Interaction protein MORF8 of rice wild abortion type cytoplasmic male sterility recovery protein Rf4 and application of interaction protein MORF8

Abstract

The invention belongs to the field of gene engineering and molecular breeding, and particularly discloses interaction protein MORF8 of rice wild abortion type cytoplasmic male sterility recovery protein Rf4 and application of the interaction protein MORF8, the amino acid sequence of the interaction protein MORF8 is shown as SEQ ID NO.2, the interaction protein MORF8 is coded by an MORF8 gene, and the nucleotide sequence of the MORF8 gene is shown as SEQ ID NO.1; the application of the interaction protein MORF8 of the rice wild abortion type cytoplasmic male sterility restoring protein Rf4 in rice breeding comprises the following steps: constructing a rice wild abortion type cytoplasmic male sterility line overexpressing the MORF8, and hybridizing the rice wild abortion type cytoplasmic male sterility line overexpressing the MORF8 with a rice wild abortion type cytoplasmic male sterility restoring line containing the restoring protein Rf4, hybrid seeds which synergistically enhance the fertility recovery capability of the recovery protein Rf4 are generated. Compared with the prior art, the invention discloses a synergistic enhancement function of MORF8, obviously improves the maturing rate of rice, and provides an efficient and safe novel molecular breeding tool for creating high-yield and stable-yield hybrid rice.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular breeding, and in particular to MORF8, the interacting protein of rice wild-type cytoplasmic male sterility restorer protein Rf4, and its application. Background Technology

[0002] Rice is one of the world's most important food crops, and increasing its yield is crucial for food security. The application of three-line hybrid rice fully utilizes the heterosis of rice, significantly increasing rice yield. The three-line hybrid system consists of a sterile line, a restorer line, and a maintainer line. Crossing the sterile line and the restorer line produces hybrid varieties with heterosis. Cytoplasmic male sterility (CMS) is caused by mitochondrial sterility genes, and its negative effects can be communicated by the nuclear-encoded restorer gene. ） Eliminate. Analysis The fertility restoration mechanism is of significant economic value for increasing rice yield and optimizing rice production. Currently, the wild-type cytoplasmic male sterility (CMS-WA) system is the most widely used type in three-line hybrid rice production. The sterility gene in this system... WA352 and the main effect of the recovery gene All have been cloned, and By reducing WA352 The expression level restores fertility. For example, the rice cytoplasmic male sterility restoration gene and its application disclosed in Chinese patent application number CN201410090900.X provide a wild-type cytoplasmic male sterility restoration gene isolated and cloned from rice. The nucleotide sequence and its encoded amino acid polypeptide sequence are described. This gene belongs to the PPR gene family and is a constitutively expressed gene that negatively regulates the sterility gene in the mitochondrial genome. WA352 To restore fertility by adjusting the expression level. Transforming rice varieties that do not originally contain the restorer gene into new restorer lines can cultivate new restorer lines. Crossing transgenic restorer lines with wild-type sterile lines can produce fertile hybrids for production.

[0003] Against this backdrop, the present invention discovered and identified MORF8, a cooperating factor of Rf4, which can interact and synergistically participate in fertility restoration. Furthermore, research has revealed... MORF8 Increased expression can significantly improve The ability to restore fertility. This discovery not only reveals... The molecular mechanisms mediating fertility restoration provide insights into addressing the homogenization of restorer line resources and ensuring their safe and efficient utilization. This core germplasm resource offers new strategies. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides MORF8, an interacting protein of rice wild-type cytoplasmic male sterility restorer protein Rf4, and its applications.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a protein MORF8, an interaction protein with the rice wild-type cytoplasmic male sterility restorer protein Rf4, the amino acid sequence of which is shown in SEQ ID NO.2. The protein MORF8 is composed of... MORF8 Gene encoding, the MORF8 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The second technical solution provided by this invention is the application of MORF8, an interaction protein of rice wild-type cytoplasmic male sterility restoration protein Rf4, in rice breeding, including: constructing an overexpression... MORF8 The wild-type cytoplasmic male sterile rice line will overexpress... MORF8 Rice wild-type cytoplasmic male sterile lines and lines containing recovery proteins Hybridization of wild-type cytoplasmic male sterility restorer lines in rice produces synergistically enhancing restorer proteins. Hybrid seeds that restore fertility.

[0007] Furthermore, construct overexpression MORF8 The process of establishing a wild-type cytoplasmic male sterile line in rice is as follows: Amplification MORF8 The encoded sequence, and cloned into a string with Ubi Overexpression vectors were constructed in pOX plant expression vectors containing promoters. MORF8 -OE; the overexpression vector MORF8 -OE was transformed into Agrobacterium EHA105, and the vector was introduced into a wild-type cytoplasmic male sterile rice line using an Agrobacterium-mediated stable transformation system. Overexpression was obtained after hygromycin resistance screening and PCR identification. MORF8 Rice wild-type cytoplasmic male sterile line plants.

[0008] Compared with existing technologies, this invention overexpresses [the gene] in wild-type cytoplasmic male sterile rice lines. MORF8 It has no significant effect on sterile lines, but it does have an effect on lines containing the restorer gene. After hybridization of restorer lines, MORF8 Overexpression significantly enhanced This invention discloses for the first time the ability to restore fertility, improve pollen viability and spikelet seed setting rate, without negatively impacting plant growth.MORF8 Synergistic enhancement Its function significantly improves the seed setting rate of rice, providing a new, efficient, and safe molecular breeding tool for creating high-yield and stable-yield hybrid rice. Attached Figure Description

[0009] Figure 1 for MORF8 Expression analysis: A is MORF8 Expression analysis in rice roots, stems, leaves, and anthers; B is... MORF8 Subcellular localization analysis.

[0010] Figure 2 To verify the interaction between MORF8 and Rf4: A shows the pull-down experiment results of MORF8-MBP and Rf4-MBP-His proteins; B shows the results of the co-immunoprecipitation (Co-IP) experiment using MORF8 autoantibody to pull down Rf4 protein; C shows the results of Vc- MORF8 and Results of bimolecular fluorescence complementation (BiFC) experiments on -Vn protein.

[0011] Figure 3 for MORF8 Genetic analysis of overexpression lines: A and B are under the backgrounds of ZS97A and ZS97A×ZSRf4I, respectively. MORF8 - The pollen (top) and spikelet (bottom) phenotypes of the OE overexpression lines are shown, with scale bars of 50 μm and 5 cm, respectively. FS indicates total sterility, PF indicates partial fertility, and the numbers in the upper left corner of the figure represent pollen staining rate or spikelet seed setting rate; C represents... MORF8 -OE strains MORF8 Relative expression level detection; D and E represent the expression levels under ZS97A and ZS97A×ZSRf4I backgrounds, respectively. MORF8 -OE strains WA352 Detection of relative expression levels; MORF8 use UFC1 For internal reference genes, WA352 use atp6 This is an internal reference gene; the data are the mean ± standard deviation of three biological replicates; a two-tailed method was used between two samples. t -test to perform significant difference analysis (** P < 0.01). Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0013] Example 1 MORF8Cloning of genes Obtained from the rice genome database MORF8 The complete nucleotide sequence of the gene: AAACCCTCAGCCCCCCTCATTCCCAATCTAGGGTTTTCCCC ATGTAG AACACTGCTAGAATAAACGATATGTAGAATCTAGTACGAGCGTAACTTCATTTGTTTCCTGTTGTTATCGCATGTATACTCTTATGCTGCTTTGTGAACTCGGTTTCGTATGTGTCGAGCAATCACTTCAATGTGTCTGGAACTAGCGGTTGTTTGAAGCACTTAAAACATTATAAATGTTTGGTCAACTAGTGCATTTTGTCC (SEQ ID NO. 2). The ones in bold are MORF8 The exons, start codon, and stop codon are indicated by underscores.

[0014] Amplification of rice using RT-PCR technology MORF8 The cDNA sequence of the gene, which encodes the protein MORF8, has the following amino acid sequence: MASASRFLLLSRLPAAAASSTSRLLRPLSAAGSLLPAALAPSAPRAAAAAARCFATQPATSSLRDSSPNWSNRPPKETILLDGCDFEHWLVVVEPPPGDPSNPEPTRDEIIDGYIKTLAQVVGSEEEARHKIYSVSTRHYFAFGALVSEELSYKLKELPKVRWVLPDSYLDVRNKDYGGEPFINGEAVPYDPKYHEEWVR NNARANERSRRNDRPRNFDRSRNFERRRENMQNFQNRDVPPGQGFNSPPPPGQGPVPPRDAPPMHHAQGNVPPPPPPNAGPPNYQPHAPNPQGYTNYQQG GAPGYQGGPPGYQGSNQGYQGPPPPPPSAYQGNNPGYQGGGPGYQGGNPPPYQGGNPGYAPGYHGQGGNPSYQQGGDNYNAGVPAYERDGQGRNYQ (SEQ ID NO.1).

[0015] Example 2 MORF8 Gene expression pattern analysis RNA was extracted from sporogenous cells, pollen mother cells, meiotic cells, and early uninucleus cells of rice roots, stems, leaves, and anthers using the Trizol method. Reverse transcription was performed using the Novizan Reverse Transcription Rate Kit, following the instructions.

[0016] MORF8 The primer sequences used for gene expression detection are shown in SEQ ID NO. 3~SEQ ID NO. 4: SEQ ID NO. 3: 5'-CCCCACCATCTGCTTACCAA-3', SEQ ID NO. 4: 5'-TTGCTGGTAGCTAGGGTTGC-3'; Internal reference gene UFC1 The primer pair sequences are shown in SEQ ID NO. 5~SEQ ID NO. 6: SEQ ID NO. 5: 5'-GATGGCAAGACCCACAAG-3', SEQ ID NO. 6: 5'-TCCCGAACCTTGGGCAGT-3'.

[0017] Detection using qRT-PCR method MORF8 The expression levels of genes in different tissues and developmental stages. The results showed that... MORF8 The gene is expressed at different developmental stages of rice roots, stems, leaves, and anthers, exhibiting a constitutive expression pattern (see [link to gene expression]). Figure 1 (A in the middle).

[0018] Example 3 MORF8 Gene subcellular localization Obtained by DNA polymerase amplification MORF8 The complete coding sequence of the gene was seamlessly cloned into the plant expression vector pYL322d1-eGFP via homologous recombination to construct... MORF8 -GFP subcellular localization vector. Protoplasts were prepared from the stems of etiolated rice seedlings using an enzymatic digestion method. Equal amounts of... MORF8 -GFP recombinant plasmid and mitochondrial localization marker plasmid COX11 mCherry was co-transformed into protoplasts using a PEG-mediated method. After transformation, cells were cultured at 28°C in the dark for 12–16 h. Fluorescence signals were observed using a confocal laser scanning microscope: GFP fluorescence was excited by a 488 nm laser (green channel), and mCherry fluorescence was excited by a 561 nm laser (red channel). Cell morphology was captured using a bright field imager, and images from each channel were overlaid for co-localization analysis.

[0019] The results showed that after conversion MORF8 In the protoplasts of GFP and mitochondrial marker plasmids, the green fluorescent signal exhibits a granular network distribution that completely overlaps with the red fluorescent signal, resulting in yellow fluorescence (see [link to original text]). Figure 1 (B in the original text). This result indicates that the protein MORF8 is located in the mitochondria of rice cells.

[0020] Example 4: Interaction analysis of proteins MORF8 and Rf4 (1) Pull-down in vitro interaction verification Build MORF8 -MBP and -MBP-His fusion expression vector, the above plasmids were transformed respectively E . coli BL21(DE3) competent cells were induced to express the target protein using IPTG. Rf4-MBP-His protein was incubated with MBP empty vector protein and MORF8-MBP protein with Ni-NTA agarose protein purification beads at 4°C for 2 hours, followed by washing. solution The beads were washed to remove unbound protein, and SDS-PAGE loading buffer was added. The mixture was boiled for 5 minutes and then subjected to Western blotting. The target protein was detected using MBP and His antibodies. Western blotting showed that Rf4-MBP-His successfully pulled down MORF8-MBP, but failed to pull down the empty MBP-carried protein (see [link to article]). Figure 2 (A) demonstrates that the protein MORF8 can interact with the protein Rf4 in vitro.

[0021] (2) Co-IP verification interaction Will Instantaneous expression carriers incorporating FLAG tags -FLAG, transferred into rice protoplasts for protein expression. Total protein was extracted from rice protoplasts using a plant protein extraction kit. An appropriate amount of total plant protein was taken, and anti-MORF8 autoantibody and Protein A / G agarose beads were added. A control group was established, using wild-type mouse IgG instead of anti-MORF8 antibody. The mixture was incubated at 4°C with slow rotation for 2 hours. After incubation, the protoplasts were repeatedly washed with rinsing buffer to remove non-specifically bound proteins. SDS-PAGE loading buffer was then added for SDS-PAGE electrophoresis, followed by Western blotting analysis. Results showed... MORF8 Rf4-FLAG was detected in the gene-drop complex, but not in the negative control group (IgG). -FLAG indicates that proteins MORF8 and Rf4 can form specific immune complexes in rice cells (see [link]). Figure 2 (B in the middle).

[0022] (3) BiFC verification interaction Will MORF8 The gene was cloned into the Vc vector, and Cloned into the Vn vector, Vc- MORF8 and -Vn BiFC vector. Equal amounts of the two plasmids were mixed and co-transformed into rice protoplasts via PEG-mediated transformation. After incubation at 28℃ in the dark for 16–20 hours, fluorescence observation was performed using a confocal laser scanning microscope, with YFP signals excited by a 514 nm laser. The results showed that the experimental group (Vc- MORF8 + A strong yellow fluorescence signal was observed in the co-transformed protoplasts, exhibiting a clear punctate structure (see [link to original text]). Figure 2 (C in the text). The above results demonstrate that in living rice cells, proteins MORF8 and Rf4 can interact directly in the mitochondria.

[0023] Example 5 MORF8 Creation and phenotypic identification of gene overexpression vector lines (1) Creation and identification of overexpression lines Amplification using high-fidelity DNA polymerase MORF8 The complete coding sequence of the gene was cloned into a pOX plant expression vector with a Ubi promoter using homologous recombination technology to construct an overexpression vector. MORF8 -OE. After construction, the vector's correctness is verified by sequencing. MORF8 -The primer sequence pairs for OE vector amplification are shown in SEQ ID NO. 7~SEQ ID NO. 8: SEQ ID NO. 7:ACTTCTGCAGGGTACCATGGCGTCGGCGTCGCGC, SEQ ID NO. 8: AACGCGTACTAGTAAGCTCTGGTAATTCCTCCCTG.

[0024] Will MORF8 The -OE overexpression vector was introduced into Agrobacterium EHA105, and then, through Agrobacterium-mediated stable transformation, the vector was introduced into the CMS-WA male-sterile line ZS97A. Positive transgenic plants were obtained by screening using hygromycin resistance and PCR identification, and were denoted as follows: MORF8 -OE#1 and MORF8 -OE#2 strain; will MORF8 -OE#1 and MORF8 The -OE#2 strain was crossed with the restorer line ZSRf4I to obtain ZS97A×ZSRf4I. MORF8 -OE plants, denoted as MORF8 -OE#3 and MORF8-OE#4 strain. Simultaneously, a hybrid combination ZS97A×ZSRf4I was constructed, consisting of the sterile line ZS97A and the restorer line ZSRf4I (existing technology, not described further in this embodiment). Hygromycin resistance screening primer sequence pairs are shown in SEQ ID NO. 9~SEQ ID NO. 10; MORF8 The primer sequence pairs for PCR identification of transgenic organisms are shown in SEQ ID NO. 11~SEQ ID NO. 12: SEQ ID NO. 9: 5'-ATTTGTGTACGCCCGACAGT-3'; SEQ ID NO. 10: 5'-GTGCTTGACATTGGGGAGTT-3'; SEQ ID NO. 11: 5'-GGATGATGGCATATGCAGCAG-3'; SEQ ID NO. 12: 5'-GAGGTGGAGGAACATTACCC-3'.

[0025] Extract ZS97A, ZS97A×ZSRf4I, MORF8 -OE#1、 MORF8 -OE#2、 MORF8 -OE#3 and MORF8 Total RNA from spikelets of the -OE#4 strain was reversed and paired with specific primers. MORF8 Genes and WA352 Quantitative analysis of gene expression, MORF8 The primer sequence pairs for quantitative detection of the gene are shown in SEQ ID NO. 3~SEQ ID NO. 4; internal reference. UFC1 The quantitative detection primer sequence pairs are shown in SEQ ID NO. 5~SEQ ID NO. 6. WA352 The primer sequence pairs for quantitative detection of the gene are shown in SEQ ID NO. 13~SEQ ID NO. 14; internal reference gene. atp6 The quantitative detection primer sequence pairs are shown in SEQ ID NO.15~SEQ ID NO.16: SEQ ID NO. 13: 5'-CATGCAAATGTCCCGGATTCAAGC-3'; SEQ ID NO. 14: 5'-GTAAGCGGACTCTTTCGACCAAG-3'; SEQ ID NO. 15: 5'-GGCATTACGATCGTTGGATTTC-3'; SEQ ID NO. 16: 5'-TTGATGGAGATTTATAGCATCATTC-3'.

[0026] qPCR was performed using the 2× ChamQ Universal SYBR qPCR Master Mix kit (Novizan). Each 20 μL reaction mixture was prepared according to the manufacturer's instructions, and each sample was repeated three times. The reaction program was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 10 s, annealing at 60℃ for 10 s, extension at 72℃ for 10 s, for a total of 40 cycles.

[0027] MORF8 -OE#1 and MORF8 -OE#2 strain MORF8 The relative expression levels of genes are shown in Figure 3C. As can be seen from the figure... MORF8 The relative expression level of the gene was higher than that of the CMS-WA sterile line ZS97A, indicating that... MORF8 The gene is overexpressed in the CMS-WA sterile line ZS97A; MORF8 -OE#1 and MORF8 -OE#2 strain WA352 The relative expression levels are shown in Figure 3D. Compared with the control ZS97A, WA352 No significant change in gene expression indicates MORF8 Gene overexpression does not affect the sterility of sterile lines; such as Figure 3 As shown in E, when MORF8 -OE#1 and MORF8 -OE#2 was crossed with the restorer line ZSRf4I, and the F1 generation of the hybrid ( MORF8 -OE#3 and MORF8 -OE#4) WA352 The expression level of [the substance] was significantly lower than that of the control hybrid combination (ZS97A×ZSRf4I), indicating that... MORF8 Overexpression of genes can enhance right WA352 The inhibitory effect.

[0028] (2) Analysis of pollen fertility and seed set rate Collect rice spikelets nearing flowering in the field and preserve them in 75% ethanol. Observe 3 to 5 spikelets from each plant. Add 1% I2-KI solution to a glass slide, remove the anthers of the spikelets with tweezers, place them in the 1% I2-KI solution, stir, and remove the residue. Observe under a 10x microscope, examining 4 fields of view for each spikelet. Fertile pollen is stained black by iodine stain, while sterile pollen is shriveled and cannot be stained. Figure 3 As shown in A–B, the ZS97A strain and MORF8 -OE#1 and MORF8The pollen of the -OE#2 strain is shriveled and unable to color, while MORF8 -OE#3 and MORF8 The pollen staining rate of the -OE#4 strain was significantly increased by nearly 20% compared with ZS97A×ZSRf4I (see [link to original text]). Figure 3 (B in the middle). MORF8 -OE#3 and MORF8 The spikelet setting rate of the OE#4 line was nearly 20% higher than that of the ZS97A×ZSRf4I line; and in terms of plant growth and development... MORF8 The -OE strains showed no significant difference from the wild type (Fig. 3A–B) and had no negative impact on plant growth and development.

[0029] In summary, MORF8 Overexpression of genes can significantly enhance The CMS-WA-mediated fertility restoration ability improves the pollen staining rate and spikelet seed setting rate of rice.

[0030] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A protein MORF8 that interacts with the rice wild-type cytoplasmic male sterility restorer protein Rf4, characterized in that, Its amino acid sequence is shown in SEQ ID NO.2, and the protein MORF8 is composed of... MORF8 Gene encoding, the MORF8 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application of MORF8, an interaction protein with the rice wild-type cytoplasmic male sterility restorer protein Rf4 as described in claim 1, in rice breeding, characterized in that... including: Construct overexpression MORF8 The wild-type cytoplasmic male sterile rice line will overexpress... MORF8 Rice wild-type cytoplasmic male sterile lines containing the gene and those containing the restorer gene By hybridizing wild-type cytoplasmic male sterile restorer lines of rice, hybrid seeds were produced that synergistically enhance the fertility restoration ability of the restorer protein Rf4.

3. The application according to claim 2, characterized in that, Construct overexpression MORF8 The process of establishing a wild-type cytoplasmic male sterile line in rice is as follows: Amplification MORF8 The coding sequence of the gene, and clone it into a gene with... Ubi Overexpression vectors were constructed in pOX plant expression vectors containing promoters. MORF8 -OE; the overexpression vector MORF8 -OE was transformed into Agrobacterium EHA105, and the vector was introduced into a wild-type cytoplasmic male sterile rice line using an Agrobacterium-mediated stable transformation system. Overexpression was obtained after hygromycin resistance screening and PCR identification. MORF8 The gene-derived wild-type cytoplasmic male sterile rice line.

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