Application of Scutellum-specifically Expressed Gene OsMYB80 in Inhibiting Rice Panicle Germination

Abstract

The present invention discloses the application of the scutellum-specific expression gene OsMYB80 in inhibiting rice panicle germination, and relates to the technical field of plant genetic engineering. The present invention discloses the application of the scutellum-specific expression gene OsMYB80 in regulating rice panicle germination, and achieves the purpose of regulating rice panicle germination by regulating the expression of the OsMYB80 gene; the CDS nucleotide sequence of the OsMYB80 gene is shown in SEQ ID NO.2, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.3. The present invention constructs a knockout vector of the rice OsMYB80 gene; transforms the constructed knockout vector into rice; cultivates, screens and obtains transgenic rice plants with significantly reduced panicle germination rate. The results show that knocking out the OsMYB80 gene significantly reduces the panicle germination rate of transgenic rice.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and more specifically, relates to the application of scutellum-specific expression gene OsMYB80 in inhibiting rice panicle germination. Background Art

[0002] Rice is the main food crop in my country and plays a decisive role in ensuring national food security. The dormancy period of rice seeds is of great significance to agricultural production and seed preservation. For agricultural production, mastering the dormancy law of seeds can make the seeds germinate and grow in the most suitable time and increase the harvest; for seed preservation, understanding the dormancy law of seeds can help select the best storage temperature, humidity and other factors to extend the shelf life of seeds.

[0003] When rice seeds germinate, GA in the embryo regulates the scutellum epithelial cells or the endosperm aleurone layer to produce amylase, breaking down the storage starch in the endosperm into glucose, which is transported to the embryonic cells and produces ATP through respiration to provide energy for germination. Therefore, the scutellum becomes the switch for regulating seed germination.

[0004] Seed dormancy refers to the phenomenon that seeds cannot germinate under conditions suitable for their growth. However, in the process of crop domestication, due to more consideration of high yield, high quality, disease and pest resistance, the retention of seed moderate dormancy traits is often neglected, resulting in spike sprouting in many crops such as rice and wheat. Spike sprouting, also known as preharvest sprouting (PHS), refers to the phenomenon that wheat grains sprout on the wheat ears before harvest. This is an abnormal phenomenon, usually caused by continuous rain and humid environment during the period from maturity to harvest. It not only affects the quality of grains, but also affects the storage of wheat and the quality of sowing in the next season or the following year, causing great economic losses to wheat production. Insufficient rice dormancy leads to spike sprouting (preharvest sprouting), which seriously affects the grain yield and quality in my country and even the world, and then affects the production efficiency and economic benefits, especially the high temperature and high humidity environment in the Yangtze River Basin in my country, which brings great losses to agricultural production. Rice seed dormancy is a complex quantitative trait. At present, the regulatory network identified at the molecular level is very limited, which hinders the selection and breeding of seed dormancy varieties. Therefore, the research and identification of genes related to rice dormancy regulation has important application value. Using modern biotechnology to transform and regulate rice dormancy and create rice germplasm resources with different dormancy intensities has important theoretical and practical significance for guiding the selection and breeding of rice varieties adapted to specific regions and improving rice adaptability and yield. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of the scutellum-specific expression gene OsMYB80 in inhibiting rice panicle germination, for inhibiting rice panicle germination (germination before harvest).

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Application of the scutellum-specific expression gene OsMYB80 in regulating rice panicle germination, wherein the CDS nucleotide sequence of the OsMYB80 gene is shown in SEQ ID NO.2.

[0008] The application is to reduce rice panicle germination by inhibiting the expression of OsMYB80 gene.

[0009] The application described, the steps are as follows:

[0010] 1) Construction of a knockout vector for the rice OsMYB80 gene;

[0011] 2) Transforming the constructed knockout vector of rice OsMYB80 gene into rice;

[0012] 3) Cultivate, screen and obtain transgenic rice plants with significantly reduced ear germination rate.

[0013] In the application, the knockout vector is a plant expression vector.

[0014] The application, the method for constructing the rice OsMYB80 gene knockout vector is: designing oligo primers according to the gRNA target sequence that inhibits the expression of the OsMYB80 gene, configuring the reaction system to synthesize oligo dimers; constructing the oligo dimers into the CRISPR / cas vector to obtain the rice OsMYB80 gene knockout vector.

[0015] In the application, the gRNA target sequence for inhibiting the expression of the OsMYB80 gene is:

[0016] ①CRISPR-OsMYB80: 5'-GCAAGCTCTAGCCATGGCACCGG-3';

[0017] Or ②CRISPR-OsMYB80: 5'-CACCATGGCAGTCCCGGACATGG-3'.

[0018] The oligo primer sequence is as follows:

[0019] ①Oligo-F: 5'-TGTGTGGCAAGCTCTAGCCATGGCACCGG-3';

[0020] Oligo-R: 5'-AAACCCGGTGCCATGGCTAGAGCTTGCCA-3'.

[0021] ②Oligo-F: 5'-TGTGTGCACCATGGCAGTCCCGGACATGG-3';

[0022] Oligo-R: 5'-AAACCCATGTCCGGGACTGCCATGGTGCA-3'.

[0023] The rice OsMYB80 gene knockout vector is used in inhibiting rice panicle germination.

[0024] The invention discloses an application of a mutant of the scutellum-specific expression gene OsMYB80 in regulating rice panicle germination, wherein the CDS nucleotide sequence of the OsMYB80 gene is shown in SEQ ID NO.2; the mutant is mutated at any position of the sequence shown in SEQ ID NO.2, and the mutation can cause the termination of protein expression of the OsMYB80 gene.

[0025] The mutations include point mutations, deletion mutations, and insertion mutations.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1) The rice OsMYB80 gene disclosed in the present invention has a CDS nucleotide sequence as shown in SEQ ID NO.2, and an amino acid sequence of its expressed protein as shown in SEQ ID NO.3. The present invention constructs a knockout vector of the rice OsMYB80 gene; transforms the constructed knockout vector into rice; and cultivates, screens, and obtains transgenic rice plants with significantly reduced panicle germination rate. The results show that knocking out the OsMYB80 gene significantly reduces the panicle germination rate of transgenic rice.

[0028] 2) The present invention finally obtains transgenic homozygous plants myb80-11 and myb80-176 with site-directed mutations at the target site of the rice OsMYB80 gene. The myb80-11 homozygous mutant shows an insertion of a base at the target site ①, resulting in a protein coding frameshift; the myb80-176 homozygous mutant shows a deletion of eight bases at the target site ②, resulting in a protein coding frameshift. Both homozygous mutation forms cause protein coding frameshifts, resulting in protein function loss.

[0029] 3) Compared with the control group, the ear germination rate of the myb80-11 and myb80-176 homozygous mutant lines was significantly reduced, and the ear germination phenomenon was almost non-existent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The quantitative results of the expression pattern of OsMYB80 gene in different parts are shown in Figure 1 (A is the expression pattern analysis of OsMYB80 gene in different parts; B is the expression pattern analysis of OsMYB80 gene in seeds after pollination);

[0031] Figure 2 This is the GUS staining image of OsMYB80 gene in seed scutellum;

[0032] Figure 3 This is the location map of the OsMYB80 gene target site;

[0033] Figure 4 This is the sequencing result of the homozygous mutant of the OsMYB80 gene;

[0034] Figure 5 Figure 2 shows the spike germination phenotype observation (A) and statistical results (B) of the myb80 homozygous mutant. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0036] The materials used in the tissue expression pattern analysis experiment of the present application are 6-day-old roots, 30-day-old stems, 30-day-old leaves, 2nd leaf, spikelets at different stages, seeds soaked for different times after maturity (24h, 48h), seeds at 14 days of development, and seeds at 30 days of development of the rice variety Zhonghua 11 (ZH11).

[0037] The material used for the intraspecific expression profile at different stages in the present application is the seeds of the rice variety Zhonghua 11 (ZH11), which are collected every three days starting from the 7th day of pollination and ending on the 34th day.

[0038] Example 1

[0039] 1. Total RNA extraction and cDNA acquisition

[0040] The Tiangen polysaccharide and polyphenol plant total RNA extraction kit was used to extract RNA from different parts of rice (6-day roots, 30-day stems, 30-day leaves, 2nd leaves, spikelets at different stages, seeds soaked for different times after maturity (24h, 48h), seeds at 14 days of development, and seeds at 30 days of development). The extracted total RNA was used as a template, and the reverse transcription reagent HiScriptⅢ1stStrand cDNASynthesis Kit (Nanjing Novozyme Biotechnology Co., Ltd.) was used for reverse transcription experiments to obtain cDNA.

[0041] 2. Analysis of the expression pattern of OsMYB80 gene in seeds at different locations and stages

[0042] The expression pattern of OsMYB80 gene in different parts of rice and seeds at different stages was analyzed, and quantitative primers of OsMYB80 gene were designed. The primer sequences are shown below:

[0043] Q-OsMYB80-F: 5'-CGGACCTCAACGCCTACAAC-3';

[0044] Q-OsMYB80-R: 5'-GCAACCGAAATCCAGGCTAAC-3'.

[0045] The corresponding cDNA (added in an amount of 50 ng) was used, rice Ubiquitin was used as an internal reference, and ChamQ Universal SYBR qPCR Master Mix (Nanjing Novozyme Biotech Co., Ltd.) was used for quantitative experiments.

[0046] The results are as follows Figure 1 As shown, the OsMYB80 gene was specifically expressed in seeds, and its expression trend decreased during seed development.

[0047] 3. Target gene transformation into rice and DNA level identification of mutant plants

[0048] The upstream 2500 bp promoter of the OsMYB80 coding gene (as shown in SEQ ID NO.1) was amplified from the ZH11 genomic DNA. The promoter amplification primer sequence is as follows:

[0049] proMYB80-GUS-F: 5'-AACTGCAGAGAGGCAAAGAAATCGTCGTC-3';

[0050] proMYB80-GUS-R: 5'-CGGGATCCAGATTTTTGATTGTGGGTGGAG-3'.

[0051] The fragment was connected to the 5' end of the GUS coding region of the XB-4 vector by T4 ligation, and mixed according to the reaction system shown (Vector 1 μL, proMYB80 7 μL, Buffer 1 μL, T4 enzyme 1 μL), and reacted at 16°C for 4 hours.

[0052] The ligated fragments were transformed into competent Escherichia coli cells using the CaCl2 heat shock transformation method and then identified. After obtaining positive recombinants, the plasmid was extracted and then transformed into Agrobacterium tumefaciens GV3101 strain. The target gene was transferred into the rice variety Zhonghua 11 using the Agrobacterium infection method.

[0053] Through hygromycin resistance screening, T0 generation transgenic lines were obtained. After two generations of plant identification, proMYB80-GUS homozygous transgenic plants were obtained.

[0054] 4. GUS staining

[0055] Use Coolaber GUS staining kit (SL7160) to stain rice seeds. The specific steps are as follows:

[0056] Rice seeds 10 days after flowering were cut open longitudinally in the middle with a scalpel to obtain the embryo-endosperm section. The samples were directly immersed in 1×GUS dye solution and vacuum-infiltrated for 30 min under dark conditions. They were then transferred to a 33°C incubator for 6 h and photographed under a microscope.

[0057] The results are as follows Figure 2 As shown, the OsMYB80 gene was specifically expressed in the seed scutellum.

[0058] Example 2

[0059] 1. Target gene cloning

[0060] The OsMYB80 gene is located on rice chromosome 1 and contains 3 exons and 2 introns. Due to its expression specificity, the cDNA of rice ZH11 seeds 14 days after fertilization was used as a template to amplify the OsMYB80 coding region;

[0061] The amplification primers are:

[0062] MYB80-F: 5'-ATGCTTCCAACCTCAAAGAAC-3';

[0063] MYB80-R: 5'-TCACTTGAACCTGGTCAGGTC-3';

[0064] The PCR product obtained by the above amplification was subjected to first generation sequencing, and the CDS sequence of the OsMYB80 gene was finally cloned as shown in SEQ ID NO.2, and the amino acid sequence of the expressed protein was shown in SEQ ID NO.3.

[0065] 2. Selection of OsMYB80 gene target sites

[0066] The OsMYB80 gene is located on rice chromosome 1 and contains 3 exons and 2 introns. The target site selected for the knockout vector construction is located in the first exon region ( Figure 3). One of the target double strands knocked out using CRISPR technology has the following structure: 5'-NGG-3', and N in the PAM sequence NGG represents any one of A, T, C, and G. The gRNA target sequence of the OsMYB80 gene is as follows:

[0067] ①CRISPR-OsMYB80: 5'-GCAAGCTCTAGCCATGGCACCGG-3'.

[0068] ②CRISPR-OsMYB80: 5'-CACCATGGCAGTCCCGGACATGG-3'.

[0069] 3. Construction of OsMYB80 gene editing knockout vector and acquisition of gene editing mutants

[0070] gRNA target sequence of OsMYB80 gene ①Synthetic oligo:

[0071] Oligo-F: 5'-TGTGTGGCAAGCTCTAGCCATGGCACCGG-3';

[0072] Oligo-R: 5'-AAACCCGGTGCCATGGCTAGAGCTTGCCA-3'.

[0073] gRNA target sequence of OsMYB80 gene②Synthetic oligo:

[0074] Oligo-F: 5'-TGTGTGCACCATGGCAGTCCCGGACATGG-3';

[0075] Oligo-R: 5'-AAACCCATGTCCGGGACTGCCATGGTGCA-3'.

[0076] The synthesized primers were dissolved in water to 10 μmol, mixed according to the reaction system shown (Buffer Aneal 18 μL, FPrimer 1 μL, R Primer 1 μL), heated at 95°C for 3 minutes using a PCR instrument, and then the temperature was slowly lowered to 20°C at about 0.2°C / second to prepare oligo dimers.

[0077] According to the reaction system shown (H2O 6μL, CRISPR / Cas Vector 2μL, Oligo dimer 1μL, EnzymeMix 1μL), mix well and react at 20°C for 1 hour. The oligo dimers were constructed into the CRISPR / cas vector BGK032 by homologous recombination.

[0078] The ligated fragments were transformed into competent Escherichia coli cells using the CaCl2 heat shock transformation method and then identified. After obtaining positive recombinants, the plasmids were extracted and then transformed into Agrobacterium tumefaciens GV3101 strains. The target gene was transferred into the rice variety Zhonghua 11 using the Agrobacterium infection method.

[0079] The T0 transgenic lines were obtained by hygromycin resistance screening. The DNA of the resistant plants was extracted, and the genomic DNA was amplified by PCR using specific primers containing the target site. The PCR products were sequenced for the first generation to obtain transgenic homozygous plants with site-directed mutations in the target site of the rice OsMYB80 gene.

[0080] Finally, two homozygous mutants were identified and named myb80-11 and myb80-176. The sequencing results of the two mutants are shown in Figure 4 As shown, the myb80-11 homozygous mutant showed an insertion of a base "A" at the target site ① (the sequence of the myb80-11 homozygous mutant is shown in SEQ ID NO.4; the translated protein sequence is shown in SEQ ID NO.5), resulting in a protein coding frameshift; the myb80-176 homozygous mutant showed a deletion of eight bases "GTCCCGAA" at the target site ② (the sequence of the myb80-176 homozygous mutant is shown in SEQ ID NO.6; the translated protein sequence is shown in SEQ ID NO.7), resulting in a protein coding frameshift. Both homozygous mutant forms cause protein coding frameshifts, resulting in protein function loss.

[0081] The primer sequences used to amplify the OsMYB80 gene target sites ①② are as follows:

[0082] OsMYB80-F: 5'-ATGCTTCCAACCTCAAAGAACA-3';

[0083] OsMYB80-R: 5'-CCCAAATCGTGACGACTGCT-3'.

[0084] 4. Identification of OsMYB80 gene-edited rice panicle germination resistance phenotype

[0085] In 2023, the homozygous mutant plants myb80-11 and myb80-176 were subjected to panicle germination experiments in the experimental field of the Biotechnology Park of Huaiyin Normal University (119°01′E / 33°36′N). The rice panicles 30 days after flowering (30DAP) were taken for phenotypic observation and panicle germination statistics, with three individual plants each time and three independent replicates.

[0086] The results are as follows Figure 5As shown, compared with the control group, the ear germination rate of myb80-11 and myb80-176 lines was significantly reduced, and the ear germination phenomenon was almost non-existent.

[0087] The results showed that knocking out the OsMYB80 gene significantly reduced the panicle germination rate of gene-edited rice.

[0088] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. Scutellum-specific expression genes OsMYB80 The application of the invention in regulating rice ear germination, OsMYB80 The CDS nucleotide sequence of the gene is shown in SEQ ID NO. 2; the application is to inhibit the OsMYB80 Gene expression to reduce rice panicle germination.

2. The use according to claim 1, characterized in that: Here are the steps: 1) Construction of rice OsMYB80 Gene knockout vectors; 2) The constructed rice OsMYB80 The gene knockout vector was transformed into rice; 3) Cultivate, screen and obtain transgenic rice plants with significantly reduced ear germination rate.

3. The use according to claim 2, characterized in that: The rice OsMYB80 The gene knockout vector is a plant expression vector.

4. The use according to claim 3, characterized in that: The rice OsMYB80 The method for constructing a gene knockout vector is as follows: OsMYB80 Design oligo primers for the gRNA target sequence of gene expression and configure the reaction system to synthesize oligo dimers; The oligo dimer was constructed into the CRISPR / cas vector to obtain rice OsMYB80 Gene knockout vectors.

5. The use according to claim 4, characterized in that: The inhibition OsMYB80 The gRNA target sequences for gene expression are: ①CRISPR- OsMYB80 :5'-GCAAGCTCTAGCCATGGCACCGG-3'; or ② CRISPR- OsMYB80 : 5'-CACCATGGCAGTCCCGGACATGG-3'.

6. The use according to claim 4, characterized in that: The oligo primer sequence is as follows: ①Oligo-F: 5'-TGTGTGGCAAGCTCTAGCCATGGCACCGG-3'; Oligo-R: 5'-AAACCCGGTGCCATGGCTAGAGCTTGCCA-3'; ②Oligo-F: 5'-TGTGTGCACCATGGCAGTCCCGGACATGG-3'; Oligo-R: 5'-AAACCCATGTCCGGGACTGCCATGGTGCA-3'.

7. The rice according to claim 4 OsMYB80 Application of gene knockout vector in inhibiting rice panicle germination.

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