Gene OsFLO17 related to rice starch synthesis as well as encoding protein and application thereof

Abstract

The invention discloses a gene OsFLO17 related to rice starch synthesis as well as an encoding protein and application of the gene OsFLO17. Through phenotypic analysis of a rice endosperm powder mutant flo17 and preliminary positioning of a target gene, the protein OsFLO17 related to starch synthesis is finally obtained through cloning, and the related protein is composed of an amino acid sequence shown in SEQ ID NO.3. The starch granule development related protein provided by the invention influences the synthesis process of rice endosperm starch, and a transgenic plant with normal starch filling can be obtained by introducing the coding gene of the protein into a plant with abnormal starch granules. Therefore, the protein and the gene coded by the protein can be applied to plant genetic improvement.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a rice starch synthesis-related gene OsFLO17, its encoded protein and application. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop. With economic growth and rising living standards, the demand for high-quality rice is increasing. Therefore, improving rice quality while maintaining high yields has become a key goal of rice research and industrial development in my country.

[0003] The rice endosperm is a key tissue that determines rice yield and quality. The endosperm contains approximately 80% starch, 8%-10% protein, and the remainder consists of fat, dietary fiber, and vitamins. Starch is the primary storage substance in the rice endosperm and is directly related to rice yield and quality. Furthermore, starch stored in the endosperm is the core energy source for seed germination and early seedling growth, playing a key role in rice growth and development. Therefore, understanding the molecular regulatory mechanisms of rice starch synthesis is of great significance for the genetic improvement of rice quality.

[0004] Cloning key genes for endosperm development through endosperm-abnormal mutants is an effective means of deeply analyzing the formation of rice grain quality. After long-term research, scientists have gained a certain understanding of the regulatory network for the accumulation of storage substances in rice. However, given the complexity of endosperm development and its internal material transport processes, further in-depth research is still needed to reveal its underlying mechanisms. Summary of the Invention

[0005] The present inventors screened the floury endosperm mutant flo17 from the Ningjing 3 MNU mutagenesis library and cloned the gene OsFLO17, which is responsible for the mutant's starch synthesis and endosperm development defects. Therefore, the present invention aims to provide a rice starch synthesis-related gene, OsFLO17, its encoded protein, and its applications.

[0006] The nucleotide sequence of the gene OsFLO17 provided by the present invention is shown in 1) or 2) below:

[0007] 1) The nucleotide sequence shown in SEQ ID NO. 1;

[0008] 2) The nucleotide sequence shown in SEQ ID NO.2.

[0009] SEQ ID NO. 2 in the sequence listing consists of 3747 nucleotides.

[0010] The present invention also provides a protein encoded by the above gene OsFLO17. Specifically, the protein provided by the present invention is selected from any one of (a) or (b):

[0011] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3;

[0012] (b) A protein derived from SEQ ID NO. 3 that is related to starch synthesis and has one or more amino acid residues substituted, deleted, or added to the amino acid sequence of SEQ ID NO. 3. SEQ ID NO. 3 in the sequence listing consists of 1248 amino acids.

[0013] To facilitate purification of OsFLO17 in (b), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO. 3.

[0014] Table 1 Tag sequences

[0015]

[0016] The OsFLO17 protein in (b) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. The gene encoding the OsFLO17 protein in (b) above can be obtained by deleting one or more amino acid residue codons from the nucleotide sequence of SEQ ID NO. 1, and / or performing one or more base pair missense mutations, and / or attaching the coding sequence of the tag shown in Table 1 to its 5' and / or 3' end.

[0017] The recombinant expression vector containing any of the above genes also falls within the scope of protection of the present invention.

[0018] Existing plant expression vectors can be used to construct a recombinant expression vector containing the gene.

[0019] Plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. These vectors may also contain the 3' untranslated region of the exogenous gene, specifically a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal directs the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the 3' untranslated region of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the rouge synthase Nos gene) and plant genes (such as the soybean storage protein gene) all have similar functions.

[0020] When using the gene to construct a recombinant plant expression vector, any enhancing promoter or constitutive promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter, can be added before the transcription initiation nucleotide. These promoters can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are diverse and can be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or a structural gene.

[0021] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For the safety of transgenic plants, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0022] The recombinant overexpression vector can be a recombinant plasmid obtained by inserting the gene (OsFLO17) into the recombination site of the vector pCAMBIA1390, which is double-digested with the restriction endonucleases HindIII and BamHI. The pCAMBIA1390 containing OsFLO17 was designated pCAMBIA1390-OsFLO17. pCAMBIA1390-OsFLO17 was generated by inserting the OsFLO17 genomic coding sequence, along with a 2007-bp upstream promoter region and a 9671-bp downstream fragment, into the pCAMBIA1390 multiple cloning sites between HindIII and BamHI via recombination techniques (Clontech, Infusion Recombination Kit).

[0023] The expression cassette, transgenic cell line and recombinant bacteria containing any of the above genes (OsFLO17) all fall within the scope of protection of the present invention.

[0024] Another object of the present invention is to provide a method for filling normal rice endosperm starch synthesis in transgenic plants.

[0025] The present invention provides a method for cultivating transgenic plants with normal rice endosperm starch synthesis and filling, comprising introducing the gene into a plant with abnormal endosperm starch synthesis and filling to obtain a transgenic plant with normal endosperm starch synthesis and filling; the transgenic plant with normal endosperm starch synthesis and filling is a transgenic plant with normal endosperm starch synthesis and filling, capable of forming a mature, transparent endosperm. Specifically, the gene is introduced into a plant with abnormal endosperm starch synthesis and filling via the recombinant expression vector; the plant with abnormal endosperm starch synthesis and filling can be a flo17 mutant.

[0026] The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria or the method can all be applied to rice breeding.

[0027] By introducing the gene encoding the protein into plant cells using any vector capable of directing exogenous gene expression in plants, transgenic cell lines and transgenic plants can be obtained. Expression vectors carrying the gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transfection, and the transformed plant tissues can be cultivated into plants. The transformed plant hosts can be either monocots or dicots, such as tobacco, Lotus japonicus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0028] The rice starch synthesis-related protein of the present invention influences starch accumulation in the rice endosperm. Transforming the gene encoding the protein into rice plants with floury endosperms can yield transgenic plants with normal endosperm starch filling. Therefore, the protein and its encoding gene of the present invention can be used in plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The phenotypes of wild-type Ningjing 3 and mutant flo17 are shown. Figures a and c show the dry seed and endosperm cross-section phenotypes of Ningjing 3, b and d show the dry seed and endosperm cross-section phenotypes of mutant flo17, and e and f are scanning electron micrographs of endosperm cross-sections of Ningjing 3 and mutant flo17, respectively.

[0030] Figure 2 Shown are the results of grain shape and quality analysis for wild-type Ningjing 3 and mutant flo17. (a) represents seed length, width, and thickness; (b) represents the total starch content, amylose content, sucrose content, fructose content, and glucose content; (g) represents the starch chain length distribution; and (h) represents the starch viscosity profile (RVA) of the seeds.

[0031] Figure 3Semi-thin sections of developing endosperm from wild-type Ningjing 3 and mutant flo17 are shown. (a) An iodine-potassium iodide-stained endosperm section from wild-type Ningjing 3, with a1 and a2 being magnified views of the region in a. (b) An iodine-potassium iodide-stained section from mutant flo17, with b1 and b2 being magnified views of the region in b.

[0032] Figure 4 Showing positional cloning of the mutant gene. Figure a shows the fine-grained mapping of flo17; figure b shows the cloned gene and its mutation site; figure c shows the cross-section phenotype of dry seeds from Ningjing 3 (WT) and transgenic complementation lines (L1, L2); and figure d shows a scanning electron micrograph of a cross-section of endosperm from Ningjing 3 (WT) and transgenic complementation lines (L1, L2). DETAILED DESCRIPTION

[0033] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0034] Example 1: Discovery of Rice Starch Synthesis-Related Sites and Their Encoding Genes

[0035] 1. Phenotypic Analysis of the Rice Starch Biosynthesis-Filling Abnormality Mutant flo17

[0036] The mutant line flo17 with powdery and wrinkled grains was screened from the MNU chemical mutagenesis mutant library of the japonica rice variety Ningjing 3. The wild type has transparent grains (see Figure 1 a, c), while flo17 showed a powdery and wrinkled grain phenotype (see Figure 1 The endosperm of flo17 is floury and concave at the belly, indicating that the genetic mutation affects the filling of starch, resulting in changes in the structure and properties of starch granules, resulting in differences in the appearance of the endosperm. Scanning electron microscopy was performed on cross-sections of wild-type and mutant flo17 seeds. The starch granules in the cross-sections of wild-type seeds are regular and closely arranged (see Figure 1 e), while the starch granules in the center of the mutant flo17 were loosely arranged with a large number of gaps between the granules (see Figure 1 f).

[0037] The grain shape traits of mature seeds of wild type and mutant flo17 were examined. Compared with wild type, flo17 had reduced grain thickness, but no significant difference in grain length and width (see Figure 2 The total starch and amylose content of the seeds were determined to be significantly decreased in the endosperm of flo17 (see Figure 2 Compared with the mature seeds of the wild type, the sucrose content, fructose content, and glucose content of flo17 seeds were significantly increased (see Figure 2 (d, e, f).

[0038] The chain length distribution of endosperm starch showed that the mutant had a significant increase in short-chain starch and a significant decrease in the content of branched chains with a degree of polymerization greater than 16 (see Figure 2 The rice quality of wild type and mutants was determined and it was found that the RVA spectrum of mutant flo17 (see Figure 2 Middle H) A dramatic change compared to wild type.

[0039] Semi-thin sections of the endosperm in the early stages of development were observed, and it was found that the starch granules of the wild type developed normally and were uniform in size, and the tortoise shell structure of the amyloplast was clear (see Figure 3 In the mutant flo17, however, abnormally developed complex starch granules can be seen, with most granules being small, loose, and irregular (see Figure 3 b, b1, and b2). This indicates that the FLO17 gene mutation in the mutant leads to abnormal starch filling in the rice endosperm and affects the development of starch granules, resulting in a floury, wrinkled endosperm phenotype.

[0040] 2. Map-based cloning of mutant gene sites

[0041] 1. Preliminary positioning of target genes

[0042] The mutant flo17 was crossed with the indica rice variety N22. Ten individuals with the recessive extreme flo17 phenotype (shrunken grains) were selected from the flo17 / N22 F2 segregating population, and seed DNA was extracted. Linkage analysis using genome-wide molecular markers mapped the mutant gene responsible for the flo17 mutant phenotype to a region between InDel markers Y1 and Y2 on chromosome 2, a distance of 0.82 Mb at the end of the chromosome long arm.

[0043] 2. Fine positioning of target genes

[0044] Based on the initial mapping results, molecular markers on the public map were searched between InDel markers Y1 and Y2. Based on the rice genome sequence information published by NCBI, SSR markers were developed in this interval (see Table 1). The SSR markers on the public map were integrated with the rice genome sequence, and BAC / PAC clone sequences near the mutation site were downloaded. Potential SSR sequences (number of repeats ≥ 6) in the clones were searched using SSRHunter (Li Qiang et al., Heredity, 2005, 27(5):808-810) or SSRIT online software (http: / / archive.gramene.org / db / markers / ssrtool). These SSRs and their adjacent 400-500 bp sequences were compared with the corresponding indica rice sequences using the BLAST program at NCBI online. If the SSR repeat numbers differed between the two, it was preliminarily inferred that the PCR product of the SSR primer was polymorphic between indica and japonica. SSR primers were then designed using Primer Premier 5.0 software and synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Self-designed SSR primer pairs were mixed in equal proportions and tested for polymorphism between N22 and Ningjing 3. Polymorphic variants were used as molecular markers for fine mapping. Fine mapping of the target gene was performed using 1000 recessive extreme individuals (see Table 2 for molecular markers used for fine mapping).

[0045] Table 2 Molecular markers used for fine mapping

[0046]

[0047] The target gene OsFLO17 was finally finely mapped between markers Y5 and Y6, with a physical distance of 56 kb (see Figure 4 After sequencing the genes in this region, it was found that there was a single base substitution in the 7th exon of the 6th ORF, which caused the translation of the target protein to terminate prematurely ( Figure 4 Middle b).

[0048] 3. Acquisition of the powdery gene OsFLO17

[0049] cDNA was extracted from leaves of japonica rice variety Ningjing 3. PCR amplification was performed using the cDNA as a template with primers Primer1 and Primer2. The amplified product was sequenced. The sequencing result is shown in SEQ ID NO.2, and the encoded protein is shown in SEQ ID NO.3.

[0050] primer1: 5'-ATGATGTTCACGGAGGGGC-3' (SEQ ID NO. 19);

[0051] primer2: 5'-TCAGTAATAGAAGCTAGAGTTC-3' (SEQ ID NO. 20).

[0052] The protein shown in SEQ ID NO.3 of the sequence listing is named OsFLO17 protein, which consists of 1248 amino acid residues. The gene encoding OsFLO17 protein is named OsFLO17 gene, and its open reading frame is shown in SEQ ID NO.2.

[0053] Example 2: Application of OsFLO17 protein and its encoding gene

[0054] 1. Construction of recombinant expression vector (cutting the vector with HindⅢ and BamHI)

[0055] The OsFLO17 gene was amplified by PCR using the genomic DNA of Ningjing 3 (from the Germplasm Resource Bank of the Rice Research Institute of Nanjing Agricultural University) as a template. The PCR primer sequences are as follows:

[0056] Primer3: 5'-TCGGTGGAGCGTCTTGATATTGTTTTGTACAGGT-3' (SEQ ID NO. 21);

[0057] primer4: 5'-ATACTCCATGAACTTCGCCAGAATCTCTAGCATAGT-3' (SEQ ID NO. 22);

[0058] The product amplified by these primers encompassed the entire gene genome and the 2007-bp promoter region (SEQ ID NO. 1 in the sequence listing). The PCR product was cloned into the pCAMBIA1390 vector (cut with HindIII and BamHI) using the INFUSION Recombination Kit (Takara, Japan). The infusion recombination reaction (10 μL) consisted of 1.0 μL of PCR product, 6.0 μL of pCAMBIA1390, 2.0 μL of 5× infusion buffer, and 1 μL of infusion enzyme mix. After brief centrifugation, the mixture was incubated at 50°C for 20 minutes. A 2.5 μL aliquot of the reaction mixture was used to transform competent Escherichia coli DH5α cells (Tiangen, Beijing) using the heat shock method. All transformed cells were evenly plated onto LB solid medium supplemented with 50 mg / L kanamycin. After incubation at 37°C for 16 hours, positive clones were selected for sequencing. Sequencing results showed that a recombinant expression vector containing the gene shown in SEQ ID NO.1 was obtained. pCAMBIA1390 containing OsFLO17 was named pCAMBIA1390-OsFLO17, and the OsFLO17 gene fragment was inserted between the HindIII and BamHI restriction sites of the vector using an Infusion recombination kit.

[0059] 2. Obtaining recombinant Agrobacterium

[0060] pCAMBIA1390-OsFLO17 was transformed into Agrobacterium tumefaciens EHA105 (purchased from Innovent Biologics, Inc., USA) to obtain a recombinant strain. The plasmid was extracted and identified by PCR and enzyme digestion. The recombinant strain that was correctly identified by PCR and enzyme digestion was named EH-pCAMBIA1390-OsFLO17.

[0061] 3. Obtaining genetically modified plants

[0062] EH-pCAMBIA1390-OsFLO17 was used to transform the rice mutant flo17 with abnormal amyloplast development. The specific method was as follows:

[0063] (1) Cultivate EH-pCAMBIA1390-OsFLO17 at 28°C for 16 hours, collect the cells, dilute them into N6 liquid medium (Sigma, C1416), resuspend them, and adjust the OD value of the cell suspension. 600nm is 0.5;

[0064] (2) The embryonic callus tissue of mature embryos of flo17 (mutant) rice cultured for one month was mixed with the bacterial solution of step (1) and infected for 30 minutes. The bacterial solution was dried with filter paper and then transferred to co-cultivation medium (N6 solid co-cultivation medium, Sigma) and co-cultivated at 24°C for 3 days.

[0065] (3) The callus from step (2) was inoculated on N6 solid screening medium containing 100 mg / L hygromycin for the first screening (16 days);

[0066] (4) Select healthy calli and transfer them to N6 solid screening medium containing 100 mg / L hygromycin for a second screening, subculture every 15 days;

[0067] (5) Select healthy calli and transfer them to N6 solid screening medium containing 50 mg / L hygromycin for the third screening, subculture every 15 days;

[0068] (6) Select resistant calli and transfer them to differentiation medium for differentiation; obtain T0 generation positive plants that differentiate into seedlings.

[0069] 4. Identification of transgenic plants

[0070] 1. PCR molecular identification

[0071] In this study, hygromycin marker was used to identify transgenic plants.

[0072] PCR reaction system for labeling analysis: DNA (20 ng / μL) 2 μL, Primer5 (10 pmoL / μL) 2 μL, Primer6 (10 pmol / μL) 2 μL, 10xBuffer (MgCl2 free) 2 μL, dNTP (10 mM) 0.4 μL, MgCl2 (25 mM) 1.2 μL, rTaq (5 U / μL) 0.4 μL, ddH2O 10 μL, total volume 20 μL.

[0073] The amplification reaction was performed on a PCR instrument: 94°C for 3 min; 94°C for 30 sec, 56°C (adjusted for different primers) for 30 sec, 72°C for 1.5 min, 34 cycles; 72°C for 5 min.

[0074] The PCR products were analyzed by agarose gel electrophoresis (1% agarose), and the transgenic positive plants were identified based on the presence or absence of bands.

[0075] Primer5: 5'-TAGGAGGGCGTGGATATGTC-3' (SEQ ID NO. 23);

[0076] Primer6: 5'-TACACAGCCATCGGTCCAGA-3' (SEQ ID NO. 24).

[0077] 2. Phenotypic identification

[0078] T0 generation pCAMBIA1390-OsFLO17 positive plants, mutant flo17, and Ningjing 3 were planted in the transgenic experimental field of the Chinese Academy of Agricultural Sciences. After the seeds matured, the seeds of each material were collected. It was observed that transparent seeds appeared in the seeds of the pCAMBIA1390-OsFLO17 plants (see Figure 4 (c, d) This demonstrates that the mutant phenotype of flo17 is caused by a mutation in OsFLO17. pCAMBIA1390-OsFLO17 can restore the transparent phenotype of flo17 seeds.

Claims

1. A rice starch synthesis-related protein, or its encoding gene, or a recombinant expression vector containing the encoding gene, or a recombinant bacterium containing the encoding gene, for use in plant breeding; the application is for cultivating high-quality transgenic plants with normal starch synthesis; the plant being tobacco, Lotus japonicus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, turf grass, or alfalfa; The rice starch synthesis-related protein is a protein consisting of the amino acid sequence shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: A tag sequence is added to the end of the rice starch synthesis-related protein, and the tag sequence is Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in 1) or 2) below: 1) The nucleotide sequence shown in SEQ ID NO. 1; 2) The nucleotide sequence shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The recombinant expression vector is a recombinant plasmid obtained by inserting the coding gene between the multiple cloning sites HindIII and BamHI of the pCAMBIA1390 vector.

5. A method for cultivating transgenic rice plants with normal starch synthesis pathways, characterized by: A gene encoding a protein related to rice starch synthesis is introduced into a plant with abnormal starch synthesis to obtain a transgenic plant with normal starch synthesis; wherein the abnormal starch synthesis is insufficient endosperm filling; the encoding gene is introduced into the plant with abnormal starch synthesis via a recombinant expression vector, wherein the plant is tobacco, Lotus japonicus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, turf grass or alfalfa; The rice starch synthesis-related protein is a protein consisting of the amino acid sequence shown in SEQ ID NO.

3.

6. The method according to claim 5, characterized in that: The nucleotide sequence of the gene encoding the rice starch synthesis-related protein is shown in 1) or 2) below: 1) The nucleotide sequence shown in SEQ ID NO. 1; 2) The nucleotide sequence shown in SEQ ID NO.2.

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