OsGPA17, a plant gluten sorting-related protein, its encoding gene, and its applications

Abstract

This invention discloses a plant glutenin sorting-related protein, OsGPA17, its encoding gene, and its applications. This invention utilizes the rice glutenin 57H mutant. gpa17 Phenotypic analysis and preliminary localization of the target gene led to the cloning of the protein sorting-related protein OsGPA17. This protein consists of the amino acid sequence shown in SEQ ID NO.1, and the nucleotide sequence of the gene is shown in SEQ ID NO.2 or SEQ ID NO.3. The gluten sorting-related protein of this invention affects the gluten sorting process in rice endosperm. Introducing the encoding gene of this protein into plants with reduced mature protein content yields transgenic plants with normal mature protein content. Therefore, the protein and its encoding gene of this invention can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a plant gluten sorting-related protein OsGPA17, its encoding gene, and its applications. Background Technology

[0002] Starch is the most abundant component in rice seeds, composed of amylose and amylopectin, and their ratio significantly affects the eating quality of rice. Therefore, current rice eating quality improvement mainly focuses on starch improvement. Of particular note is storage protein, the second most abundant nutrient in rice after starch, and a crucial source of plant protein in the human diet. Its content and composition also play a vital role in the formation of rice eating quality. Mutations in any gene involved in the synthesis, transport, processing, and accumulation of storage proteins can severely impact rice quality. Therefore, in-depth analysis of the molecular mechanisms of rice storage protein synthesis and sorting is of great significance for the genetic improvement of rice quality.

[0003] Rice gluten is the most important storage protein in rice seeds, accounting for approximately 60%-80% of the total protein content. Its content and composition affect the eating and processing quality of rice. Improving the amino acid composition of gluten to enhance its nutritional value, or regulating its content and structure to improve rice quality, through genetic engineering and other methods, is an important direction in current rice research. The gluten precursor accumulation (57H) mutant is an ideal genetic material for elucidating the mechanism of gluten synthesis and transport. Several key genes regulating gluten transport have been cloned, but given the complexity of gluten sorting, the fine-tuned regulatory network of gluten synthesis and sorting still requires further investigation. The inventors obtained a novel 57H mutant from a chemical mutagenesis mutant library of the japonica rice variety Kitaake. gpa17 The protein OsGPA17, which encodes gluten-sorting-related protein, has not yet been reported in studies on its involvement in rice gluten sorting. Summary of the Invention

[0004] The inventors sorted abnormal mutants from rice glutenin. gpa17 Phenotypic analysis and map-based cloning were conducted, and the gluten sorting-related protein OsGPA17 was finally cloned, thus providing a gluten sorting-related protein, its encoding gene, and its applications.

[0005] The glutenin sorting-related protein (OsGPA17) provided by this invention is derived from rice (Oryza sativa). Oryza sativa var.Kitaake), is a protein that is either (a) or (b) as follows:

[0006] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0007] (b) A derived protein having the amino acid sequence shown in SEQ ID NO.1 modified by substitution and / or deletion and / or addition of one or more amino acid residues and still having the function described.

[0008] SEQ ID NO.1 consists of 953 amino acid residues.

[0009] To facilitate the purification of OsGPA17 in (a), 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.1.

[0010] Table 1. Sequence of Labels

[0011]

[0012] The OsGPA17 mentioned in (b) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. The encoding gene of OsGPA17 mentioned in (b) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID NO.2, 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.

[0013] Furthermore, this invention also provides a gene encoding the aforementioned glutenin sorting-related proteins. OsGPA17 .

[0014] The gene OsGPA17 The nucleotide sequence can be as follows: 1) or 2):

[0015] 1) The nucleotide sequence shown in SEQ ID NO.2;

[0016] 2) The nucleotide sequence shown in SEQ ID NO.3.

[0017] SEQ ID NO.2 consists of 2862 nucleotides.

[0018] Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0019] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0020] 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).

[0021] 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.

[0022] 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.

[0023] The recombinant expression vector can be a multiple cloning site of the pCAMBIA2300 vector. EcoRI and SmaI Recombination insertion of the gene between ( OsGPA17 The recombinant plasmid obtained is pCAMBIA2300-. OsGPA17 The pCAMBIA2300- OsGPA17 It will be by OsGPA17 The genome coding sequence, along with the upstream 2173 bp promoter region and the downstream 1410 bp fragment, was inserted into the pCAMBIA2300 multiple cloning site using recombination technology. EcoRI and SmaI The results were obtained from (Takara, In-fusion Recombinant Kit).

[0024] Will contain OsGPA17 The pCAMBIA2300 is named pCAMBIA2300- OsGPA17 .

[0025] Contains any of the genes mentioned above ( OsGPA17 The expression cassette, transgenic cell line, and recombinant bacteria are all within the scope of protection of this invention.

[0026] This invention also provides a method for cultivating transgenic plants with normal gluten sorting. The method involves introducing the gene into plants with abnormal gluten sorting to obtain transgenic plants with normal gluten sorting. The plants with abnormal gluten sorting are those with a dramatic increase in gluten precursors in the endosperm accompanied by a decrease in mature gluten content. The transgenic plants with normal gluten sorting are those where gluten precursors can be normally processed into mature gluten. Specifically, the gene is introduced into plants with abnormal gluten sorting via the recombinant expression vector; the plants with abnormal gluten sorting can be GPA17 protein functional defect mutants.

[0027] 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.

[0028] 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.

[0029] Meanwhile, this invention also provides a method for detecting whether rice gluten sorting is normal. Several mature rice seeds to be identified are used to extract total seed protein. The protein is then analyzed by SDS-PAGE gel electrophoresis to determine whether the gluten composition of the identified strain differs from that of the wild type. If separation is observed, the gluten sorting is considered abnormal; if no separation is observed, the gluten sorting is considered normal. This method specifically includes the following steps:

[0030] (1) Take one wild-type control seed and randomly select several mature rice seeds to be identified. After removing the shells, observe the seed phenotype.

[0031] (2) Crush each seed with needle-nose pliers and place it into a 2.0 ml Eppendorf tube (note that each tube should be numbered). Add 600 μl of seed total protein extract, vortex for 30 minutes to ensure the extract is fully in contact with the sample, and place in a 60°C oven overnight. Remove the tube the next day.

[0032] (3) After vortexing to homogenize, centrifuge at 12000 rpm for 5 min, and take 2 μl of supernatant for spotting (the control sample is used as the first sample of each gel).

[0033] (4) Use a 0.75mm SDS-PAGE gel for sample loading. For electrophoresis, first run the stacking gel at 80V. After the proteins enter the separating gel, increase the voltage to 120V and continue running until the bromophenol blue indicator is close to the bottom of the gel. Then, peel the gel off the glass plate, stain with Coomassie Brilliant Blue for 1 hour, remove the staining solution, add an appropriate amount of destaining solution, and destain until the protein bands are clearly distinguishable. Finally, use a gel imaging device to scan and image the gel.

[0034] (5) Analyze the SDS-PAGE spectrum to identify whether the gluten composition of the strain seeds is separated from that of the wild type. If there is separation, it can be preliminarily judged that the gluten sorting is abnormal; if there is no separation, it can be preliminarily judged that the gluten sorting is normal.

[0035] The gluten-sorting related protein of this invention affects the gluten sorting process in rice endosperm. Introducing the encoding gene of this protein into plants with reduced mature gluten content yields transgenic plants with normal mature gluten content. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0036] Figure 1 Wild-type Kitaake and mutants gpa17 Seed appearance phenotype. Where A represents Kitaake and... gpa17 Dry seeds and endosperm cross-section phenotypes, B for Kitaake and gpa17 Scanning electron microscope image of a cross section of the endosperm.

[0037] Figure 2 Wild-type Kitaake and mutants gpa17 SDS-PAGE gel mapping and Western blot analysis were performed. A represents Kitaake protein gel imaging and Western blot analysis. gpa17 SDS-PAGE gel image of endosperm storage protein components, B represents Kitaake and gpa17 Western blotting analysis of glutenin.

[0038] Figure 3 Wild-type Kitaake and mutants gpa17Observation of a semi-thin section of the developing endosperm. In this section, A and B represent Kitaake and... gpa17 Coomassie brilliant blue staining results of semi-thin sections of endosperm during mid-developmental stage; C and D are Kitaake and... gpa17 Immunofluorescence analysis of semi-thin sections of endosperm during mid-development. In the figure, triangles indicate protein body I (PBI), and arrows indicate protein body II (PBII).

[0039] Figure 4 Wild-type Kitaake and mutants gpa17 Immunogold observation of developing endosperm. AD ​​represents Kitaake and... gpa17 Phenotypes of protein body I (PBI) and protein body II (PBII) in mid-developmental endosperm, EF is gpa17 Novel protein body structures appearing in the mid-development endosperm; GI is Kitaake and gpa17 Morphology of the Golgi apparatus in the endosperm during mid-development.

[0040] Figure 5 Map-based cloning of mutant genes. Where A represents... gpa17 A detailed positioning map, B is... gpa17 The mutation sites and early termination diagram.

[0041] Figure 6 Phenotypic analysis of transgenic complementary families. Where A represents Kitaake and... gpa17 And the dry seeds and endosperm cross-section phenotypes of transgenic complementary families, B being Kitaake and gpa17 And SDS-PAGE diagrams of complementary family storage protein components.

[0042] Figure 7 pCAMBIA2300 vector map. Detailed Implementation

[0043] 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.

[0044] Example 1: Discovery of glutenin sorting-related proteins and their encoding genes in rice

[0045] I. Rice Glutelin Sorting Mutants gpa17 Phenotypic analysis

[0046] Mutant lines with mealy grains were screened from the chemical mutagenesis mutant library of the japonica rice variety Kitaake. gpa17Compared to the wild type, gpa17 The main characteristic is that the seeds are powdery and opaque (see...). Figure 1 (A). Scanning electron microscopy analysis confirmed... gpa17 The loose arrangement of mid-starch granules is likely the main reason for the opaque texture of the endosperm (see [link]). Figure 1 (B) gpa17 SDS-PAGE analysis of seed proteins showed an increase in glutenin 57kDa precursor, with a corresponding decrease in the content of mature glutenin acidic and basic subunits, and a decrease in prolysin content (see [link to study]). Figure 2 (A). Western blot analysis confirmed that... gpa17 The content of acidic subunits of mature glutenin is reduced (see...) Figure 2 (B)

[0047] Observation of semi-thin sections of endosperm in mid-developmental stage after Coomassie brilliant blue staining revealed two types of protein bodies in the wild type: globular protein body I and irregularly shaped protein body II. Protein body II is slightly larger than protein body I (see...). Figure 3 In the diagram, triangle A indicates protein body I, and arrow indicates protein body II. Meanwhile... gpa17 The morphology of the intermediate protein body II was not significantly different from that of the wild type (see...). Figure 3 (B in the middle, indicated by the arrow). Protein body I exhibits numerous and smaller protein body structures (see...). Figure 3 (B in the middle, marked with a triangle). Further immunofluorescence experiments on semi-thin sections of mid-developing endosperm also verified the above results (see...). Figure 3 (C, D). The above results indicate that... gpa17 The mutant protein body I exhibited abnormal development, resulting in numerous small, novel protein body structures. To elucidate the formation process of these novel protein body structures, transmission electron microscopy combined with immunogold assay was used to observe the mid-developing endosperm (see...). Figure 4 Consistent with the cytological results observed in the semi-thin sections, gpa17 The middle protein body I exhibits a large number of smaller protein body structures ( Figure 4 (AD). Studies have shown that glutenin, prolysin, and other proteins are synthesized and processed in the endoplasmic reticulum. However, mutants... gpa17 Abnormal morphology of the middle endoplasmic reticulum and Golgi apparatus ( Figure 4 (EI), and accompanied by the appearance of a novel abnormal protein body structure of glutenin encapsulated by prolysins ( Figure 4 (Middle F).

[0048] In conclusion, the above results confirm that... gpa17 Abnormal morphology of the endoplasmic reticulum and Golgi apparatus, which are responsible for gluten sorting, significantly affects the formation of protein body I. Gluten synthesis is also affected by these abnormalities, ultimately resulting in an increase in gluten precursors and a decrease in prolamins.

[0049] II. Target Gene Localization

[0050] 1. Preliminary localization of the target gene

[0051] Using mutants gpa17 Crossed with the widely compatible variety Dular (purchased from the germplasm resource bank of the Institute of Crop Science, Chinese Academy of Agricultural Sciences), in gpa17 / Ten grains were selected from the F2 segregating population of Dular. gpa17 Recessive extreme individuals exhibiting the phenotype (opaque grains with increased gluten precursors) were used to extract seed DNA. Linkage analysis was performed using primers covering the entire rice genome to identify the responsible individuals. gpa17 The mutant gene in the mutant phenotype is located on chromosome 11 and is linked between marker R11-3 and F1.

[0052] 2. Fine mapping of target genes

[0053] Based on the initial mapping results, molecular markers on the common map were searched for in linkage markers R11-3 and F1, and linkage markers were developed independently in this region based on the rice genome sequence information published by NCBI. Polymorphism between Kitaake and Dular was detected using self-designed marker primers, and those exhibiting polymorphism were used as molecular markers for fine mapping. Fine mapping of the target gene was performed using 243 recessive extreme individuals (molecular markers are shown in Table 2).

[0054] Table 2 Molecular markers used for fine localization

[0055]

[0056] Ultimately, the target gene OsGPA17 The precise location is between interlocking markers F3 and F4, with a physical distance of 33.3 kb. Figure 5 (A). After sequencing the genes within this region, the gene was found. OsGPA17 A single base substitution occurred in the first exon, causing premature termination of mutant gene expression. Figure 5 (B)

[0057] III. Target Gene OsGPA17 The acquisition

[0058] cDNA was extracted from leaves of the japonica rice variety kitaake. 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.1.

[0059] primer1: 5'-ATGGAGAAGCCATGCACCC-3' (SEQ ID NO. 20);

[0060] primer2: 5'-TTAACTGCTGCCTCCCTTCTGT-3' (SEQ ID NO. 21).

[0061] The protein shown in SEQ ID NO.1 was named OsGPA17 protein, which consists of 953 amino acid residues. The gene encoding OsGPA17 protein was named... OsGPA17 The gene, whose open reading frame is shown in SEQ ID NO.2.

[0062] Example 2: Application of OsGPA17 protein and its encoding gene

[0063] I. Construction of Genomic Complementation Vector

[0064] pCAMBIA2300 vector EcoRI and SmaI The small fragments between the restriction enzyme sites are replaced with the double-stranded DNA molecule shown in SEQ ID NO.3 of the sequence listing (including...). OsGPA17 The promoter sequence (2173 bp upstream) and the terminator sequence (1410 bp downstream) of the gene were used to obtain pCAMBIA2300- OsGPA17 Genomic complementation vector (sequencing verified), pCAMBIA2300 vector map can be found here. Figure 7 .

[0065] II. Obtaining Complementary Transgenic Plants

[0066] 1. Take the pCAMBIA2300 obtained in step one... OsGPA17 The complementary vector was introduced into Agrobacterium strain EHA105 (American Junction Company) to obtain recombinant Agrobacterium.

[0067] 2. Transform the japonica rice mutant using the recombinant Agrobacterium obtained in step 1. gpa17 The specific steps are as follows:

[0068] (1) Take the recombinant Agrobacterium cells obtained in step 1, resuspend them in N6 liquid medium (Sigma, C1416) and adjust the OD of the bacterial solution. 600nm It is 0.5.

[0069] (2) The japonica rice mutant cultured for one month gpa17 Mature embryonic callus was infected in the bacterial solution obtained in step (1) for 30 min. After the bacterial solution was dried with filter paper, it was transferred to solid N6 medium (Sigma, C1416) containing 10 g / L agar and cultured at 24°C for 3 days.

[0070] (3) The callus cultured in step (2) was inoculated on solid screening N6 solid medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L kanamycin and cultured for 16 days (first screening).

[0071] (4) The healthy callus cultured in step (3) was inoculated on solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L kanamycin and cultured for 15 days (second screening).

[0072] (5) The healthy callus cultured in step (4) was inoculated on solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L kanamycin and cultured for 15 days (third screening).

[0073] (6) The healthy callus cultured in step (4) was inoculated on differentiation medium (Phyto Technology Laboratories, M524) to differentiate and obtain T0 generation plants.

[0074] 3. Identify the T0 generation plants obtained in step 2. Extract total DNA from the leaves of the plants to be tested, and perform PCR amplification using primers primer3 and primer4. Sequencing the amplification products will identify transgenic positive plants with bimodal mutation sites.

[0075] primer3: 5'-CTGAGTTCCACAGAATCTCAG (SEQ ID NO. 22)-3';

[0076] Primer4: 5'-AGCTGATACCCACTATTAGCC-3' (SEQ ID NO. 23).

[0077] III. Phenotypic Identification

[0078] Convert T0 to pCAMBIA2300 respectively OsGPA17 Plants, wild-type Kitaake and mutants gpa17 Planted in the transgenic experimental field of the Chinese Academy of Agricultural Sciences. Results showed that transparent grains appeared in the seeds of the transgenic line T2. Figure 6 In the middle A), SDS-PAGE analysis showed that transparent seeds (L1, L2, L3) exhibited the same characteristics as wild-type seeds. Figure 6 (B). This verifies that the opaque starch and increased gluten precursor traits before transgenication are caused by... OsGPA17 Genetically controlled, that is OsGPA17The gene is related to gluten sorting. pCAMBIA2300- OsGPA17 Transformed rice gpa17 The mutant can increase the content of mature glutenin to a normal level.

Claims

1. The application of a glutenin sorting-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 being the use of transgenic rice that is a GPA17 protein functional defect mutant to be bred into a transgenic rice with normal storage protein processing and maturation. The amino acid sequence of the glutenin sorting-related protein is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, A tag sequence, either FLAG or GFP, is added to the front or end of the gluten-sorting-related protein.

3. The application as described in claim 2, 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.2; 2) The nucleotide sequence shown in SEQ ID NO.

3.

4. The application according to claim 1, characterized in that: The recombinant expression vector is located at the multiple cloning site of the pCAMBIA2300 vector. EcoRI and SmaI The encoded gene was inserted between the two.

5. A method for breeding transgenic rice with normal gluten sorting, comprising introducing a gene encoding a protein related to gluten sorting into rice with abnormal gluten sorting to obtain transgenic rice with normal gluten sorting; wherein the abnormal gluten sorting is due to abnormal accumulation of gluten precursors; wherein the gene is introduced into rice with abnormal gluten sorting via a recombinant expression vector; wherein the rice with abnormal gluten sorting is a GPA17 protein functional defect mutant; The amino acid sequence of the glutenin sorting-related protein is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that: The nucleotide sequences of the genes encoding the gluten-sorting-related proteins are shown in 1) or 2) below: 1) The nucleotide sequence shown in SEQ ID NO.2; 2) The nucleotide sequence shown in SEQ ID NO.3.

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