CN116731145B - Plant gluten sorting related protein OsGPA11 and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a plant gluten sorting related protein OsGPA11, and a coding gene and application thereof. The invention selects mutant through rice glutengpa11Finally cloning to obtain the gluten sorting related protein OsGPA11, wherein the related protein consists of an amino acid sequence shown as SEQ ID NO.1, and the nucleotide sequence of the gene is shown as SEQ ID NO.2 or SEQ ID NO. 3. The related gluten sorting proteins influence the sorting process of gluten in rice endosperm, and the coding genes of the proteins are introduced into plants with reduced mature gluten content, so that transgenic plants with normal mature gluten content can be obtained. Therefore, the protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Plant gluten sorting related protein OsGPA11 and encoding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a plant gluten sorting related proteinOsGPA11And coding genes and application thereof.

Background

Rice is the first large grain crop in China. At present, the total yield of the rice in China can meet the total demand of the rice in China, and along with the continuous improvement of the resident living standard in China, higher demands are put on high-quality edible rice, but the high-quality rate of the rice in China is not high at present, and the great demand of people on high-quality rice cannot be met. In order to improve the high-quality rate of rice, enhance the international competitiveness of rice in China, ensure the national grain safety, and greatly require molecular basic research for enhancing the formation of rice quality, and provide gene resources and theoretical basis for the cultivation of new varieties of high-quality rice. Starch is the most important nutrient in rice, and its content, composition and structure have an important effect on rice quality, so that the improvement of taste quality of rice is usually mainly based on starch improvement. It is important to note that storage proteins are the second major nutrient class in rice other than starch, whose content and composition directly affect various quality indicators and nutritional values of rice, but which are not well appreciated. Mutation of any key gene in the synthesis, transport and deposition processes of storage proteins can seriously affect rice quality. Therefore, the analysis of the molecular regulation mechanism of rice storage protein synthesis and separation has important significance for the genetic improvement of rice quality.

Gluten accounts for 60-80% of the rice storage protein content, belongs to high-quality protein, is easy to digest and absorb by human bodies, and is an important target for improving the rice protein quality by controlling the gluten content within a certain range. Gluten precursor accumulation (57H) mutants are good genetic material for studying gluten synthesis sorting. By cloning key genes in the processes of gluten synthesis, transportation, processing and accumulation, a gluten sorting network is constructed, and important theoretical guidance can be provided for improving the quality of rice proteins. At presentSeveral key node genes regulating gluten transport have been cloned, but in view of the complexity of gluten sorting, a fine regulatory network for gluten synthesis sorting has yet to be studied intensively. The inventor screens from the chemical mutagenesis mutant library of japonica rice variety TC65 to obtain a new 57H mutantgpa11There is no report on the study of the OsGPA11 protein involved in rice storage protein synthesis.

Disclosure of Invention

The present inventors selected mutants by rice glutengpa11The invention provides a gluten sorting related protein OsGPA11, and a coding gene and application thereof.

The gluten sorting related protein (OsGPA 11) provided by the invention is derived from rice of the genus oryzaOryza sativavar.taichung 65), is a protein of the following (a) or (b):

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

(b) And (3) the derivative protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO.1 and still has the functions.

SEQ ID NO.1 consists of 500 amino acid residues.

To facilitate purification of OsGPA11 in (a), a tag as shown in Table 1 may be attached to the amino-terminus or the carboxyl-terminus of a protein consisting of the amino acid sequence shown in SEQ ID NO. 1.

TABLE 1 sequence of tags

The OsGPA11 in the (b) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing. The coding gene of OsGPA11 in (b) above can be obtained by deleting one or several amino acid residues in the DNA sequence shown in SEQ ID NO.2, and/or performing one or several base pair missense mutation, and/or ligating the coding sequences of the tags shown in Table 1 at the 5 '-end and/or the 3' -end thereof.

Genes encoding the above-mentioned proteins related to the separation of storage proteinsOsGPA11) And also falls within the scope of the present invention.

The geneOsGPA11The nucleotide sequence of (a) may be 1) or 2) or 3) or 4) as follows:

1) A nucleotide sequence shown as SEQ ID NO. 2;

2) A nucleotide sequence shown as SEQ ID NO. 3;

3) A nucleotide sequence which hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and which codes for said protein;

4) A nucleotide sequence which has more than 90% homology with the DNA sequence defined in 1), 2) or 3) and which encodes a gluten sorting related protein.

SEQ ID NO.2 consists of 1503 nucleotides.

The stringent conditions may be those of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS in solution at 65 ^o Hybridization and washing the membrane under C.

Recombinant expression vectors containing any of the above genes are also within the scope of the present invention.

Recombinant expression vectors containing the genes can be constructed using existing plant expression vectors.

The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal directs the addition of polyadenylation to the 3 'end of the mRNA precursor, and may be similarly functional in the untranslated regions transcribed from the 3' end of, for example, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes) and plant genes (e.g., soybean storage protein genes).

When the gene is used for constructing a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter such as a cauliflower mosaic virus (CAMV) 35S promoter and a Ubiquitin promoter (Ubiquitin) of corn can be added before transcription initiation nucleotide, and the recombinant plant expression vector can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

The recombinant expression vector may be a multiple cloning site in the pCAMBIA1305.1 vector (http:// www.cambia.org/daisy/cammbia/585)EcoRI andNcorecombination between I and insertion of the genes [ (]OsGPA11) The recombinant plasmid obtained. The recombinant plasmid can be pCAMBIA1305.1-OsGPA11The method comprises the steps of carrying out a first treatment on the surface of the The pCAMBIA1305.1-OsGPA11Will be composed ofOsGPA11The genome coding sequence is inserted into pCAMBIA1305.1 multiple cloning site by recombination technique together with upstream 1996bp promoter region and downstream 581bp fragmentEcoRI andNcoi (Clontech, information recombination kit).

Will containOsGPA11Named pCAMBIA1305.1-OsGPA11。

Comprising any one of the above genesOsGPA11) The expression cassette, the transgenic cell line and the recombinant bacteria belong to the protection scope of the invention.

Another object of the present invention is to provide a method for breeding a transgenic plant having normal gluten sorting, which comprises introducing the gene into a plant having abnormal gluten sorting to obtain a transgenic plant having normal gluten sorting; the abnormal gluten sorting plant is a plant with the gluten precursor in endosperm increased sharply and the content of mature gluten reduced; the transgenic plant with normal gluten sorting is a transgenic plant with gluten precursors which can be normally processed into mature gluten. Specifically, the gene is introduced into a gluten sorting abnormal plant through the recombinant expression vector; the abnormal gluten sorting plant may be a GPA11 protein function deficient mutant.

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

The gene encoding the protein is introduced into plant cells by using any vector capable of guiding the expression of exogenous genes in plants, and a transgenic cell line and a transgenic plant can be obtained. The expression vector carrying the gene may be transformed into plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues are cultivated into plants. The plant host to be transformed may be either a monocot or a dicot, such as: tobacco, radix et rhizoma Baimai, arabidopsis thaliana, rice, wheat, corn, cucumber, tomato, poplar, turf grass, alfalfa, etc.

The gluten sorting related proteins of the invention affect the sorting process of gluten in rice endosperm. The coding gene of the protein is introduced into a plant with reduced mature gluten content, so that a transgenic plant with normal mature gluten content can be obtained. Therefore, the protein and the coding gene thereof can be applied to plant genetic improvement.

Drawings

FIG. 1 wild type TC65 and mutantgpa11Is a visual phenotype of (a). Wherein A is TC65 andgpa11the cross-cut phenotype of the dry seed and endosperm, B is TC65 andgpa11endosperm transection scanning electron microscope pictures.

FIG. 2 wild type TC65 and mutantgpa11 SDS-PAGE and Western blot analysis. Wherein A is TC65 andgpa11SDS-PAGE of endosperm storage protein fractions, B TC65 andgpa11gluten and globulin Western-Blot analysis.

FIG. 3 wild type TC65 and mutantgpa11Semi-thin sections of developing endosperm were observed. Wherein A-C is TC65 andgpa11results of Coomassie brilliant blue staining of mid-development endosperm semi-thin sections with D-F as TC65 andgpa11mid-development endosperm semithin section immunofluorescence analysis.

FIG. 4 wild type TC65 and mutantgpa11Immune colloidal gold observation of development endosperm. Wherein A-B is TC65 andgpa11morphological structure of endoplasmic reticulum in metaphase endosperm, C-M is TC65 andgpa11morphological structure of protein body, golgi apparatus and compact vesicle in metaphase endosperm, N-O is TC65 andgpa11extracellular space structural differences in the metaphase endosperm and the process of paraparietal formation.

FIG. 5 map cloning of mutant genes. Wherein A isGPA11Fine positioning map of B isGPA11Is a mutation site of (a);

FIG. 6 phenotypic analysis of transgenic complementation lines, wherein A is TC65 andgpa11and the endosperm transection of the transgenic complementary family, B is TC65 andgpa11a map of complementary family storage protein components.

FIG. 7 pCAMBIA1305.1 vector map.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Example 1 discovery of gluten sorting-related proteins and genes encoding the same in Rice

1. Rice protein sorting mutantgpa11Phenotypic analysis of (a)

Screening mutant lines of kernel powder from chemical mutation mutant library of japonica rice variety TC65gpa11. In contrast to the wild-type species,gpa11is mainly characterized by the quality of the grain powder and the opacity (see A in figure 1). Scanning electron microscope analysis confirmsgpa11The loosening of the starch granules may be the main cause of the opalescence of the endosperm (see B in fig. 1).gpa11SDS-PAGE patterns of seed proteins showed an increase in the gluten 57kDa precursor, a corresponding decrease in the mature gluten acidic and basic subunit content, and a decrease in the globulin content (see FIG. 2A); western blot analysis further validated the above changes (see B in fig. 2).

After being stained with coomassie brilliant blue and immunofluorescence, the semi-thin endosperm slices in the middle of development are observed to show that two types of protein bodies exist in the wild type, namely, spherical protein body I and irregularly-shaped protein body II, wherein the protein body II is slightly larger than the protein body I (see A and D in fig. 3, triangular protein body I and arrow mark protein body II). Interestingly, ingpa11In addition to the two proteins mentioned above, a class of paraparietal structures filled with large amounts of protein was found in the cell wall attachment (see E, asterisk in fig. 3B and 3). In addition, in the case of the optical fiber,gpa11the protein body II is significantly smaller than the wild type (see B in FIG. 3, arrow mark), and the protein is sorted onto the vacuolar membrane (see F in FIGS. 3C and 3, box). The above results indicate thatgpa11Protein body II in the mutant is dysplasia, and a large amount of protein is filled in a wall paracorporeal structure formed near the cell wall.

To analyze the process of forming the paraparietal structure, the endosperm of the metaphase stage was observed using transmission electron microscopy in combination with immune colloidal gold technology (see fig. 4). Studies have shown that gluten is initially synthesized in the endoplasmic reticulum and transported to the golgi apparatus by COPII vesicles (see A in FIG. 4), but in mutantsgpa11In (B) the gluten fraction remains in the endoplasmic reticulum (see B in fig. 4). The prolamin is mainly stored in protein body I (see figure 4C),gpa11the middle part of gluten is also mis-sorted into protein body I (see D in fig. 4). Post-golgi transport of gluten is mediated by a class of transport means known as tight vesiclesSee E in fig. 4). Like the wild type, the mutantgpa11Medium dense vesicles can normally sprout from the golgi apparatus (see F in fig. 4). However, it is interesting that,gpa11abnormal aggregation of a large number of dense vesicles (see G in FIG. 4), erroneous separation into extracellular volume (I in FIG. 4), gradual accumulation and formation of large paraparietal structures (J-K in FIG. 4), thus leading togpa11The protein body II in (1) was smaller than that in the wild type, and the gluten was insufficiently filled (L-M in FIG. 4). In addition, gluten is also anchored to the vacuolar membrane (see N-O in FIG. 4).

Taken together, the above results confirmgpa11The dense vesicles responsible for transporting gluten undergo a false sort, which in turn results in a large amount of gluten precursor entering the extracellular space and not being able to be cleaved into mature acidic and basic subunits.

2. Targeting genes

1. Preliminary localization of target genes

By means of mutantsgpa11Hybridization with Dular, a wide variety of affinities, purchased from the germplasm resource pool of the national academy of agricultural sciences, in Chinagpa11Selecting 10 grains from F2 separation group of Dulargpa11Recessive extreme individuals with phenotype (opaque grain flour and increased gluten precursor) extract seed DNA and construct DNA pools. Linkage analysis of InDel markers using 175 covering the whole genome of rice will be responsiblegpa11The mutant gene of the mutant phenotype is located near the chromosome 2 linkage marker R2-5.

2. Fine localization of target genes

According to the initial positioning result, searching a molecular marker on a public map near the linkage marker R2-5, and automatically developing an InDel marker in the interval according to the rice genome sequence information published by NCBI. The target gene was finely mapped using 430 extreme individuals (molecular markers for fine mapping are shown in table 2).

TABLE 2 molecular markers for Fine localization

Finally, the target gene is obtainedOsGPA11Fine positioning is performed between the linkage markers T27 and T44 at a physical distance of 102 kb (see a in fig. 5). By sequencing the genes in this interval, it was found that there was a single base substitution on exon 1 of ORF 1, resulting in erroneous translation of the target protein (see B in FIG. 5).

3. Target geneOsGPA11Is obtained by (a)

Extracting cDNA of japonica rice variety kitaake leaves, carrying out PCR (polymerase chain reaction) amplification by using cDNA as a template and adopting a primer1 and a primer2, sequencing an amplification product, wherein the sequencing result is shown as SEQ ID NO.2, and the coded protein is shown as SEQ ID NO. 1.

primer1：5'-ATGGCGACCATAGCTTTCTCTCGG-3'；

primer2：5'-TCACTCTGAGGCCTCGTTCTCCGA-3'。

The protein shown in SEQ ID NO.1 is named as OsGPA11 protein and consists of 500 amino acid residues. The gene encoding the OsGPA11 protein is named asOsGPA11The open reading frame of the gene is shown as SEQ ID NO. 2.

The gene accession numbers is input into the national rice database, no related literature report exists, and the specific links are as follows: http:// www.ricedata.cn/gene/gene_info. Aspxid=loc_os 02g16820.

Example 2, osGPA11 protein and application of encoding gene thereof

1. Construction of genome complementary vector

The pcambia1305.1 vector (fig. 7) (ref: he Gao, mingnaJin, et al,Days to heading 7a major quantitative locusdetermining photoperiod sensitivity and regionaladaptation in price.Proc Natl Acad Sci USA,2014, 111 (46): 16337-16342)EcoRI andNcothe small fragment between the cleavage sites of I is replaced by a double-stranded DNA molecule shown in SEQ ID NO.3 of the sequence Listing (comprisingOsGPA11The promoter region of gene upstream 1996bp and downstream 584 bp) to give pCAMBIA1305.1-OsGPA11Genome complementary vector (verified by sequencing).

2. Acquisition of complementary transgenic plants

1. The pCAMBIA1305.1 obtained in the step oneOsGPA11The complementing vector was introduced into an agrobacterium EHA105 strain (a company of the united states of america) to obtain recombinant agrobacterium.

2. Adopting the recombinant agrobacterium obtained in the step 1 to transform a japonica rice variety TC65 (wild type), and specifically comprises the following steps:

(1) Taking recombinant Agrobacterium thallus obtained in step 1, re-suspending with N6 liquid culture medium (Sigma Co., C1416) and adjusting bacterial liquid OD _600nm 0.5.

(2) Infecting the mature embryo embryogenic callus of the japonica rice variety TC65 (wild type) cultivated for one month in the bacterial liquid obtained in the step (1) for 30min, sucking the bacterial liquid by filter paper, transferring the bacterial liquid into a solid N6 culture medium (Sigma Co., C1416) containing 10g/L agar, and co-culturing at 24 ℃ for 3 days;

(3) Inoculating the callus cultured in the step (2) on a solid screening N6 solid medium (Sigma Co., C1416) containing 10g/L agar and 100mg/L hygromycin, and culturing for 16 days (first screening);

(4) Inoculating the healthy callus cultured in the step (3) on a solid screening N6 medium (Sigma Co., C1416) containing 10g/L agar and 100mg/L hygromycin, and culturing for 15 days (second screening);

(5) Inoculating the healthy callus cultured in the step (4) on a solid screening N6 medium (Sigma Co., C1416) containing 10g/L agar and 100mg/L hygromycin, and culturing for 15 days (third screening);

(6) Inoculating the healthy callus cultured in the step (4) to a differentiation medium (PhytoTechnology Laboratories company, M524) for differentiation to obtain T ₀ And (5) replacing plants.

3. For T obtained in step 2 ₀ And identifying the generation of plants, extracting total DNA of leaves of the plants to be detected, carrying out PCR (polymerase chain reaction) amplification by adopting a primer3 and a primer4, sequencing an amplified product, and replacing sites to obtain the double-peak transgenic positive plants.

primer3：5'-TCACATCAACCAGCCCAAGT-3'；

primer4：5'- GCCAGGAGTATTGCTGTATCG-3'。

3. Phenotypic identification

T0 generation is converted into pCAMBIA1305.1 respectivelyOsGPA11The plant is used for the cultivation of the plants,gpa11and wild type TC65 were planted in the national academy of agricultural sciences transgene test field. The results showed that opaque kernels appeared in the transgenic line T2 seeds (see FIG. 6A), and SDS-PAGE detected that the clear seeds (L1, L2 and L3) performed as the wild type and that the silty seeds (L1, L2 and L3) performed as the mutant (see FIG. 6B). Thus, it was confirmed that the pre-transgenic opaque meal and gluten precursor increase trait was controlled by the OsGPA11 gene, i.e., the OsGPA11 gene was a gluten sorting related gene.

Claims (3)

1. Use of a gluten sorting related protein or a gene encoding the same in plant breeding, characterized in that it is used in the cultivation of a transgenic plant with normal gluten sorting, said plant being rice; the method is realized by introducing the coding gene of the gluten sorting related protein into a gluten sorting abnormal plant to obtain a transgenic plant with normal gluten sorting; the abnormal gluten sorting plant is GPA11 protein function defect mutant;

the amino acid sequence of the gluten sorting related protein is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the nucleotide sequence of the coding gene is shown in SEQ ID No.2 or SEQ ID No. 3.

3. A method for culturing transgenic plant with normal gluten sorting is to introduce the coding gene of related gluten sorting protein into abnormal gluten sorting plant to obtain transgenic plant with normal gluten sorting; wherein the abnormal gluten sorting is abnormal accumulation of gluten precursors and the plant is rice;

the amino acid sequence of the gluten sorting related protein is shown as SEQ ID NO. 1; the abnormal gluten sorting plant is GPA11 protein function defect mutant.