Plant glutelin sorting related protein OsGPA15 as well as coding gene and application thereof

Abstract

The invention discloses a plant glutelin sorting related protein OsGPA15 as well as a coding gene and application thereof. The glutelin sorting related protein OsGPA15 is finally cloned through phenotypic analysis of a paddy rice protein sorting mutant gpa15 and preliminary localization of a target gene, the related protein consists of an amino acid sequence as 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 invention further discloses a preparation method of the glutelin sorting related protein OsGPA15. The glutelin sorting related protein provided by the invention influences the sorting process of glutelin in rice endosperm, and a transgenic plant with normal mature glutelin content can be obtained by introducing the coding gene of the protein into a plant with reduced mature glutelin content, so that the protein and the coding gene thereof provided by the invention can be applied to plant genetic improvement.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and relates to a plant gluten sorting-related protein OsGPA15 and a coding gene and application thereof. Background Art

[0002] Rice is an important food crop in the world. It is the staple food of more than half of the world's population. In my country, more than 60% of the population relies on rice as their staple food. Although the output of rice in my country can basically meet the overall domestic demand for rice, with the improvement of the living standards of the Chinese people, the per capita rice consumption has declined, and people's demand for high-quality, nutritious and healthy rice has increased significantly. Although my country has achieved remarkable results in improving the quality of rice, there is still a certain gap compared with high-quality rice abroad. As the second largest nutrient in rice after starch, rice storage protein plays an important role in the formation of rice taste quality. Glutelin is the main component of rice storage protein, accounting for about 60%-80% of the total protein content, and is the preferred target for improving rice protein quality. Therefore, analyzing the genetic mechanism of glutenin synthesis, transport, processing and accumulation from the genetic, cellular and biochemical levels has important theoretical significance and practical value for improving rice protein quality.

[0003] Rice gluten precursor (57H) accumulation mutants are ideal genetic materials for analyzing the mechanism of gluten synthesis and transportation and improving rice quality. Through gene cloning and functional studies of gluten precursor accumulation mutants, the molecular network pathway of gluten from synthesis to deposition can be systematically elucidated. So far, several key genes regulating gluten transport have been cloned, and the molecular mechanism of gluten sorting has been preliminarily depicted, but the complete regulatory network of gluten sorting is still not clear enough, and it is necessary to continue to locate and clone more key genes to further reveal the gluten sorting mechanism. Summary of the invention

[0004] The inventors sorted mutants of rice glutelin gpa15 The gluten sorting-related protein OsGPA15 was cloned, thereby providing a gluten sorting-related protein OsGPA15 and its encoding gene and application. The gluten sorting-related protein OsGPA15 of the present invention affects the sorting process of gluten in rice endosperm. The encoding gene of the gluten sorting-related protein is introduced into a plant with reduced mature gluten content, and a transgenic plant 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.

[0005] The gluten sorting-related protein (OsGPA15) provided by the present invention is derived from rice ( Oryza sativavar. Kitaake), is a protein of (a) or (b) as follows: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A derivative protein having the above function after one or more amino acid residues are substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO.1.

[0006] SEQ ID NO. 1 consists of 70 amino acid residues.

[0007] In order to facilitate the purification of OsGPA15 in (a), a tag as shown in Table 1 may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO. 1.

[0008] Table 1. Sequence of tags

[0009] The OsGPA15 in (b) above can be synthesized artificially, or its coding gene can be synthesized first and then expressed biologically. The coding gene of OsGPA15 in (b) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO.2, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0010] At the same time, the present invention also provides a gene encoding the above storage protein sorting-related protein ( OsGPA15 ).

[0011] The gene OsGPA15 The nucleotide sequence may be as follows 1) or 2) or 3) or 4): 1) The nucleotide sequence shown in SEQ ID NO.2; 2) The nucleotide sequence shown in SEQ ID NO.3; 3) a nucleotide sequence that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein; 4) A nucleotide sequence that has more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encodes a protein related to gluten sorting.

[0012] SEQ ID NO. 2 consists of 213 nucleotides.

[0013] The recombinant expression vector containing any of the above genes also falls within the protection scope of the present invention.

[0014] Existing plant expression vectors can be used to construct a recombinant expression vector containing the gene.

[0015] The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The 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' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase Nos gene) and plant genes (such as soybean storage protein genes) have similar functions.

[0016] When using the 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 of maize (Ubiquitin). 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 start codon in the adjacent region, etc., but they must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0017] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change (GUS gene, luciferase gene, etc.), a resistant antibiotic marker (gentamicin marker, kanamycin marker, etc.) or an anti-chemical reagent marker gene (such as an anti-herbicide gene) that can be expressed in plants. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0018] The recombinant expression vector can be at the multiple cloning site of the pCUbi1390 vector Hin dⅢ and Bam the gene is recombinantly inserted between HⅠ ( OsGPA15 ) to obtain a recombinant plasmid. The recombinant plasmid can specifically be pCUbi1390- OsGPA15 ; the pCUbi1390- OsGPA15 is obtained by inserting the fragment of the genomic coding sequence together with the 1998bp promoter region upstream through recombinant technology into the multiple cloning site of pCUbi1390 OsGPA15 between dⅢ and Hin Bam ​obtained between HⅠ (Takara, In-fusion recombination kit).

[0019] will contain OsGPA15 The pCUbi1390 was named pCUbi1390- OsGPA15 .

[0020] Containing any of the above genes ( OsGPA15 )'s expression cassette, transgenic cell line and recombinant bacteria all fall within the protection scope of the present invention.

[0021] The present invention also provides a method for cultivating a transgenic plant with normal gluten sorting, which is to introduce the 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 precursors 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 gluten precursors can be normally processed into mature gluten. Specifically, the gene is introduced into the plant with abnormal gluten sorting through the recombinant expression vector; the plant with abnormal gluten sorting can be a GPA15 protein function defective mutant.

[0022] The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria or the method can be applied to rice breeding.

[0023] By using any vector that can guide the expression of foreign genes in plants, the gene encoding the protein is introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissues are cultivated into plants. The transformed plant host can be either a monocot or a dicot, such as tobacco, Lotus japonicus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, lawn grass, clover, etc.

[0024] The inventors carefully screened the chemical mutagenesis mutant library of japonica rice variety Kitaake and obtained a new 57H mutant gpa15 At present, there is no research report on the involvement of OsGPA15 protein in the synthesis of rice storage proteins. The gluten sorting-related protein OsGPA15 of the present invention affects the sorting process of gluten in rice endosperm. Transgenic plants with normal mature gluten content can be obtained by introducing the coding gene of the gluten sorting-related protein OsGPA15 into plants with reduced mature gluten content. The protein of the present invention and its coding gene can be applied to plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , wild-type Kitaake and mutant gpa15 The appearance phenotype of A is Kitaake and gpa15 Dry seed and endosperm cross-section phenotypes, B is Kitaake and gpa15 Scanning electron micrograph of cross section of endosperm.

[0026] Figure 2 , wild-type Kitaake and mutant gpa15 SDS-PAGE and Western blot analysis. gpa15 SDS-PAGE images of endosperm storage protein components, B is Kitaake and gpa15 Western-Blot analysis of glutenin.

[0027] Figure 3 , wild-type Kitaake and mutant gpa15 Semi-thin section observation of developing endosperm. A is Kitaake and gpa15 Coomassie blue staining results of semi-thin sections of endosperm in the middle stage of development. B is Kitaake and gpa15 Immunofluorescence analysis of semi-thin sections of mid-developing endosperm.

[0028] Figure 4 , wild-type Kitaake and mutant gpa15 Transmission electron microscopy observation of developing endosperm. AC is Kitaake and gpa15 The morphology and structure of protein body II in the mid-developing endosperm, DF is gpa15 The morphology and structure of protein body I in the mid-developing endosperm.

[0029] Figure 5 , map-based cloning of mutant genes. gpa15 The fine positioning map of gpa15 mutation site.

[0030] Figure 6 , phenotypic analysis of transgenic complementation families. Among them, A is Kitaake and gpa15 As well as the dry seed and endosperm cross-section phenotypes of the transgenic complementary families, B is Kitaake and gpa15 And the SDS-PAGE images of storage protein components of complementary families, C is Kitaake and gpa15 The results of rhodamine staining of thick sections of endosperm in the middle stage of development of the complementary family were observed to observe the restoration of the normal structure of protein body I.

[0031] Figure 7 , pCUbi1390 vector map. DETAILED DESCRIPTION

[0032] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0033] Example 1: Discovery of proteins related to glutenin sorting in rice and their encoding genes 1. Rice glutenin sorting mutants gpa15 Phenotypic analysis Screening of grain powdery mutants from the EMS chemical mutagenesis mutant library of japonica rice variety kitaake gpa15 Compared with the wild type, gpa15 The main characteristics of the endosperm are powdery, opaque (see Figure 1 Scanning electron microscopy analysis confirmed gpa15 The loose arrangement of starch granules and the presence of gaps in the starch granules may be the main reason for the opacity of the endosperm powder (see Figure 1 Middle B). gpa15 The SDS-PAGE profile of the seed protein showed an increase in the 57 kDa precursor of glutenin, a decrease in the content of the corresponding mature glutenin acidic and basic subunits, and a decrease in the content of globulin (see Figure 2 (A) Western blot analysis confirmed gpa15 The content of acidic and basic subunits of mature glutenin is reduced (see Figure 2 Middle B).

[0034] Semi-thin sections of mid-developmental endosperm and Coomassie Brilliant Blue staining revealed two typical protein bodies in the subaleurone cells of wild-type endosperm: lightly stained spherical protein body I and darkly stained irregularly shaped protein body II (see Figure 3 In the left picture of A, the light color is protein body I and the dark color is protein body II). gpa15 In the case of a protein body, the structure of the protein body is abnormal, with light-colored protein bodies attached to the periphery of the dark-colored protein body, which is called a composite protein body ( Figure 3 A middle figure, the structure selected by the dotted line), and compared with the wild type, the light-colored protein body I showed smaller aggregated dots (see Figure 3 The above results were also verified by immunofluorescence experiments on semi-thin sections of mid-developmental endosperm (see Figure 3 The above results show that gpa15 The protein bodies in the mutants develop abnormally.

[0035] Transmission electron microscopy and immunogold analysis were used to observe the endosperm in the middle stage of development. The results were consistent with the cytological results observed by semi-thin sections. gpa15 There are unfilled protein bodies II in the new protein body structure, which is composed of glutenin as the core, several alcohol-soluble proteins around it and the structure is wrapped by the endoplasmic reticulum membrane ( Figure 4 Compared with the wild type, gpa15 Protein bodies I become smaller and aggregate in clusters or in the lumen of the endoplasmic reticulum ( Figure 4 (in DF).

[0036] In summary, the above results confirm gpa15 There is a defect in the export of storage proteins from the endoplasmic reticulum, which results in the inability of glutenin precursors to be exported from the endoplasmic reticulum and thus to be processed into mature acidic and basic subunits.

[0037] 2. Map-based cloning of mutant gene loci 1. Preliminary localization of target genes Using mutants gpa15 It was crossed with the broad-compatibility indica rice variety Dular. gpa15 / Dular's F 2 Select 10 particles from the isolated population gpa15 The DNA of seeds of individuals with recessive extreme phenotypes (opaque grain powder and increased glutenin precursors) was extracted. Linkage analysis was performed using Indel marker primers covering the entire rice genome. gpa15 The mutant gene responsible for the mutant phenotype is located on chromosome 5 and is linked between markers 5-13 and 5-21.

[0038] 2. Fine positioning of target genes Based on the initial positioning results, molecular markers on the public map were searched between linkage markers 5-13 and 5-21, and linkage markers were developed in this interval based on the rice genome sequence information published by NCBI. Self-designed marker primers were used to detect polymorphisms between Kitaake and Dular, and those showing polymorphisms were used as molecular markers for fine positioning. The target gene was finely positioned using 292 recessive extreme individuals (molecular markers are shown in Table 2).

[0039] Table 2. Molecular markers used for fine mapping

[0040] Finally, the target gene OsGPA15 Fine mapping was performed between linkage markers Y4 and 5-16, with a physical distance of 506 kb ( Figure 5 After resequencing the genes in this interval, it was found that the gene OsGPA15There is a single base substitution in the third exon of the target protein, which causes the amino acid to be replaced ( Figure 5 Middle B).

[0041] 3. Target Gene OsGPA15 Acquisition cDNA was extracted from the leaves of japonica rice variety kitaake, and PCR amplification was performed using primers primer1 and primer2 with cDNA as a template. The amplified product was sequenced, and the sequencing result is shown in SEQ ID NO.2. The protein encoded by the protein is shown in SEQ ID NO.1.

[0042] primer1: 5'-ATGTCGGCGCTATTCAACTT-3' (SEQ ID NO. 18); primer2: 5'-TCAGAAGAAAATAGTGGATA-3' (SEQ ID NO. 19).

[0043] The protein shown in SEQ ID NO.1 was named OsGPA15 protein, which consists of 70 amino acid residues. The gene encoding OsGPA15 protein was named OsGPA15 The gene, whose open reading frame is shown in SEQ ID NO.2.

[0044] Example 2: Application of OsGPA15 protein and its encoding gene 1. Construction of genome complementation vector The pCUbi1390 vector Hin dⅢ and Bam The small fragment between the HI restriction sites is replaced with the double-stranded DNA molecule shown in SEQ ID NO.3 in the sequence list (including OsGPA15 The promoter sequence of 1998 bp upstream and the terminator sequence of 1047 bp downstream of the gene were obtained by plasmid pCUbi1390- OsGPA15 Genome complementation vector (sequencing verified), pCUbi1390 vector map see Figure 7 .

[0045] 2. Obtaining complementary transgenic plants 1. The pCUbi1390- OsGPA15 The complementation vector was introduced into Agrobacterium EHA105 strain (Invitrogen, USA) to obtain recombinant Agrobacterium.

[0046] 2. Use the recombinant Agrobacterium obtained in step 1 to transform the japonica rice variety kitaake (wild type), and the specific steps are as follows: (1) Take the recombinant Agrobacterium obtained in step 1, resuspend it in N6 liquid medium (Sigma, C1416) and adjust the OD of the bacterial solution. 600nm is 0.5; (2) One-month-old mature embryonic callus of japonica rice variety kitaake (wild type) was inoculated with the bacterial solution obtained in step (1) for 30 min, and 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; (3) The callus cultured in step (2) was inoculated on a solid screening N6 solid medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and cultured for 16 days (first screening); (4) The healthy callus cultured in step (3) was inoculated on a solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and cultured for 15 days (second screening); (5) The healthy callus cultured in step (4) was inoculated on a solid screening N6 medium (Sigma, C1416) containing 10 g / L agar and 100 mg / L hygromycin and cultured for 15 days (third screening); (6) The healthy callus cultured in step (4) was inoculated on differentiation medium (M524, PhytoTechnologyLaboratories) to differentiate and obtain T 0 Generation of plants.

[0047] 3. For T obtained in step 2 0 The plants of the next generation were identified, the total DNA of the leaves of the plants to be tested was extracted, PCR amplification was performed using primers primer3 and primer4, the amplification products were detected by electrophoresis, and the plants with bands were transgenic positive plants.

[0048] primer3: 5'-TGAGGGATTATTTCATTTGA-3' (SEQ ID NO. 20); primer4: 5'-ATTACATGTTTAGGTCTGTT-3' (SEQ ID NO. 21).

[0049] 3. Phenotypic identification T 0 Transformation pCUbi1390- OsGPA15 Plants, gpa15 and wild-type Kitaake were planted in the transgenic experimental field of the Chinese Academy of Agricultural Sciences. The results showed that the transgenic line T 2Transparent seeds appeared in the seeds ( Figure 6 Middle A), SDS-PAGE detection of transparent seeds (L1, L2) and wild-type performance ( Figure 6 Middle B), rhodamine staining results of thick sections of endosperm in the middle stage of development are also consistent with the wild type ( Figure 6 C). This confirms that the opaque flour and increased gluten precursors before transgenic were caused by OsGPA15 Gene controlled, that is, OsGPA15 The gene is a gluten sorting-related gene. OsGPA15 Transformed rice gpa15 mutant, which can increase its mature glutenin content to normal levels.

Claims

1. Application of a gene of 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 shown in SEQ ID NO.

1.

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

3.

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

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

3.

5. The method according to claim 3 or 4, characterized in that: The gene is introduced into the plant with abnormal gluten sorting through a recombinant expression vector, wherein the plant is tobacco, Lotus japonicus, Arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, lawn grass, or alfalfa.

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