxidase / dehydrogenase OsCKX2 mutant, which is based on the wild-type cytokinin oxidase / dehydrogenase OsCKX2, in which the 556th 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.

[0006] Furthermore, the present invention provides a nucleic acid encoding the mutant.

[0007] Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2; the nucleic acid is Gn1a Allele Gn1a G556D .

[0008] Gn1a Allele Gn1a G556D In the wild type Gn1a Based on the gene, the 1667th base of the coding region mutated from G to A (i.e., the 4813th base of SEQ ID NO.1), resulting in Gn1a The amino acid glycine at position 556 of the encoded OsCKX2 was replaced by aspartic acid. Gn1a The gene sequence is shown in SEQ ID NO.1.

[0009] The mutation method G556D described in the present invention is a commonly used method for expressing amino acid mutation sites in the art, where G is the amino acid before mutation, 556 is the mutation site, and D is the amino acid after mutation. One way to achieve this mutation can be rice. Gn1a The 1667th base from the start codon of the gene coding region mutated from G to A.

[0010] Further, the present application provides a biological material containing the mutant or the nucleic acid.

[0011] Preferably, the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a non-renewable cell.

[0012] Further, the present application provides an application of the mutant, the nucleic acid or the biological material in at least one of the following aspects: (1) weakening the activity of cytokinin oxidase / dehydrogenase OsCKX2 and increasing the content of cytokinin CK in the ear part of a plant; (2) increasing the plant height; (3) increasing the tiller number of a plant; (4) increasing the ear length of a plant; (5) increasing the number of primary branches and / or secondary branches of a plant; (6) increasing the grain number per ear of a plant.

[0013] Further, the present application provides an application of the mutant, the nucleic acid or the biological material in plant breeding or improvement of plant germplasm resources.

[0014] Preferably, the plant is a monocotyledon or a dicotyledon, preferably rice.

[0015] Further, the present application provides a preparation method of a transgenic plant, comprising introducing the mutant, the nucleic acid or the biological material into a plant.

[0016] Preferably, the introduction is performed by using genetic engineering means or hybridization means. Preferably, the genetic engineering means comprises: introducing an expression vector carrying the nucleic acid (allele) into a plant cell by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection or electroporation technology. Gn1a allele Gn1a G556D Preferably, the hybridization means comprises: hybridizing plant seeds with plant seeds containing the nucleic acid (allele). allele Gn1a Gn1a G556D Preferably, the hybridization means comprises: hybridizing plant seeds with plant seeds containing the nucleic acid (allele).

[0017] The above preparation method is especially suitable for breeding of japonica rice.

[0018] Compared with the prior art, the present application has the following beneficial effects: The present application provides a new allele of a rice gene Gn1a gene Gn1a G556D ​And its encoded cytokinin oxidase / dehydrogenase OsCKX2 mutant, the allele can improve the structure of ear type and increase yield, can be used for germplasm improvement of plants and breeding of new plant varieties with high yield, and has important application value in the field of plant breeding. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Wild type Huai rice No. 5, spikelet mutant provided for example 1 of the present application pal1 And pal1 Phenotype of suppressor pal1s35 ; wherein A is the plant morphology at the mature stage, B is the ear type in the closed state, C is the ear type in the expanded state, and D-I are the statistical analysis of plant height, tiller number, ear length, primary branch number, secondary branch number and grain number per ear, respectively.

[0020] Figure 2 Mutation site analysis of suppressor in pal1s35 Gene Gn1a ; wherein A is pal1s35 Base mutation and amino acid change schematic diagram, B is the conservation analysis of mutated amino acid G556 in the homologous proteins in different species, C is the activity analysis of the mutant protein, D is the comparison of CK content in the young ear of wild type Huai rice No. 5, pal1 Mutant and suppressor pal1s35 .

[0021] Figure 3 Phenotype comparison chart of wild type, pal1 Mutant and Gn1a Knockout line plant provided for example 3 of the present application; wherein A is Gn1a Target site schematic diagram of knockout line, B is the plant morphology at the mature stage, C is the ear type in the closed state, D is the ear type in the expanded state, E-J are the statistical analysis of plant height, tiller number, ear length, primary branch number, secondary branch number and grain number per ear, respectively.

[0022] Figure 4 Phenotype analysis of pure mutant carrying Gn1a G556D New allele in the background of Huai rice No. 5 provided for example 4 of the present application gn1a G556D ; wherein A is the plant morphology at the mature stage, B is the statistical analysis of plant height, C is the statistical analysis of tiller number, D is the ear type in the expanded state, E is the ear type in the closed state, and F-J are the statistical analysis of ear length, primary branch number, secondary branch number, grain number per ear and plot yield, respectively. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application. In the embodiments provided in the present specification, the specific techniques or conditions not noted are implemented according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not noted the manufacturers are the conventional products that can be purchased through the regular channels.

[0024] Example 1 Suppressor mutant pal1s35 Obtaining and phenotype analysis In previous studies, by 60 Co-γ radiation mutagenesis of japonica rice variety Huaimang 5, a short panicle mutant pal1 ( panicle length 1 ) was obtained, and the mutant gene PAL1 encodes CK receptor OHK4 / OsHK4. Compared with Huaimang 5, the mutant has shorter panicles, and the number of branches and grains is significantly reduced. In the present study, a suppressor mutant (preserved in the Major Engineering Building 515 of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and the material can be obtained from the applicant only for verifying the present application) was screened from the mutant library induced by EMS mutagenesis. Compared with the mutant, the panicle length, the number of primary branches, secondary branches and grains per panicle are significantly increased, and close to the level of Huaimang 5. pal1 pal1 The present application obtains a short panicle mutant pal1s35 ( pal1 ) by 60 Co-γ radiation mutagenesis of japonica rice variety Huaimang 5, and the suppressor mutant is a long panicle mutant (phenotype suppressor mutant) screened from the mutant library induced by EMS mutagenesis (A-C of FIG. 1). Compared with the wild type, the mutant has reduced plant height and increased tiller number (A, D-E of FIG. 1). Meanwhile, statistical analysis shows that the panicle length of the mutant is reduced by 30.80% compared with the wild type, the number of primary branches and secondary branches is reduced by 20.00% and 38.51% respectively compared with the wild type, and the number of grains per panicle is reduced by 24.00% compared with the wild type (F-I of FIG. 1). pal1s35

[0025] pal1 panicle length 1 pal1s35 pal1 Figure 1 pal1 Figure 1 pal1 Figure 1 pal1 ​​​​​​​​​​​​Compared with mutants, revertants pal1s35 The plant height increased and the number of tillers decreased ( Figure 1 A, DE). Statistical analysis also showed that the revertant pal1s35 Ear length pal1 The mutant increased by 46.05%, and the number of primary and secondary branches was pal1 The mutants increased by 25.00% and 67.60%, and the number of grains per ear was pal1 Mutants increased by 34.84% ( Figure 1 of FI).

[0026] Example 2 Revertant pal1s35 Gene mapping Revertant pal1s35 and pal1 The mutants were crossed to obtain F1 lines, which were then self-pollinated to obtain an F2 segregating population. This F2 population was used for genetic analysis and gene mapping. Analysis of F2 lines segregating for the trait showed a 3:1 segregation ratio between the mutant and normal individual plants, indicating that the mutant trait is controlled by a pair of dominant genes.

[0027] Analysis of revertant mutants using the Mutmap method pal1s35 Candidate genes from pal1 and pal1s35 From the F3 progeny produced by the hybridization, 30 individuals with wild-type phenotype and 30 individuals with mutant phenotype were selected to extract leaf genomic DNA, which was evenly mixed to form two DNA pools. The DNA of the mixed pool was then subjected to whole-genome sequencing. Candidate gene screening was based on the criteria of SNP index1 ≥ 0.9 and index2 ≤ 0.5. A candidate SNP was identified, and the gene annotation showed pal1s35 The SNP identified in the study is located at the end of the fourth exon of LOC_Os01g10110. The 1667th base G in the coding region mutates to A, resulting in the 556th amino acid glycine (G) being replaced by aspartic acid (D). Figure 2 Functional annotation showed that LOC_Os01g10110 was previously reported as Gn1a ( Grain Number 1a ) gene, which encodes the cytokinin oxidase / dehydrogenase OsCKX2 that catalyzes the degradation of active cytokinin (CK), Gn1a Rice yield is controlled by regulating cytokinins, panicle length, and the number of spikelets.

[0028] Further, the amino acid sequence of wild type OsCKX2 was subjected to Blast alignment in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, and homologous proteins of OsCKX2 in different plant species were downloaded. The conservation of the mutant amino acid in different species was analyzed using MEGA11 and GENEDOC. The results showed that the amino acid at position 556 of OsCKX2 in rice was extremely conserved (B), indicating that this residue is important for the function of OsCKX2 protein. After consulting literature and related databases, the G1667A mutation site has not been reported and there is no natural variation, so Figure 2 Gn1a the G1667A mutation site is a new allele. Gn1a G556D Gn1a

[0029] Further, to construct pCold-MBP-OsCKX2 plasmid, the coding region fragment of OsCKX2 was amplified from the japonica rice variety Nipponbare, and the size of the PCR product was 1698 bp. Then, the In-Fusion HD Cloning Kit (Clontech) was used to clone it into the Ndel and EcoRI sites of the pCold-MBP vector. To produce the recombinant protein, the expression vector constructed above was introduced into Escherichia coli BL21(DE3)pLysS cells. The bacterial cells were cultured in Luria-Bertani (LB) medium at 37°C to 0.6-0.8 (OD 600 ​​​), and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and the continuous culture was continued at 16°C for 10 hours. The bacterial solution was centrifuged at 4000 rpm for 10 minutes at 4°C to collect the cells, and then the cells were lysed at 4°C using an ultrasonic disrupter. The lysate was centrifuged at 12000 rpm for 30 minutes at 4°C, and amylase resin (NEB) was added to the supernatant and rotated gently at 4°C for 1 hour, centrifuged at 2000 rpm for 1 minute, and then discarded. The eluent was added for elution, and finally the purified recombinant protein was desalted using a BeyoDesalt™ G-25 Spin desalting column (Beyotime). The determination of the degradation activity of CKX2 on cytokinin was performed using a modified endpoint method. The reaction system was composed of 75 mM Tris / HCl buffer (pH 8.5), 500 μM 2,6-dichlorophenol indophenol (Sigma-Aldrich) (as an 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 then the sample was centrifuged at 12000 rpm for 5 minutes to remove the protein precipitate. Then, 0.2 mL of 4-aminophenol [2% (m / v) in 6% (m / v) trichloroacetic acid] was added to the supernatant, and then the absorption spectrum of 300 to 700 nm wavelength was immediately scanned (within 3 minutes) to determine the concentration of the Schiff base product of the specific substrate (ε 352 = 15.2 mM -1 cm -1 ). In the control experiment, 15 μL of DMSO was used instead of the substrate for the blank control. The detection results showed that, compared with the wild-type protein (OsCKX2), the mutant protein OsCKX2 G556D activity was reduced by 73.36% ( Figure 2 C).

[0030] Further, we detected the CK content in the young panicles of wild-type Huai rice No. 5, pal1 mutant, and pa1ls35 mutant, and the results showed that the concentrations of tZ and iP were significantly increased in the pa1ls35 mutant compared with the pal1 mutant (D of Figure 2 ).

[0031] Example 3 Knockout using the CRISPR / Cas9 system Gn1a To confirm pa1ls35 The phenotypic reversion of the mutant is determined by Gn1a mutations, and constructed using the CRISPR / Cas9 gene editing system. Gn1a exist pal1 Loss-of-function mutants in the background ( Figure 3 A). The construction method is as follows: Gn1a The target was subcloned into the sgRNA intermediate vector and then cloned into the destination vector pYLCRISPR / Cas9Pubi-H. The construct was introduced into rice callus via Agrobacterium-mediated transformation (primers used are shown in Table 1).

[0032] Table 1 Primer sequences related to Gn1a gene knockout

[0033] and pal1 Compared with the homozygous mutant lines with two gene function knockouts ( pal1 gn1a-ko1 , pal1 gn1a-ko2 ) showed the characteristics of increased plant height and decreased tiller number ( Figure 3 At the same time, the ear length, number of primary branches and number of secondary branches of the knockout line increased significantly ( Figure 3 CD and GI), the number of grains per ear also increased significantly ( Figure 3 J), which is consistent with pa1ls35 The mutants showed similar performance. Except for the number of tillers, the knockout lines had no significant differences in other phenotypic numbers compared with the WT, which indicated that pa1ls35 The phenotype is determined by Gn1a caused by loss of function.

[0034] Example 4 Gn1a G556D Breeding applications To further clarify Gn1a G556D The contribution of alleles to rice yield is Gn1a G556D of pa1ls35 The mutant was hybridized with wild type Huaidao 5 and backcrossed with Huaidao 5 as the recurrent parent. Gn1a G556D Allele strains were named gn1a G556D .

[0035] Compared with Huaidao No. 5, gn1a G556D Plant height and tiller number increased (Figure 4 The statistical data show that, compared with Huaimi 5, gn1a G556D The plant ear length increased by 5.60%, the first branch number and the second branch number increased by 22.38% and 20.32% respectively, the ear grain number increased by 33.59%, and finally led to the plot yield increased by 25.71%-28.74% (P<0.01) Figure 4 (D-J).

[0036] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, rather than limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A cytokinin oxidase / dehydrogenase OsCKX2 mutant, characterized in that The mutant is based on the wild-type cytokinin oxidase / dehydrogenase OsCKX2, with the 556th amino acid glycine (G) replaced by aspartic acid (D). The amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. A nucleic acid encoding the mutant according to 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 G556D .

4. A biological material containing the mutant according to claim 1 or the nucleic acid according to 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, virus vector, engineered bacteria or non-renewable cells.

6. Use of the mutant according to claim 1, the nucleic acid according to claim 2 or 3, or the biomaterial according to claim 4 or 5 in at least one of the following aspects: (1) Weaken the activity of cytokinin oxidase / dehydrogenase OsCKX2 and increase the content of cytokinin CK in the panicle of plants; (2) Increase plant height; (3) Increase the number of plant tillers; (4) Increase plant spike length; (5) Increase the number of primary and / or secondary branches of the plant; (6) Increase the number of grains per ear of the plant.

7. Use of the mutant according to claim 1, the nucleic acid according to claim 2 or 3, or the biological material according to claim 4 or 5 in plant breeding or plant germplasm improvement.

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

9. A method for preparing a transgenic plant, characterized in that: The method comprises introducing the mutant according to claim 1, the nucleic acid according to claim 2 or 3, or the biological material according to claim 4 or 5 into a plant.

10. The preparation method according to claim 9, characterized in that The introduction is carried out by genetic engineering or hybridization; Preferably, the genetic engineering means comprises: introducing an expression vector carrying the nucleic acid according to claim 2 or 3 into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, or electroporation technology; Preferably, the hybridization means comprises: hybridizing plant seeds with plant seeds containing the nucleic acid according to claim 2 or 3.

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