A rice starch synthesis-related gene, OsFLO17, its encoded protein, and its applications.

Abstract

This invention discloses a rice starch synthesis-related gene, OsFLO17, its encoded protein, and its applications. Through phenotypic analysis of the rice endosperm mealy mutant flo17 and preliminary localization of the target gene, the starch synthesis-related protein OsFLO17 was ultimately cloned. This protein consists of the amino acid sequence shown in SEQ ID NO.3. The starch granule development-related protein of this invention affects the rice endosperm starch synthesis process. Introducing the encoding gene of this protein into plants with abnormal starch granules can yield transgenic plants with normal starch filling. Therefore, the protein and its encoded gene of this invention can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a rice starch synthesis-related gene OsFLO17, its encoded protein, and its applications. Background Technology

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

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

[0004] Cloning key genes involved in endosperm development using abnormal endosperm mutants is an effective method for deeply understanding the formation of rice grain quality. Through long-term research, scientists have gained some understanding of the regulatory network of rice storage substance accumulation; however, given the complexity of endosperm development and its internal substance transport processes, further in-depth research is needed to reveal its underlying mechanisms. Summary of the Invention

[0005] The inventors screened a starchy endosperm mutant, flo17, from the Ningjing 3 MNU mutagenesis library and cloned the gene OsFLO17, which causes defects in starch synthesis and endosperm development in this mutant. Therefore, the purpose of this invention is to provide a rice starch synthesis-related gene, OsFLO17, its encoded protein, and its applications.

[0006] The gene OsFLO17 provided by this invention has the following nucleotide sequence as shown in 1) or 2):

[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 aforementioned gene OsFLO17. Specifically, the protein provided by the present invention is selected from any one shown in (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 associated with starch synthesis and involves substitution and / or deletion and / or addition of one or more amino acid residues of 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 the purification of OsFLO17 in (b), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.3.

[0014] Table 1. Sequence of Labels

[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 encoding gene of the OsFLO17 protein in (b) above can be obtained by deleting one or more amino acid residues from the codons in the nucleotide sequence shown in SEQ ID NO.1, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0017] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0018] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0019] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the Nos gene for lipase) and plant genes (such as the soybean storage protein gene).

[0020] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing or constitutive promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0021] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[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 restriction endonucleases HindIII and BamHI. pCAMBIA1390 containing OsFLO17 is named pCAMBIA1390-OsFLO17. pCAMBIA1390-OsFLO17 is obtained by inserting the OsFLO17 genomic coding sequence, along with an upstream 2007 bp promoter region and a downstream 9671 bp fragment, into the multiple cloning site of pCAMBIA1390 between HindIII and BamHI using recombination technology (Clontech, Infusion Recombinant Kit).

[0023] Expression cassettes, transgenic cell lines, and recombinant bacteria containing any of the genes (OsFLO17) described above are all within the scope of protection of this invention.

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

[0025] The present invention provides a method for culturing transgenic plants with normal endosperm starch synthesis and filling in rice. This involves introducing the gene into plants with abnormal endosperm starch synthesis and filling to obtain transgenic plants with normal endosperm starch synthesis and filling. The transgenic plants with normal endosperm starch synthesis and filling are transgenic plants capable of forming mature, transparent endosperm. Specifically, the gene is introduced into plants with abnormal endosperm starch synthesis and filling through the recombinant expression vector; the plants with abnormal endosperm starch synthesis and filling can be flo17 mutants.

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

[0027] By using any vector capable of guiding the expression of exogenous genes in plants, the gene encoding the stated protein can be introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the stated 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, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The plant host being transformed can be either monocotyledonous or dicotyledonous, such as tobacco, birdsfoot, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0028] The rice starch synthesis-related protein of this invention affects the starch accumulation process in rice endosperm. Introducing the coding gene of this protein into rice plants with mealy endosperm yields transgenic plants with normal endosperm starch filling. Therefore, the protein and its coding gene of this invention can be applied to plant genetic improvement. Attached Figure Description

[0029] Figure 1 The images show the morphological characteristics of wild-type Ningjing 3 and the mutant flo17. Images a and c show the cross-sectional phenotypes of dry seeds and endosperm of Ningjing 3, images b and d show the cross-sectional phenotypes of dry seeds and endosperm of the mutant flo17, and images e and f are scanning electron microscope images of the cross-sectional endosperm of Ningjing 3 and the mutant flo17, respectively.

[0030] Figure 2 This table shows the results of grain type determination and quality analysis for wild-type Ningjing 3 and the mutant flo17. Specifically, a represents the determination of seed length, width, and thickness; b, c, d, e, and f represent the total starch content, amylose content, sucrose content, fructose content, and glucose content of the seeds; g represents the starch chain length distribution; and h represents the starch viscosity spectrum of the seeds, i.e., the Relative Value Aspect Ratio (RVA) spectrum.

[0031] Figure 3This image shows the observation results of semi-thin sections of the endosperm of wild-type Ningjing 3 and mutant flo17. In the image, a is an iodine-potassium iodide staining image of the endosperm of wild-type Ningjing 3, a1 and a2 are magnified views of the region in image a, b is an iodine-potassium iodide staining image of mutant flo17, and b1 and b2 are magnified views of the region in image b.

[0032] Figure 4 Map-based cloning of the mutant gene is shown. Among them, a is the fine mapping of flo17, b is the gene obtained by cloning flo17 and its mutation site; c is the cross-sectional phenotypic map of dry seeds of Ningjing 3 (WT) and transgenic complementary lines (L1, L2); d is the scanning electron microscope image of the endosperm cross section of Ningjing 3 (WT) and transgenic complementary lines (L1, L2). Detailed Implementation

[0033] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0034] Example 1: Discovery of rice starch synthesis-related sites and their encoding genes

[0035] I. Phenotypic Analysis of flo17, a Rice Starch Synthesis Filler Mutant

[0036] A mutant line flo17 with floury 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 exhibits a phenotype of floury and wrinkled grains (see a). Figure 1 (b, d). The endosperm of flo17 is mealy and concave on the ventral side, indicating that the gene mutation affected starch filling, leading to 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-section of wild-type seeds were regular, orderly, and densely packed (see [reference]). Figure 1 In contrast, the starch granules in the central part of mutant flo17 are loosely arranged with numerous gaps between them (see [reference]). Figure 1 (f)

[0037] The grain shape traits of mature seeds of wild-type and mutant flo17 were investigated. Compared with the wild type, flo17 had reduced grain thickness, while grain length and width showed no significant differences (see...). Figure 2 (a). Determination of total starch and amylose content in seeds revealed a significant decrease in both total starch and amylose content in the endosperm of flo17 (see [reference]). Figure 2 (b, c). Compared with mature wild-type seeds, flo17 seeds showed significantly higher sucrose, fructose, and glucose contents (see [reference]). Figure 2 (d, e, f).

[0038] The chain length distribution of endosperm starch showed 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 in the mutant (see...). Figure 2 (g). Measurements of wild-type and mutant rice quality revealed that the RVA spectrum of the mutant flo17 (see...) Figure 2 The 'h' type has undergone significant changes compared to the wild type.

[0039] Semi-thin sections of the endosperm in early development were observed, revealing that the starch granules in the wild type were normally developed, uniform in size, and exhibited a clear tortoise-shell structure for starch production (see...). Figure 3 (a, a1, a2). In the mutant flo17, abnormally developed complex starch granules can be observed; most starch granules are small, loose, and irregular (see...). Figure 3 (b, b1, b2). It can be seen that the mutant caused abnormal starch filling in the rice endosperm due to the FLO17 gene mutation, and the development of starch granules was also affected, resulting in the endosperm exhibiting a powdery and wrinkled phenotype.

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

[0041] 1. Preliminary localization of the target gene

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

[0043] 2. Fine mapping of target genes

[0044] Based on the initial localization results, molecular markers on the public map were searched between InDel markers Y1 and Y2, and SSR markers were developed in this interval based on the rice genome sequence information published by NCBI (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 sites were downloaded. Potential SSR sequences (repetition count ≥ 6) in the clones were searched using SSRHunter (Li Qiang et al., Genetics, 2005, 27(5):808-810) or the 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 online in NCBI using the BLAST program. If there was a difference in the number of SSR repeats between the two, it was preliminarily inferred that the PCR product of the SSR primers was polymorphic between indica and japonica rice. 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 their polymorphism between N22 and Ningjing 3 was detected. Polymorphic individuals were used as molecular markers for fine mapping. The target gene was finely mapped using 1000 recessive extreme individuals (the molecular markers used for fine mapping are shown in Table 2).

[0045] Table 2 Molecular markers used for fine localization

[0046]

[0047] Finally, the target gene OsFLO17 was finely mapped between markers Y5 and Y6, with a physical distance of 56 kb (see...). Figure 4 (a). Sequencing of the genes within this region revealed a single-base substitution in the 7th exon of the 6th ORF, causing premature termination of translation of the target protein. Figure 4 (b)

[0048] III. Obtaining the starch gene OSFLO17

[0049] cDNA was extracted from the leaves of the japonica rice variety Ningjing 3. Using the cDNA as a template, PCR amplification was performed using primers primer1 and primer2. The amplified products were sequenced, and the sequencing results are shown in SEQ ID NO.2. 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] I. Construction of recombinant expression vector (vector digestion with HindIII and BamHI enzymes)

[0055] Using genomic DNA from Ningjing 3 (from the germplasm resource bank of the Rice Research Institute of Nanjing Agricultural University) as a template, the OsFLO17 gene was obtained by PCR amplification. 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 primer amplification products included the complete genome and the 2007 bp promoter region of the gene (SEQ ID NO. 1 in the sequence listing). The PCR product was cloned into the vector pCAMBIA1390 (digested with HindIII and BamHI) using the INFUSION recombination kit (Takara, Japan). The infusion recombination reaction system (10 μL) consisted of: 1.0 μL PCR product, 6.0 μL pCAMBIA1390, 2.0 μL 5× infusion buffer, and 1 μL infusion enzyme mix. After brief centrifugation, the mixture was incubated at 50°C for 20 minutes. 2.5 μL of the reaction mixture was then transformed into *E. coli* DH5α competent cells (Tiangen, Beijing) using the heat shock method. All transformed cells were evenly spread on LB agar containing 50 mg / L kanamycin. After incubation at 37°C for 16 h, positive clones were picked and sequenced. 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. The OsFLO17 gene fragment was inserted between the HindIII and BamHI restriction sites of the vector using the Infusion recombination kit.

[0059] II. Obtaining Recombinant Agrobacterium

[0060] pCAMBIA1390-OsFLO17 was transformed into Agrobacterium tumefaciens strain EHA105 (purchased from Ingenium Biotech, USA) to obtain a recombinant strain. Plasmids were 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] III. Obtaining Transgenic Plants

[0062] The EH-pCAMBIA1390-OsFLO17 was transformed into the rice mutant flo17, which has abnormal powder-forming development. The specific method is as follows:

[0063] (1) Incubate EH-pCAMBIA1390-OsFLO17 at 28℃ for 16 hours, collect the bacterial cells, dilute them in N6 liquid medium (Sigma, C1416), resuspend them, and adjust the OD of the bacterial culture. 600nm It is 0.5;

[0064] (2) Mix the embryogenic callus of mature embryo of flo17 (mutant) rice cultured for one month with the bacterial solution in step (1) and infect for 30 min. After the bacterial solution is dried with filter paper, transfer it to co-culture medium (N6 solid co-culture medium, Sigma) and co-culture at 24℃ for 3 days.

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

[0066] (4) Select healthy callus and transfer it to N6 solid selection medium containing 100 mg / L hygromycin for a second selection. Subculture every 15 days.

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

[0068] (6) Select resistant callus and transfer it to differentiation medium for differentiation; obtain T0 generation positive plants that have differentiated into seedlings.

[0069] IV. Identification of Transgenic Plants

[0070] 1. PCR Molecular Identification

[0071] In this study, hygromycin markers were used to identify transgenic plants.

[0072] PCR reaction system for label analysis: DNA (20 ng / μL) 2 μL, Primer 5 (10 pmol / μL) 2 μL, Primer 6 (10 pmol / μL) 2 μL, 10x Buffer (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℃ for 3 min; 94℃ for 30 sec, 56℃ (adjusted for different primers) for 30 sec, 72℃ for 1.5 min, 34 cycles; 72℃ for 5 min.

[0074] PCR products were analyzed by agarose gel electrophoresis (1% agarose), and 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, the mutant flo17, and Ningjing 3 were planted in the transgenic experimental field of the Chinese Academy of Agricultural Sciences. After seed maturity, seeds from each material were harvested, and transparent seeds were observed in the seeds of the pCAMBIA1390-OsFLO17 plants (see...). Figure 4 (c, d). Therefore, it is proven that the mutant phenotype in flo17 is caused by the mutation of OSFLO17. pCAMBIA1390-OsFLO17 can restore the transparent phenotype of seeds in the flo17 line.

Claims

1. The application of 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 in rice breeding; The application described is in the cultivation of rice with abnormal starch synthesis caused by the gene mutation shown in SEQ ID NO.2 into a transgenic plant with normal starch synthesis and superior quality; The rice starch synthesis-related protein is a protein composed of the amino acid sequence shown in SEQ ID NO.

3.

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

3. The application as described in claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in either 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 application 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 with a normal starch synthesis pathway, characterized in that: The gene encoding a protein related to rice starch synthesis was introduced into rice with abnormal starch synthesis to obtain transgenic rice with normal starch synthesis; wherein the abnormal starch synthesis refers to insufficient endosperm filling, and the rice with abnormal starch synthesis is caused by the gene mutation shown in SEQ ID NO.2; The rice starch synthesis-related protein is a protein composed of the amino acid sequence shown in SEQ ID NO.

3.

6. The method according to claim 5, characterized in that: The nucleotide sequences of the genes encoding the proteins related to rice starch synthesis are 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.

7. The method according to claim 5, characterized in that: The encoding gene was introduced into rice with abnormal starch synthesis via a recombinant expression vector.

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