Application of HIRA gene in regulation and control of plant male fertility

Abstract

The invention discloses application of an HIRA gene in regulation and control of plant male fertility, and belongs to the technical field of plant gene functions. According to the invention, CRISPR-Cas9 gene editing vector construction is carried out on a rice gene OsHIRA1, a rice variety Nipponbare is transfected by the vector, an obtained gene editing plant is identified and observed, a homozygous mutant cannot be obtained, in-vitro and in-vivo pollen tube germination experiments are carried out on heterozygous mutant pollen, and half of pollen tubes germinate abnormally. It can be known that the rice OsHIRA1 participates in the pollen tube germination process. The method can be used for breeding hybrid rice and provides a new reference thought for breeding strategies of other species.

Description

Technical Field

[0001] The present invention relates to the technical field of plant gene functions, and particularly relates to the application of a HIRA gene in regulating plant male fertility. Background Art

[0002] Rice belongs to the Gramineae family, and rice provides key carbohydrates, proteins, vitamins, and minerals.

[0003] Rice hybrid breeding is a revolutionary agricultural technology. It creates hybrid offspring with excellent characteristics by artificially pollinating two rice varieties with different genetic characteristics. The core of this technology lies in the utilization of "heterosis", that is, the hybrid offspring are often superior to their parents in terms of growth potential, yield, disease resistance, etc. The traditional hybrid rice breeding process usually includes several key steps. First, select parents with excellent characteristics; second, ensure the purity of hybridization through artificial emasculation and pollination; then, conduct strict screening and testing on the hybrid offspring to determine whether their performance meets the expected goals. However, this also leads to defects such as higher seed production costs and the need to purchase new seeds every year. Therefore, when exploring new breeding technologies, researchers created the three-line breeding method. Three-line breeding includes three different genetic lines: male sterile line, maintainer line, and restorer line. Among them, sterility is usually a male sterile line, which only provides female gametes, accepts foreign pollen for fertilization, while male gametes are abnormal and cannot pollinate other rice. The adoption of the male sterile line avoids the cumbersome steps of artificial emasculation and reduces production costs, which is the core of this technical system. Therefore, the discovery of male gamete sterility genes is particularly important for the development and innovation of the three-line breeding system. However, the application of the HIRA gene in regulating plant male fertility has not been reported in the existing technology. Summary of the Invention

[0004] In order to solve the above-mentioned deficiencies in the existing technology, the purpose of the present invention is to provide the application of a HIRA gene in regulating plant male fertility, which is used in the rice hybrid breeding process and provides a reference for the breeding of other crops.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: Provide the application of a HIRA gene in regulating plant male fertility; wherein, the nucleotide sequence of the HIRA gene is as shown in SEQ ID NO:1.

[0006] Further, the plant is rice.

[0007] Further, by knocking out the HIRA gene, the plant cannot produce homozygous plants.

[0008] Further, by knocking out the HIRA gene, a heterozygous mutant is obtained. The heterozygous mutant can produce offspring when the female gametes are fertilized, while the male gametes are abnormal.

[0009] A preparation for regulating plant male fertility, which preparation comprises the above-mentioned HIRA gene or the protein encoded by this gene.

[0010] A method for regulating plant male fertility, comprising the following steps:

[0011] Obtaining male gamete sterile plants by knocking out the HIRA gene.

[0012] The present invention has the following beneficial effects:

[0013] The gene edited in the present invention is the OsHIRA1 gene (LOC_Os09g39420) in rice. The full length of this gene is 4318 bp, and the cDNA is 2928 bp. The protein encoded by this gene is a molecular chaperone of histone H3.3, with a full length of 975 amino acids. By knocking out this gene, that is, by constructing a CRISPR-Cas9 gene editing vector for the rice gene OsHIRA1, transfecting the vector into the rice variety Nipponbare, identifying and observing the obtained gene-edited plants, it was found that the heterozygous mutants could not produce homozygous plants. By statistically analyzing the genotypes of the offspring produced by the heterozygous mutants, it was found that homozygous mutants could not be produced. By performing reciprocal crosses between the heterozygous mutants and the control Nipponbare (Nip), it was found that when the heterozygous mutants were used as male parents, the ratio of wild-type and heterozygous mutants in the offspring was 1:1. When the heterozygous mutants were used as female parents, only wild-type plants could be produced in the offspring, indicating that only wild-type female gametes of the heterozygous mutants could be fertilized to produce offspring, and there were abnormalities in the mutant male gametes.

[0014] In the present invention, through in vitro germination experiments on the gametes produced by the heterozygous mutants, it was found that only half of the gametes could germinate. By performing aniline blue staining on the gametes produced by the heterozygous mutants after fertilization to detect pollen activity, it was found that only half of the male gametes had activity, indicating that there were problems with the activity of the mutant gametes. Therefore, the OsHIRA1 gene can be used as a fertility regulation gene and has high application value in rice genetic breeding. Description of the Drawings

[0015] Figure 1 It is a diagram of the experimental results of in vitro pollen tube germination.

[0016] Figure 2 It is a diagram of the experimental results of in vivo pollen tube germination. Detailed Embodiments

[0017] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0018] Example 1:

[0019] I. Construction of OsHIRA1 gene knockout vector and acquisition of gene-edited mutants

[0020] Through the reference genome of NCBI, the present invention obtained the CDS nucleic acid sequence information and protein sequence information of the OsHIRA1 gene. The nucleic acid sequence information is shown as SEQ ID NO:1, and the protein sequence information is shown as SEQ ID NO:2. Based on this information as a reference, guide RNA was designed and the CRISPR-Cas9 gene editing vector was constructed.

[0021]

[0022] MITEKPSWIRHEGLQIFSIDIQPGGIRFATGGGDQKIRIWSMKSVAKDNDSDDSSQRLLATIRDHFGTVNCVRWAHHGRYLASGSDDQVIQIHERKAGTGTSEFGSGEPPDVENWKVVMTLRGHTADVVDLNWSPDDSTLASGSLDNTVHIWSMANGICTAVLRGHSSLVKGVTWDPIGSFIASQSDDKTVIIWRTSDWSLAHRTEGHWSKSLGSTFFRRLAWSPCGHFITTTHGFQKPRHSAPVLERGEWSATFDFLGHNAPVVVVKFNHSMFRKHLSSGQDAKAAPAGWANGASKASSKEHQPYNVIAIGSQDRTITVWTTASARPLFVAKHFFTQSVVDLSWSPDGYSLFACSLDGSVATFHFEAKELGYRLRDAELDELKKNRYGDVRGRQSNIAESPAQLLLEEASAKQSASKKVSSVQQFQSPPKVSTDAPNPSTSVPNQKAPEALPEDEKKTAGSTADDINKAPRLSSPVKQREYRRPDGRKRIIPEAVGFPSNQDMSNRSQNQGVDFSSLDQRMILGENGTRPSYSASGNCNNCGVRERSGITARTNISESLVIQKASAGAGSDGRLSIEQSGSVVPGSLASCSSLSIHVFNKKDNEDSLPVRLEAKPVERSAGDMIGLGGAFSTKETEITCTRGTETLWSDRISAKVTVLAGNANFWAVGCEDGCLQVYTKCGRRAMPAMMMGSAAVFIDCDECWKLLLVTRRGLMYIWDLYTRTCVLHDSLASLVTSPDEAAGKDTGTVKVISAKFSRCGSPLVVLASRHAFLYDTSLKCWLRIADDCFPASNFASSFSSTQGGELGKLQIDIGKFMARKPIWSRVTDDGVQTRSHLETQLAASLALKSPQEYRQCLLSYIRFLAREADESRLREVCESFLGPPMGMVDAASSADLKNPSWDPDVLGMKKHKLLREDILPSMATNRKVQRLLNEFMDLLSEYEAAETNVEQMDVTPTPPPPPPAAATEGNNNGAS(SEQ ID NO:2).

[0023] OsHIRA1 guide RNA1:

[0024] 5’-TCTATCGACATCCAGCCGGG-3’(SEQ ID NO:4);

[0025] OsHIRA1 guide RNA2:

[0026] 5’-TCAGCCCCTGTGCTTGAACG-3’(SEQ ID NO:5).

[0027] The primer dimer of the guide RNA was ligated to the CRISPR-Cas9 vector pYLCRISPR / Cas9, amplified and propagated in Escherichia coli DH5α, the plasmid was extracted, and then the plasmid was transferred into Agrobacterium tumefaciens AH105. Subsequently, AH105 carrying the plasmid and wild-type Nipponbare rice seeds were sent to the company for subsequent rice infection work.

[0028] The above gene-edited materials were subjected to gene identification. The DNA of the materials was extracted using the conventional CTAB method. Primers were designed at both ends of the guideRNA, and the amplified primers were sent to a first-generation sequencing company for sequencing. A material with a 14-bp deletion was found. This 14-bp deletion caused a frameshift in the OsHIRA1 protein, resulting in premature termination of translation and only encoding a truncated OsHIRA1. The protein sequence is shown in SEQ ID NO:3.

[0029] MITEKPSWIRHEGLQIFSIDIQPGGIRFATGGGDQKIRIWSMKSVAKDNDSDDSSQRLLATIRDHFGTVNCVRWAHHGRYLASGSDDQVIQIHERKAGTGTSEFGSGEPPDVENWKVVMTLRGHTADVVDLNWSPDDSTLASGSLDNTVHIWSMANGICTAVLRGHSSLVKGVTWDPIGSFIASQSDDKTVIIWRTSDWSLAHRTEGHWSKSLGSTFFRRLAWSPCGHFITTTHGFQKPRHSAG(SEQ ID NO:3).

[0030] OsHIRA1-geno-F: 5’-CATGGTTTTCAGAAACCTAGG-3’(SEQ ID NO:6);

[0031] OsHIRA1-geno-R: 5’-CAGAAAATCAAAGGTTGCTGAC-3’(SEQ ID NO:7).

[0032] II. Heritability Analysis of the Offspring of the Heterozygous Mutant

[0033] The obtained heterozygous mutant was planted in the field, and the genotypes of the harvested seeds were identified. It was found that there was no homozygous mutant genotype. By analyzing the ratio of the genotypes of the offspring, it was found that the ratio of the wild type to the heterozygous mutant approached 1:1. The heterozygous mutant and the wild type were reciprocally crossed, and the genotypes of the seeds obtained from the reciprocal crosses were identified. It was found that when the heterozygous mutant was used as the male parent, the ratio of the wild type to the heterozygous mutant in the offspring was 1:1. When the heterozygous mutant was used as the female parent, only the wild type could be produced in the offspring (see Table 1 for details).

[0034] Table 1 Statistical Results of the Genotypes of the Hybrid Offspring of the Heterozygous Mutant

[0035]

[0036] III. In Vitro Pollen Tube Germination Experiment

[0037] Pollen from the wild type and the heterozygous mutant in the field was collected, and a conventional in vitro pollen tube germination medium was prepared. The mature pollen was shaken onto the medium and cultured under dark and humid conditions at 30 °C for 20 min, and then observed under an optical microscope and the germination rate was counted. It was found that the germination rate of the pollen in the heterozygous mutant was significantly lower than that of the wild type, only about 40% (see Figure 1 for details).

[0038] IV. In Vivo Pollen Tube Germination Experiment

[0039] The stigmas of the wild type were pollinated with pollen from the wild type and the heterozygous mutant respectively. The stigmas 2 h after pollination were placed in FAA fixative, vacuumed for 1 - 2 h, taken out and rinsed with sterile water, then placed in 5% NaOH solution and left overnight at 4 °C. The next day, they were taken out and placed in aniline blue staining for overnight treatment at 4 °C. The day after that, the number of pollen elongations was observed and counted under a microscope, and the germination rate was counted. It was found that the germination rate of the pollen of the heterozygous mutant was only half of that of the wild type (see Figure 2 for details).

[0040] In summary, the segregation ratio of the genotypes of the offspring of the heterozygous mutant with the rice gene OsHIRA1 knocked out is abnormal, and mutant male gametes cannot be produced by hybridization. Further, through in vivo and in vitro pollen tube germination experiments, it was found that there are problems with the activity of the male gametes of the mutant. Therefore, OsHIRA1 can be used as a potential candidate gene for hybrid rice breeding.

[0041] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of the HIRA gene in regulating plant male fertility; wherein, The nucleotide sequence of the HIRA gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The plant is rice.

3. The application according to claim 1 or 2, characterized in that, Knocking out the HIRA gene makes it impossible for the plant to produce homozygous plants.

4. The application according to claim 1 or 2, characterized in that, By knocking out the HIRA gene, a heterozygous mutant is obtained. The female gametes of the heterozygous mutant can be fertilized to produce offspring, while the male gametes are abnormal.

5. A preparation for regulating plant male fertility, characterized in that, The preparation comprises the HIRA gene described in claim 1 or the protein encoded by the gene.

6. A method for regulating plant male fertility, characterized in that, Comprising the following steps: Obtaining male gamete sterile plants by knocking out the HIRA gene.

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