Rice OsMATE7 gene and application thereof

Abstract

The invention discloses a rice OsMATE7 gene and application thereof, and belongs to the technical field of molecular biology. According to the application disclosed by the invention, knockout plants KO1 and KO2 of the OsMATE7 gene are constructed, then phenotypes of the knockout plants are identified and analyzed, and the knockout plants of the OsMATE7 gene are found to be almost not firm. Then analyzing the fertility of floral organs and pollen grains of the knockout plant to find that the pollen fertility of the knockout plant of the OsMATE7 gene is not influenced, but the length of a pollen tube is reduced, so that fertilization cannot be normally completed. Therefore, the OsMATE7 gene can regulate and control the male sterility of the rice. According to the research, the rice OsMATE7 gene is analyzed in rice for the first time, and a transgenic experiment proves that the gene can regulate and control rice fruiting by influencing the length of a rice pollen tube, so that a new male sterility gene resource is provided for rice breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a rice OsMATE7 gene and an application thereof. Background Art

[0002] Rice is the staple food of more than half of the world's population and an important staple food in my country. The three-line and two-line breeding methods based on male sterility are of great significance to increasing rice yields and are also the main methods for utilizing hybrid advantages of rice in my country. Therefore, finding new male sterility genes has important theoretical significance and application value for broadening the genetic basis of hybrid rice and preventing production risks caused by single germplasm.

[0003] The process of male gamete development is a delicate process. Mutations in key genes controlling microspore development at various stages may lead to abnormal pollen development and male sterility. At present, the male sterility genes cloned in rice can be roughly divided into two categories according to their regulatory periods: one is the genes related to microspore meiosis, tapetum degradation and pollen exine formation, such as the OsMSP1 gene encoding a leucine-rich repeat receptor protein serine / threonine kinase, which controls the number of microspore mother cells by inhibiting the re-differentiation of peripheral cells of the mother cell; the gene OsMS1 encoding a PHD zinc finger protein is involved in regulating the programmed cell death of the tapetum; and the gene RMS2 encoding a lipase regulates the development of the anther exine by mediating lipid balance in the anther. The other is that key genes affecting pollen tube elongation are involved in the rice fertilization process. Among them, the gene encoding chalcone synthase OsCHS1 regulates flavonoid biosynthesis. The mutation of this gene leads to insufficient energy in the germination process of pollen grains and inability to penetrate maternal tissues, resulting in fertilization failure. The gene encoding cytoplasmic dismutase DPE2 mutation leads to reduced levels of starch, glucose, fructose and ATP in pollen grains, causing pollen tube elongation disorders. However, there are currently no reports on the regulation of rice male sterility by MATE proteins in rice. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a rice OsMATE7 gene and its application, so as to provide a new gene for regulating rice male sterility.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: a rice OsMATE7 gene is provided, and the nucleotide sequence of the coding region of the rice OsMATE7 gene is shown in SEQ ID NO.1.

[0006] The amino acid sequence of the protein encoded by the rice OsMATE7 gene is shown in SEQ ID NO.2.

[0007] The present invention provides an application of a rice OsMATE7 gene in regulating rice male sterility.

[0008] Furthermore, rice male sterility was regulated by knocking out the rice OsMATE7 gene, which weakened the vigor of rice pollen.

[0009] Furthermore, rice male sterility was regulated by knocking out the rice OsMATE7 gene, which reduced the proportion of rice pollen tubes reaching the micropyle.

[0010] The present invention provides a preparation for regulating rice male sterility, wherein the preparation comprises the rice OsMATE7 gene or the encoded protein of the rice OsMATE7 gene.

[0011] The present invention also provides a method for preparing a male sterile rice variety, and the male sterile rice variety is prepared by knocking out the rice OsMATE7 gene.

[0012] The present invention has the following beneficial effects: This study first analyzed the rice OsMATE7 gene in rice, and by constructing knockout plants KO1 and KO2 of the OsMATE7 gene, and then identifying and analyzing the phenotype of the knockout plants, it was found that the knockout plants of the OsMATE7 gene were almost not fruitful. Then, by analyzing the fertility of the flower organs and pollen grains of the knockout plants, it was found that the pollen fertility of the knockout plants of the OsMATE7 gene was not affected, but the pollen tube length was reduced, resulting in the inability to complete fertilization normally. Therefore, the OsMATE7 gene can regulate the male sterility of rice. This study first analyzed the rice OsMATE7 gene in rice, and proved through transgenic experiments that the gene can regulate rice fruiting by affecting the length of the rice pollen tube, which provides a new male sterility gene resource for rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the CRISPR / Cas9 vector of rice OsMATE7 gene;

[0014] Figure 2 The target site and plant phenotype of OsMATE7 gene knockout are shown;

[0015] Figure 3 The fertility of OsMATE7 knockout plants was restored to wild type;

[0016] Figure 4 Observation of floral organ and pollen grain fertility in wild-type and OsMATE7 knockout plants;

[0017] Figure 5 For OsMATE7 gene expression profiling analysis;

[0018] Figure 6Pollen tube staining images of wild-type and OsMATE7 knockout plants. DETAILED DESCRIPTION

[0019] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] Example 1: Construction of OsMATE7 gene editing vector and phenotype observation of transformed plants

[0021] (1) Construction and transformation of OsMATE7 gene editing vector

[0022] The gRNA sequence of the target site for gene editing was selected using an online target site design tool ( http: / / skl.scau.edu.cn / ), and the gRNA sequence was recombined into the pYLCRISPR / Cas9 Pubi-H vector with plant resistance to hygromycin to obtain the OsMATE7-CRISPR / Cas9 vector (see the vector map for details). Figure 1 The specific operation method is referred to Liu et al., Current Protocols in Molecular Biology, 2016). The gene editing vector was used as the recipient material for transformation of japonica rice ZH11, and the Agrobacterium transformation and transgenic experiments were completed by Wuhan Boyuan Company.

[0023] (2) Phenotypic analysis of OsMATE7 knockout plants

[0024] After DNA was extracted from the obtained transgenic plants using the CTAB method, amplification and sequencing primers across the target sites were designed (F primer: 5'-CTGACGGTCGTATGGTTGTC-3' (SEQ ID NO. 3); R primer: 5'-GCACCGTCACCTTGCTCT-3' (SEQ ID NO. 4)) to amplify the target sequence in each individual plant and send it to Qingke Sequencing Company for sequencing.

[0025] The results are as follows Figure 2 As shown in Figure 2, the amplified sequences were analyzed by DNAMAN7 software and showed that two transgenic plants with premature termination of protein translation were obtained, named KO1 and KO2. The two OsMATE7 loss-of-function plants harvested from the T0 generation were sown in plots to form T1 transgenic lines and phenotypes were observed. Figure 2 As shown, knockout transgenic plants produced almost no fruit.

[0026] (3) Fertility restoration experiment of OsMATE7 knockout plants

[0027] OsMATE7 knockout plants KO1 and KO2 were used as female parents and ZH11 as male parent, and artificial pollination hybridization was performed to observe the fruiting of spikelets, and 30 spikelets were repeated. The specific operation steps are as follows:

[0028] ① Emasculation: Select the female spike for trimming, cut off the immature or opened spikelets, and then use scissors to cut off the upper 1 / 3 of the husk of the remaining spikelets. After cutting, spray enough water, and then remove the anthers in each spikelet to achieve the purpose of emasculation;

[0029] ②Bagging: Put the emasculated female parent into a hybridization bag to isolate the surrounding pollen, and note the name of the male and female parent, hybridization time and other information on the hybridization bag;

[0030] ③ Pollination: When the ears of the male parent begin to bloom, cut off enough ears of the male parent and cut off the bloomed lemmas, spray with an appropriate amount of water, and then put them in a hybrid bag, and put the hybrid bag in a black plastic bag to keep warm to promote flowering. Pollination can begin after most of the lemmas have cracked;

[0031] ④ Observation after pollination: 3 days after pollination, preliminarily check the fruit setting situation. Harvest the hybrid ears two weeks later and collect photos of the ear phenotypes.

[0032] The results are as follows Figure 3 As shown, after the mutant was awarded pollen from the wild-type ZH11, the mutant's fruit set basically returned to normal, indicating that the mutant's fruitless phenotype was caused by fertilization failure following abnormal pollen grains.

[0033] (4) Observation of floral organs

[0034] The pre-flowering florets of the wild-type ZH11 and the two knockout lines KO1 and KO2 were first dissected using a dissecting needle under a stereo microscope, and then the integrity of the male and female organs was observed and photographs were taken. Ten samples were taken for each material.

[0035] The results are as follows Figure 4 As shown above, the male and female organs of the mutant developed normally and were no different from the wild type.

[0036] (5) Pollen grain fertility analysis

[0037] Pollen viability was determined using iodine-potassium iodide staining, which included the following steps:

[0038] ① At the heading stage of the plant, take the spikelets that bloomed on the same day and were located at the same position of the ear from different test materials;

[0039] ② Add a drop of distilled water on the slide, take out the anthers from the spikelets with tweezers, place them in the distilled water and gently crush them to fully release the pollen grains;

[0040] ③Add 2 drops of I2 -KI staining solution, then cover the slide and observe with an optical microscope. Make sure to observe more than 3 fields of view for each slide, and keep the number of pollen grains in each field of view basically the same. Round, dark-stained pollen grains are recorded as vigorous pollen grains, and deformed, weakly stained or unstained pollen grains are recorded as weak pollen grains.

[0041] Fertility results showed Figure 4 As shown, both the mutant and the wild type were successfully stained with iodine, indicating that the fertility of the mutant pollen grains was not affected.

[0042] Example 2: Analysis of OsMATE7 gene function

[0043] (1) Expression profile analysis

[0044] ① Plant total RNA extraction: Different tissue samples of rice at different developmental stages were collected in the field, placed in 2 mL RNase-free centrifuge tubes, quickly frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted according to the instructions of the Plant Total RNA Isolation Kit of Chengdu Fuji Biotechnology Co., Ltd.;

[0045] ② Obtaining cDNA template: Refer to the instructions of RT EasyTMII (With gDNase) kit of Chengdu Fuji Biotechnology Co., Ltd. for synthesis operation. The obtained cDNA product can be added with 40 μL RNase-free ddH 2 O was diluted 3 times and stored at -20°C;

[0046] ③ Real-time fluorescence quantitative PCR: The qPCR primer sequence was queried from the database qPrimerDB (https: / / qprimerdb.biodb.org / index) to obtain the OsMATE7-specific qRT-PCR primers (Fprimer: 5'-GTCATCTTCACCGGAGACAG-3' (SEQ ID NO.5); R primer: 5'-CCGTAGTAACACGCCAGATTAA-3' (SEQ ID NO.6)). The UBQ5 and FhaB genes were used as internal references, and the real-time quantitative system was used to react to detect the relative expression of the genes.

[0047] The results are as follows Figure 5 As shown, the results of fluorescence quantitative analysis showed that the OsMATE7 gene was specifically expressed in anthers, which was consistent with the phenotypic data that it regulated rice fruiting through pollen.

[0048] (2) Pollen tube staining experiment

[0049] To observe the growth of pollen tubes in the pistil, aniline blue dye can be used. Since pollen tubes contain a large amount of callose, aniline blue dye can be detected under a fluorescence microscope after staining. Therefore, it can be used to observe the growth of pollen tubes in the pistil. The specific steps are as follows:

[0050] ① Prepare FAA fixative. The formula of 100 mL FAA fixative is: 90 mL 70% alcohol, 5 mL acetic acid, and 5 mL formalin (37%-40% formaldehyde).

[0051] ② Observe the flowering of the test materials in the field, record the flowering time, take a sufficient amount of spikelets at different time points after flowering and place them in the fixative, vacuum for 30 minutes to allow the fixative to fully penetrate the tissue, fix for 24 hours, transfer the spikelets to 70% alcohol and store at 4°C.

[0052] ③ Take out the spikelets and peel off the ovary under a stereo microscope, and rehydrate it with gradient alcohol (50%, 30%, 10%) and distilled water (2 times), staying in each gradient for 10 minutes.

[0053] ④ After rehydration, transfer the ovary to 5M NaOH solution and soften and make transparent at 56℃ for 15 minutes. Wash it with distilled water for 3 times. Be gentle during the process to avoid damaging the ovary. Then add 0.1% aniline blue dye solution, vacuum for 30 minutes, and dye for 8 hours.

[0054] ⑤ Before observation, wash the dye solution with distilled water for 3 times, drop a drop of 50% glycerol on the slide, place the ovary in the glycerol, carefully position it with tweezers, cover it with a coverslip and press gently, and then observe it under the DAPI channel of the fluorescence microscope. Collect pictures and count the proportion of pollen tubes reaching the micropyle of different test materials.

[0055] The results are as follows Figure 6 As shown, the pollen tubes of the wild type normally reached the ovary 6 hours after flowering to complete the fertilization process, while the pollen tubes of the mutant elongated and did not reach the stigma, which was consistent with the poor fruit set of the mutant.

[0056] The CDS nucleotide sequence of the OsMATE7 gene and its encoded protein sequence in the present invention are as follows:

[0057] (1) Nucleotide sequence of the coding region of the OsMATE7 gene:

[0058]

[0059] (2) Amino acid sequence of the protein encoded by the OsMATE7 gene:

[0060] MVVAVQDDETGALVAVGKGDEDDDDDDAVAGEEDEDEDDAPVVRTARGAWEVFAAESRRLWAIGAPIAFNVICLYGTNSTTQIFVGHIGNRELSAVAIGLSVVSNFSFGFLLGMGSALETLCGQAFGAGQ VAMLGIYMQRSWIILAASAALLSPLYVFAGPILRLLGQEESIAAAAGEFTVRIIPQMFALAINFPTQKFLQAQSKVTVLAWIGFAALLAHVGLLALFVSALGWGIAGAAAAYDVSSWLTALAQVAYVVGW CRDGWTGLSRKAFNELWAFVKLSLASAVMLCLEIWYMMVLVVLTGHLDDAEIAVDSISICMNINGWEGMLFIGLNAAISVRVSNELGSGRPRATMHAVVVVLVQSLAFGLLAMVLILATRNHFAVIFTGD RHLQKAVANIAYMLAVTMVLNSIQPVISGVAVGGGWQGVVAYINLACYYGFGLPLGFIFFGYLFRWGVKGIWAGMLCGTAMQTAILMYMVCKTDWEAESVQALERVRLWGGQPEHEKLPTSEPEETII(SEQ IDNO.2).

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rice OsMATE7 gene, characterized in that: The nucleotide sequence of the rice OsMATE7 gene coding region is shown in SEQ ID NO.

1.

2. The protein encoded by the rice OsMATE7 gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by the rice OsMATE7 gene is shown in SEQ ID NO.

2.

3. Use of the rice OsMATE7 gene according to claim 1 in regulating rice male sterility.

4. The use according to claim 3, characterized in that: The regulation of rice male sterility is to knock out the rice OsMATE7 gene to weaken the vigor of rice pollen.

5. The use according to claim 3, characterized in that: The regulation of rice male sterility is to reduce the length of rice pollen tube after knocking out the rice OsMATE7 gene.

6. A preparation for regulating rice male sterility, characterized in that: The preparation comprises the rice OsMATE7 gene according to claim 1 or the protein encoded by the rice OsMATE7 gene according to claim 2.

7. A method for preparing a male sterile rice variety, characterized in that: Preparation of male-sterile rice varieties by knocking out the rice OsMATE7 gene.

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