CN116715741B - Plant gluten sorting related protein OsGPA16, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a plant gluten sorting related protein OsGPA16, and a coding gene and application thereof. The invention selects mutant through rice glutengpa16Finally cloning to obtain the gluten sorting related protein OsGPA16, 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 protein related to gluten sorting influences the sorting process of gluten in rice endosperm, and 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, and therefore, the protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Plant gluten sorting related protein OsGPA16, and coding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a plant gluten sorting related protein OsGPA16, and a coding gene and application thereof.

Background

Rice is an important grain crop in the world, is a staple food for more than half of the population in the world, and takes rice as a staple food for more than 60% of the population in China. The rice breeding in China is subjected to three leaps of dwarf breeding, heterosis utilization and super rice cultivation, so that the basic self-sufficiency of seed sources is realized, the yield is greatly improved, and the international leading position of the rice breeding scientific research level is also laid. However, as the living standard of people in China is improved, the consumption of rice per person is reduced, and the demand of people for high-quality taste, nutrition and health rice is obviously increased. Although remarkable results are achieved in the rice quality improvement in China, a certain gap still exists between the rice quality improvement in China and the foreign high-quality rice.

Starch is the main nutritional ingredient of rice, and accounts for about 80% of the dry weight of rice seeds, and the content, structure and physicochemical properties of the starch are important factors for determining the quality of rice, so that the genetic improvement of the taste quality of rice is usually mainly carried out by starch at present. The rice storage protein is used as the second most important nutrient substance next to starch in rice, and the role in the formation of the taste quality of rice is not ignored. Gluten is a main component of rice storage protein, and accounts for about 60% -80% of the total protein content, and is a preferred target for improving the rice protein quality. Therefore, genetic mechanisms for analyzing gluten synthesis, transportation, processing and accumulation from the aspects of genetics, cells, biochemistry and the like have important theoretical significance and practical value for improving the quality of rice protein.

Gluten precursor accumulation (57H) mutants are a type of genetic resource that gluten precursors cannot enter protein body II or even if they enter, cannot be cleaved by vacuole processing enzymes, and are ideal genetic materials for resolving the mechanism of gluten synthesis and transport. By gene cloning and functional studies of pre-gluten volume accumulation mutants, the molecular network pathway of gluten from synthesis to deposition can be systematically elucidated. To date, a number of key genes regulating gluten transport have been cloned, and the molecular mechanism of gluten sorting has been primarily depicted, but the complete regulatory network of gluten sorting remains unclear, requiring us to continually locate and clone more key genes to further reveal the gluten sorting mechanism. The inventor screens from a chemical mutagenesis mutant library of japonica rice variety Kitaake to obtain a novel 57H mutantgpa16There is no report on the study of the OsGPA16 protein involved in rice storage protein synthesis.

Disclosure of Invention

The present inventors selected mutants by rice glutengpa16The related gluten sorting protein OsGPA16 is obtained through cloning, so that the related gluten sorting protein, and the encoding gene and the application thereof are provided. 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. The protein and the coding gene thereof can be applied to plant genetic improvement.

The gluten sorting related protein (OsGPA 16) provided by the invention is derived from rice of the genus oryzaOryza sativavarKitaake) 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 for the amino acid sequence shown in SEQ ID NO.1 and still has the functions.

SEQ ID NO.1 consists of 695 amino acid residues.

To facilitate purification of OsGPA16 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 OsGPA16 in the (b) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing. The coding gene of OsGPA16 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.

Meanwhile, the invention also provides a gene encoding the storage protein sorting related proteinOsGPA16）。

The geneOsGPA16The 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 2088 nucleotides.

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 vectorEcoRI, and RI systemNcoRecombination between I and insertion of the genes [ (]OsGPA16) The recombinant plasmid obtained. The recombinant plasmid can be pCAMBIA1305.1-OsGPA16The method comprises the steps of carrying out a first treatment on the surface of the The pCAMBIA1305.1-OsGPA16Will be composed ofOsGPA16The genome coding sequence is inserted into pCAMBIA1305.1 multiple cloning site by recombination technology together with upstream 2113bp promoter region and downstream 891bp fragmentEcoRI, and RI systemNcoI (Takara Co., in-fusion recombination kit).

Will containOsGPA16Named pCAMBIA1305.1-OsGPA16。

Comprising any one of the above genesOsGPA16) The expression cassette, the transgenic cell line and the recombinant bacteria belong to the protection scope of the invention.

The invention also provides a method for cultivating transgenic plants with normal gluten sorting, which comprises the steps of introducing the genes into plants with abnormal gluten sorting to obtain transgenic plants with 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 GPA16 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. The protein and the coding gene thereof can be applied to plant genetic improvement.

Drawings

FIG. 1 wild type Kitaake and mutantgpa16Wherein A is Kitaake andgpa16the cross-cut phenotype of the dry seed and endosperm, B is Kitaake andgpa16cross-cutting endosperm to obtain a scanning electron microscope picture;

FIG. 2 wild type Kitaake and mutantgpa16SDS-PAGE and Western blot analysis, wherein A is Kitaake andgpa16SDS-PAGE of endosperm storage protein fractions, B Kitaake andgpa16gluten Western-Blot analysis;

FIG. 3 wild type Kitaake and mutantgpa16Semi-thin section observation of developing endosperm, wherein A is Kitaake andgpa16coomassie brilliant blue staining of mid-development endosperm semi-thin sections, B is Kitaake andgpa16mid-development endosperm semi-thin slice immunofluorescence analysis;

FIG. 4 wild type Kitaake and mutantgpa16Immune colloidal gold observation of developing endosperm, wherein A-C is Kitaake andgpa16protein shape structure in metaphase endosperm, D-E is Kitaake and D-E are respectivelygpa16Morphology of golgi in mid-development endosperm; F-J is Kitaake andgpa16extracellular space structural differences in the metaphase endosperm and the process of paraparietal formation;

FIG. 5 map-based cloning of mutant genes, wherein A isgpa16Fine positioning map of B isgpa16Is a mutation site of (a);

FIG. 6 phenotypic analysis of transgenic complementation lines, wherein A is Kitaake andgpa16and transgenic complementing family dry seed and endosperm transverse phenotype, B is Kitaake andgpa16and the SDS-PAGE pattern of the storage protein components of the complementary families, C being Kitaake andgpa16dye staining of cell walls of thick endosperm sections of the metaphase of complementary pedigree development;

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: gluten sorting related protein in rice and discovery of coding gene thereof

1. Rice protein sorting mutantgpa16Phenotypic analysis of (a)

Screening mutant lines of kernel powder from chemical mutation mutant library of japonica rice variety kitaakegpa16. In contrast to the wild-type species,gpa16is mainly characterized by the quality of the grain powder and the opacity (see A in figure 1). Scanning electron microscope analysis confirmsgpa16The loosening of the starch granules may be the main cause of the opalescence of the endosperm (see B in fig. 1).gpa16SDS-PAGE patterns of seed proteins showed an increase in the 57 kDa precursor of gluten, a corresponding decrease in the acidic and basic subunit content of mature gluten, and a decrease in the globulin content (see FIG. 2A). Western blot analysis confirmedgpa16The acid subunit content of middle-aged gluten is reduced (see B in fig. 2).

The semi-thin endosperm slices in the middle of development are observed after being dyed by coomassie brilliant blue, 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 left graph in figure 3, arrow marks the protein body I and triangle marks the protein body II)). And atgpa16The protein body II is more irregular than the wild type shape (see right panel A in FIG. 3, triangular marks). Interestingly, ingpa16In addition to the two proteins, a class of paraparietal structures filled with a large amount of protein was found near the cell wall (see right panel a in fig. 3, indicated by dashed boxes). Further mid-development endosperm semi-thin slice immunofluorescence experiments also confirmed the above results (see B in fig. 3). The above results indicate thatgpa16Protein body II in the mutant is dysplasia, and a large amount of protein fills 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). Consistent with the cytological results observed with the semi-thin sections,gpa16the shape of the filling of protein II was irregular, and the content was less than that of the wild type (A-C in FIG. 4). Studies have shown that post-Golgi trafficking of gluten is mediated by dense vesicles, mutantsgpa16Medium dense vesicles can normally bud from the golgi apparatus, consistent with wild type (D and E in fig. 4). However, it is interesting that,gpa16the middle and large dense vesicles were erroneously sorted into extracellular space, gradually accumulated and formed large paraparietal structures (F-J in FIG. 4).

Taken together, the above results confirmgpa16The dense vesicles responsible for transporting gluten undergo a false sort, which in turn results in a large amount of gluten precursor entering the apoplast space that cannot be cleaved into mature acidic and basic subunits.

2. Targeting genes

1. Preliminary localization of target genes

By means of mutantsgpa16Hybridization with Dular, a wide variety of affinities, purchased from the germplasm resource pool of the national academy of agricultural sciences, in Chinagpa16F of Dular ₂ Selecting 10 grains from the segregating groupgpa16Seed DNA was extracted from recessive extreme individuals of phenotype (opaque grain flour and increased gluten precursor). Linkage analysis using primers covering the whole genome of rice will be responsible forgpa16Mutant genes of the mutant phenotype are located atChromosome 12, linked between markers I12-10 and RM 270.

2. Fine localization of target genes

According to the initial positioning result, searching a molecular marker on a public map between the linkage markers I12-10 and RM270, and automatically developing the linkage markers in the interval according to the rice genome sequence information published by NCBI. Detection of self-designed labeled primers polymorphism between Kitaake and Dular, showing that the polymorphism is used as a finely-localized molecular marker. The target gene was finely mapped using 335 recessive extreme individuals (molecular markers see table 2).

TABLE 2 molecular markers for Fine localization

Finally, the target gene is obtainedOsGPA16Fine positioning between the linked markers 1-18 and 3-3, physical distance 185 kb (A in FIG. 5). By sequencing the genes in the interval, the genes are foundOsGPA16A single base substitution exists on exon 7 of (a) to allow amino acid substitution of the target protein (B in FIG. 5).

3. Target geneOsGPA16Is 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'- ATGATTCTGTCGGCGCTCGC-3'(SEQ ID NO:16)；

primer2：5'-CTAAGACAATAGAGGTGTAG-3'(SEQ ID NO:17)。

The protein shown in SEQ ID NO.1 is named as OsGPA16 protein and consists of 695 amino acid residues. The gene encoding the OsGPA16 protein is named asOsGPA16The open reading frame of the gene is shown as SEQ ID NO. 2.

Example 2: osGPA16 protein and application of encoding gene thereof

1. Construction of genome complementary vector

The pCAMBIA1305.1 vectorEcoRI, and RI systemNcoThe 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 (comprisingOsGPA16The upstream 2113bp promoter sequence and the downstream 891bp terminator sequence of the gene to obtain pCAMBIA1305.1-OsGPA16The vector map of pCAMBIA1305.1, the complementary vector of genome (verified by sequencing), is shown in FIG. 7.

2. Acquisition of complementary transgenic plants

1. The pCAMBIA1305.1 obtained in the step oneOsGPA16The complementing vector was introduced into an agrobacterium EHA105 strain (a company of the united states of america) to obtain recombinant agrobacterium.

2. The recombinant agrobacterium obtained in the step 1 is adopted to transform japonica rice variety kitaake (wild type), and the specific steps are as follows:

(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 japonica rice variety kitaake (wild type) cultivated to one month in the bacterial liquid obtained in the step (1) for 30 min, sucking the bacterial liquid by filter paper, transferring the bacterial liquid into a solid N6 culture medium (Sigma Co., C1416) containing 10 g/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 10 g/L agar and 100 mg/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 10 g/L agar and 100 mg/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 10 g/L agar and 100 mg/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) to give 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 the amplified products, and obtaining the transgenic positive plants with double mutation sites.

primer3：5'-CCCCATGACGTTTATTGGCC-3'(SEQ ID NO:18)；

primer4：5'-TGCACGGTTGAGGAAGAGAT-3'(SEQ ID NO:19)。

3. Phenotypic identification

Respectively T ₀ Substitution of pCAMBIA1305.1-OsGPA16The plant is used for the cultivation of the plants,gpa16and wild-type Kitaake were planted in transgenic test fields of national academy of agricultural sciences. The results show that transgenic line T ₂ Transparent grains appeared in the seeds (FIG. 6A), SDS-PAGE showed that transparent seeds (L1, L2) performed as the wild type (FIG. 6B), and that the result of Coomassie blue staining of the semi-thin sections of the metaphase endosperm was also consistent with the wild type (FIG. 6C). Thus, it was confirmed that the opaque powder and gluten precursor increase characteristics before the transgene were attributed toOsGPA16Genetically controlled, i.e. theOsGPA16The gene is a gluten sorting related gene. pCAMBIA1305.1-OsGPA16Transformation of ricegpa16Mutants can raise the mature gluten content to normal levels.

Claims (4)

1. An application of a gluten sorting related protein OsGPA16 or a coding gene thereof in plant breeding is characterized in that the gluten sorting related protein OsGPA16 is used for cultivating plants with abnormal gluten sorting into transgenic plants with normal gluten sorting;

the gluten sorting related protein OsGPA16 is a protein consisting of an amino acid sequence shown in SEQ ID NO. 1;

the plant is rice; the plant with abnormal gluten sorting is an OsGPA16 gene function defect mutant.

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

3. A method for culturing transgenic plants with normal gluten sorting is to introduce a coding gene of a gluten sorting related protein OsGPA16 into plants with abnormal gluten sorting to obtain transgenic plants with normal gluten sorting; wherein the abnormal gluten sorting is abnormal accumulation of gluten precursors;

the gluten sorting related protein OsGPA16 is a protein consisting of an amino acid sequence shown in SEQ ID NO. 1;

the plant is rice; the plant with abnormal gluten sorting is an OsGPA16 gene function defect mutant.

4. A method according to claim 3, wherein the nucleotide sequence of the coding gene is as set forth in SEQ ID No.2 or SEQ ID No. 3.