A plant gluten sorting-related protein OsGPA15, its encoding gene and applications

Abstract

The present invention discloses a plant gluten sorting-related protein OsGPA15, its encoding gene and applications. Through phenotypic analysis of rice gluten sorting mutants gpa15 and preliminary mapping of target genes, the gluten sorting-related protein OsGPA15 was finally cloned. The related protein is composed of the amino acid sequence shown in SEQ ID NO.1, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2 or SEQ ID NO.3. The gluten sorting-related protein of the present invention affects the sorting process of gluten in rice endosperm. By introducing the encoding gene of the protein into plants with reduced mature gluten content, transgenic plants with normal mature gluten content can be obtained. Therefore, the protein and its encoding gene of the present invention can be applied to plant genetic improvement.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and relates to a plant glutelin sorting-related protein OsGPA15, its coding gene and applications. Background Art

[0002] Rice is an important food crop in the world. It is the staple food for more than half of the world's population. In China, more than 60% of the population takes rice as the staple food. Although the rice production in China can basically meet the overall domestic demand for rice, with the improvement of the living standards of the people in China, the per capita consumption of rice has decreased, and the demand for high-quality, nutritious and healthy rice has increased significantly. Although remarkable achievements have been made in the improvement of rice quality in China, there is still a certain gap compared with high-quality foreign rice. As the second largest nutrient in rice after starch, rice storage proteins play an important role in the formation of rice eating quality. Glutelin is the main component of rice storage proteins, accounting for about 60%-80% of the total protein content, and is the preferred target for improving the quality of rice proteins. Therefore, analyzing the genetic mechanisms of glutelin synthesis, transport, processing and accumulation at the genetic, cellular and biochemical levels has important theoretical significance and practical value for improving the quality of rice proteins.

[0003] Rice glutelin precursor (57H) accumulation mutants are ideal genetic materials for analyzing the mechanism of glutelin synthesis and transport and for improving rice quality. By cloning and studying the genes of glutelin precursor accumulation mutants, the molecular network pathway of glutelin from synthesis to deposition can be systematically elucidated. So far, multiple key genes regulating glutelin transport have been cloned, and the molecular mechanism of glutelin sorting has been preliminarily described. However, the complete regulatory network of glutelin sorting is still not clear enough, and continuous research is needed to map and clone more key genes to further reveal the glutelin sorting mechanism. Summary of the Invention

[0004] The inventor of the present invention obtained the glutelin sorting-related protein OsGPA15 by cloning from a rice glutelin sorting mutant, gpa15 thus providing a glutelin sorting-related protein OsGPA15, its coding gene and applications. The glutelin sorting-related protein OsGPA15 of the present invention affects the sorting process of glutelin in rice endosperm. Introducing the coding gene of the glutelin sorting-related protein into a plant with reduced mature glutelin content can obtain a transgenic plant with normal mature glutelin content. Therefore, the protein and its coding gene of the present invention can be applied to plant genetic improvement.

[0005] The glutelin sorting-related protein (OsGPA15) provided by the present invention is derived from Oryza sativa of the genus Oryza Oryza sativavar. Kitaake), is a protein as described in (a) or (b) below:

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

[0007] (b) A derivative protein in which one or several amino acid residues of the amino acid sequence shown in SEQ ID NO.1 are substituted and / or deleted and / or added and still have the said function.

[0008] SEQ ID NO.1 consists of 70 amino acid residues.

[0009] To facilitate the purification of OsGPA15 in (a), a tag as shown in Table 1 can be linked to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.1.

[0010] Table 1. Sequences of tags

[0011]

[0012] OsGPA15 in the above (b) can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression. The coding gene of OsGPA15 in the above (b) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO.2, and / or performing a missense mutation of one or several base pairs, and / or linking the coding sequence of the tag shown in Table 1 to its 5´ end and / or 3´ end.

[0013] Meanwhile, the present invention also provides a gene encoding the above storage protein sorting-related protein ( OsGPA15 ).

[0014] The said gene OsGPA15 may have a nucleotide sequence as follows 1) or 2) or 3) or 4):

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

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

[0017] 3) A nucleotide sequence that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes the said protein;

[0018] 4) A nucleotide sequence that has a homology of more than 90% with the DNA sequence defined in 1) or 2) or 3) and encodes a glutelin sorting-related protein.

[0019] SEQ ID NO.2 consists of 213 nucleotides.

[0020] The recombinant expression vectors containing any of the above-mentioned genes also fall within the scope of protection of the present invention.

[0021] The recombinant expression vectors containing the said gene can be constructed with existing plant expression vectors.

[0022] The said plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The said plant expression vectors may also contain the 3'-untranslated region of foreign genes, that is, contain polyadenylation signals and any other DNA fragments involved in mRNA processing or gene expression. The said polyadenylation signal can direct the addition of polyadenylic acid to the 3'-end of the mRNA precursor. For example, the non-translated regions transcribed at the 3'-ends of the genes of Agrobacterium tumefaciens Ti plasmid (such as the nopaline synthase Nos gene) and plant genes (such as soybean storage protein genes) have similar functions.

[0023] When using the said gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter (Ubiquitin) of maize. They 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 the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the said translation control signals and start codons are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0024] In order to facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vectors can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes and can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or anti-chemical reagent marker genes (such as herbicide-resistant genes). Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0025] The said recombinant expression vector can be the one with the gene recombinantly inserted between dⅢ and HⅠ in the multiple cloning site of the pCUbi1390 vector Hin dⅢ and Bam HⅠ to obtain the recombinant plasmid. The said recombinant plasmid can specifically be pCUbi1390- OsGPA15 ; The said pCUbi1390- OsGPA15 ; The said pCUbi1390- OsGPA15 is obtained by OsGPA15The fragment of the genomic coding sequence together with the 1998 bp upstream promoter region was inserted into the multiple cloning site of pCUbi1390 by recombinant technology Hin dⅢ and Bam between HⅠ (Takara, In-fusion recombination kit).

[0026] The pCUbi1390 containing OsGPA15 was named pCUbi1390- OsGPA15 .

[0027] The expression cassette, transgenic cell line and recombinant bacterium containing any one of the above-mentioned genes ( OsGPA15 ) are all within the protection scope of the present invention.

[0028] The present invention also provides a method for cultivating a transgenic plant with normal gluten sorting. The method is to introduce the said gene into a plant with abnormal gluten sorting to obtain a transgenic plant with normal gluten sorting; the plant with abnormal gluten sorting is a plant with a sharp increase in gluten precursor in the endosperm and a decrease in the content of mature gluten; the transgenic plant with normal gluten sorting is a transgenic plant in which the gluten precursor can be normally processed into mature gluten. Specifically, the said gene is introduced into the plant with abnormal gluten sorting through the said recombinant expression vector; the plant with abnormal gluten sorting can be a GPA15 protein function-deficient mutant.

[0029] The said protein, the said gene, the said recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium or the said method can all be applied to rice breeding.

[0030] Using any vector that can direct the expression of foreign genes in plants, the gene encoding the said protein is introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the said gene can be used to transform plant cells or tissues by conventional biological methods such as using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroconductivity, Agrobacterium-mediated, etc., and the transformed plant tissues are cultivated into plants. The transformed plant host can be either monocotyledonous plants or dicotyledonous plants, such as: tobacco, Lotus corniculatus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, alfalfa, etc.

[0031] The inventor of the present invention carefully screened from the chemical mutagenesis mutant library of the japonica rice variety Kitaake and obtained a brand-new 57H mutant gpa15, there is currently no research report on the participation of OsGPA15 protein in the synthesis of rice storage proteins. The glutelin sorting-related protein OsGPA15 of the present invention affects the sorting process of glutelin in rice endosperm. Introducing the coding gene of the glutelin sorting-related protein OsGPA15 into a plant with reduced mature glutelin content can obtain a transgenic plant with normal mature glutelin content. The protein and its coding gene of the present invention can be applied to plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 , the appearance phenotypes of wild-type Kitaake and the mutant gpa15 . Among them, A shows Kitaake and gpa15 dry seeds and cross-sectional phenotypes of the endosperm, and B shows Kitaake and gpa15 scanning electron microscopy images of cross-sections of the endosperm.

[0033] Figure 2 , the SDS-PAGE and Western blot analyses of wild-type Kitaake and the mutant gpa15 . Among them, A shows the SDS-PAGE map of the endosperm storage protein components of Kitaake and gpa15 , and B shows the Western-Blot analysis map of glutelin of Kitaake and gpa15 .

[0034] Figure 3 , the observation of semi-thin sections of developing endosperm of wild-type Kitaake and the mutant gpa15 . Among them, A shows the Coomassie brilliant blue staining results of semi-thin sections of developing endosperm of Kitaake and gpa15 , and B shows the immunofluorescence analysis of semi-thin sections of developing endosperm of Kitaake and gpa15 .

[0035] Figure 4 , the transmission electron microscopy observation of developing endosperm of wild-type Kitaake and the mutant gpa15 . Among them, A-C show the morphological structures of protein body II in the developing endosperm of Kitaake and gpa15 , and D-F show the morphological structures of protein body I in the developing endosperm of gpa15 .

[0036] Figure 5 , the map-based cloning of the mutant gene. Among them, A shows the fine mapping map of gpa15 , and B shows the mutation site of gpa15 .

[0037] Figure 6 , the phenotypic analysis of transgenic complementation lines. Among them, A shows Kitaake and gpa15And the phenotypes of dry seeds and cross-sections of endosperms of transgenic complementary lines. Panel B shows Kitaake and gpa15 And the SDS-PAGE gels of storage protein components of complementary lines. Panel C shows Kitaake and gpa15 And the results of observing the restored normal structure of protein body I by rhodamine staining of thick sections of mid-development endosperms of complementary lines.

[0038] Figure 7 , and the map of pCUbi1390 vector. Detailed implementation manners

[0039] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. For the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged.

[0040] Example 1: Discovery of glutelin sorting-related proteins and their encoding genes in rice

[0041] I. Phenotypic analysis of rice glutelin sorting mutants gpa15 of

[0042] A mutant line with floury grains was screened from the EMS chemical mutagenesis mutant library of the japonica rice variety Kitaake gpa15 . Compared with the wild type, gpa15 's main characteristic is that the endosperm is floury and opaque (see Figure 1 Panel A). Scanning electron microscopy analysis confirmed that gpa15 the starch granules in Panel B are arranged loosely with gaps, which may be the main reason for the floury and opaque endosperm (see Figure 1 Panel B). gpa15 The SDS-PAGE gels of seed proteins showed an increase in the 57 kDa precursor of glutelin, a decrease in the content of the corresponding mature acidic and basic subunits of glutelin, and a decrease in the content of globulin as well (see Figure 2 Panel A). Western blot analysis confirmed that gpa15 the protein contents of the mature acidic and basic subunits of glutelin in Panel B decreased (see Figure 2 Panel B).

[0043] Semi-thin sectioning and Coomassie Brilliant Blue staining of mid-development endosperms revealed that there are two typical protein bodies in the subaleurone layer cells of wild-type endosperms: spherical protein body I with lighter staining and irregularly shaped protein body II with darker staining (see the left panel of Figure 3 Panel A, where the lighter color is protein body I and the darker color is protein body II). In gpa15Among them, the protein body structure is abnormal, with light-colored protein bodies attached to the periphery of the dark-colored protein bodies, which is called a composite protein body ( Figure 3 The structure outlined by the dotted line in the middle figure A), and compared with the wild type, the light-colored protein body I shows smaller aggregated dot-like shapes (see Figure 3 The right figure of A in Figure 3 , the structure circled by the dotted line). Further immunofluorescence experiments on semi-thin sections of mid-development endosperm also verified the above results (see gpa15 B in

[0044] ). The above results indicate that gpa15 The protein body development in the mutant is abnormal. Figure 4 Using transmission electron microscopy and immunogold analysis to observe the endosperm in the middle stage of development, it was found that, consistent with the cytological results observed in semi-thin sections, gpa15 there are unfilled protein bodies II in Figure 4 The new protein body structure has glutelin as the core, several prolamins around it, and this structure is wrapped by the endoplasmic reticulum membrane (

[0045] A-C in gpa15 ). Compared with the wild type,

[0046] Protein body I in

[0047] becomes smaller and forms strings or aggregates in the endoplasmic reticulum lumen (

[0048] D-F in gpa15 ). gpa15 gpa15 gpa15 In summary, the above results confirm that

[0049]

[0050] 2. Fine mapping of the target gene

[0050] 1. Preliminary mapping of the target gene

[0048] Using the mutant gpa15 hybridized with the wide-compatibility indica rice variety Dular, 10 gpa15 recessive extreme individuals with the gpa15 phenotype (floury and opaque grains and increased proglutelin) were selected from the F2 segregation population of gpa15 / Dular, and the seed DNA was extracted. Linkage analysis was performed using Indel marker primers covering the entire rice genome, and the mutant gene responsible for

[0049] the mutant phenotype was mapped to chromosome 5, linked between markers 5-13 and 5-21.Based on the preliminary mapping results, molecular markers on the common genetic map were searched for between linkage markers 5-13 and 5-21, and linkage markers were developed independently in this interval according to the rice genome sequence information published by NCBI. The designed marker primers were used to detect the polymorphism between Kitaake and Dular, and those showing polymorphism were used as molecular markers for fine mapping. 292 recessive extreme individuals were used to finely map the target gene (the molecular markers are shown in Table 2).

[0051] Table 2. Molecular markers for fine mapping

[0052]

[0053] Finally, the target gene OsGPA15 was finely mapped between linkage markers Y4 and 5-16, with a physical distance of 506 kb ( Figure 5 in A). By re-sequencing the genes in this interval, it was found that OsGPA15 there was a single-base substitution in the third exon of the gene, resulting in an amino acid substitution in the target protein ( Figure 5 in B).

[0054] III. Obtaining of the target gene OsGPA15 The cDNA of the leaves of the japonica rice variety Kitaake was extracted. Using the cDNA as a template, PCR amplification was carried out with primer primer1 and primer primer2, and the amplified product was sequenced. The sequencing result is shown in SEQ ID NO.2, and the encoded protein is shown in SEQID NO.1.

[0055] primer1: 5'-ATGTCGGCGCTATTCAACTT-3' (SEQ ID NO.18);

[0056] primer2: 5'-TCAGAAGAAAATAGTGGATA-3' (SEQ ID NO.19).

[0057] The protein shown in SEQ ID NO.1 was named OsGPA15 protein, which consists of 70 amino acid residues. The gene encoding the OsGPA15 protein was named

[0058] gene, and its open reading frame is shown in SEQ ID NO.2. OsGPA15

[0059] Example 2: Application of OsGPA15 protein and its encoding gene

[0060] I. Construction of genomic complementary vector

[0061] For the pCUbi1390 vectorHin dⅢ and Bam the small fragment between the HI restriction sites was replaced with the double-stranded DNA molecule shown in SEQ ID NO.3 in the sequence listing (including OsGPA15 the 1998bp promoter sequence upstream of the gene and the 1047bp terminator sequence downstream), to obtain the pCUbi1390- OsGPA15 genomic complementary vector (verified by sequencing), and the vector map of pCUbi1390 is shown in Figure 7 .

[0062] II. Obtaining of Complementary Transgenic Plants

[0063] 1. The pCUbi1390- OsGPA15 complementary vector obtained in step 1 was introduced into Agrobacterium tumefaciens strain EHA105 (Invitrogen, USA) to obtain recombinant Agrobacterium tumefaciens.

[0064] 2. The recombinant Agrobacterium tumefaciens obtained in step 1 was used to transform the japonica rice variety kitaake (wild type). The specific steps are as follows:[[]]

[0065] (1) Take the recombinant Agrobacterium tumefaciens cells obtained in step 1, resuspend them with N6 liquid medium (Sigma, C1416) and adjust the OD of the bacterial solution 600nm to 0.5;

[0066] (2) Infect the mature embryo embryogenic callus of the japonica rice variety kitaake (wild type) cultured for one month in the bacterial solution obtained in step (1) for 30 min. After blotting the bacterial solution with filter paper, transfer it to solid N6 medium (Sigma, C1416) containing 10 g / L agar and co-culture at 24°C for 3 days;

[0067] (3) Inoculate the callus cultured in step (2) on solid screening N6 solid medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and culture for 16 days (the first screening);

[0068] (4) Inoculate the healthy callus cultured in step (3) on solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and culture for 15 days (the second screening);

[0069] (5) Inoculate the healthy callus cultured in step (4) on solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and culture for 15 days (the third screening);

[0070] (6) Inoculate the healthy callus after the culture in step (4) on a differentiation medium (PhytoTechnology Laboratories, M524) for differentiation to obtain T0 generation plants.

[0071] 3. Identify the T0 generation plants obtained in step 2. Extract the total DNA from the leaves of the plants to be tested, perform PCR amplification using primer primer3 and primer primer4, and perform electrophoresis detection on the amplification products. The plants with bands detected are transgenic positive plants.

[0072] primer3: 5'-TGAGGGATTATTTCATTTGA-3' (SEQ ID NO.20);

[0073] primer4: 5'-ATTACATGTTTAGGTCTGTT-3' (SEQ ID NO.21).

[0074] III. Phenotypic identification

[0075] Respectively plant the T0 generation transgenic pCUbi1390- OsGPA15 plants, gpa15 and the wild type Kitaake in the transgenic experimental field of the Chinese Academy of Agricultural Sciences. The results show that transparent grains appeared in the T2 seeds of the transgenic lines ( Figure 6 A in the figure), and the SDS-PAGE detection of the transparent seeds (L1, L2) showed the same performance as the wild type ( Figure 6 B in the figure). The results of rhodamine staining of the thick sections of the endosperm at the mid-development stage were also consistent with the wild type ( Figure 6 C in the figure). Thus, it was verified that the traits of opaque floury and increased glutenin precursors before transgenic were controlled by the OsGPA15 gene, that is, this OsGPA15 gene is a gluten sorting-related gene. Transforming the rice OsGPA15 mutant with pCUbi1390- gpa15 can increase the mature gluten content to the normal level.

Claims

1. Application of a gene encoding a gluten sorting-related protein in plant breeding, characterized in that, The plant breeding is to cultivate transgenic plants with normal gluten sorting, the plant is rice, and the amino acid sequence of the gluten sorting-related protein is as shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein , the nucleotide sequence of the gene is as shown in SEQ ID NO.2 or SEQ ID NO.

3.

3. A method for cultivating a transgenic plant with normal gluten sorting, characterized in that, The method is to introduce the gene encoding the gluten sorting-related protein with the amino acid sequence shown in SEQ ID NO.1 into plants with abnormal gluten sorting to obtain transgenic plants with normal gluten sorting; wherein the abnormal gluten sorting is the abnormal accumulation of gluten precursors; The plant is rice; the plant with abnormal gluten sorting is a mutant with a loss of function of the OsGPA15 protein.

4. The method according to claim 3, wherein: The nucleotide sequence of the gene is as shown in SEQ ID NO.2 or SEQ ID NO.3.

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