CN113683669B - Method for accumulating target protein in plant based on interaction with prolamin - Google Patents

Abstract

The invention discloses a method for accumulating a target protein in plants based on the interaction with gliadin. The present invention verifies that target proteins NZP1, NZP2 and NZP3 interact with prolamin 22-kDα-zein respectively, and express Zera-22 and target proteins NZP1, NZP2 or NZP3 in tobacco, and express Zera and target in tobacco The results of protein NZP1, NZP2 or NZP3 experiments showed that Zera‑22 induced protein body formation in tobacco, and the target protein was localized in the protein body formed by Zera‑22, which proved that the target protein NZP1, NZP2 and NZP3 could pass through the interaction with 22‑kDα ‑zein interactions accumulate in protein bodies formed by Zera‑22. Thus, based on the interaction with gliadin, the target protein can be accumulated in plants, and then applied to protein production.

Description

Method for accumulating target protein in plants based on interaction with gliadin

technical field

The invention relates to a method for accumulating a target protein in plants based on the interaction with gliadin in the field of biotechnology.

Background technique

As the demand for recombinant proteins continues to expand in industry and medicine, transgenic plants are considered to be a safe, efficient and inexpensive means of production (Ma et al., 2003). Compared with traditional expression systems (such as yeast, insect, and mammalian cell culture), transgenic plants also have the freedom from human pathogen contamination, the ability to correctly fold and assemble multimeric proteins, and the ability to directly orally administer raw or partially processed plants. material potential (Fischer et al., 2004). Over the past three decades, transgenic plants have shown promising applications as bioreactors in the production of vaccines, antibodies, and enzymes for industrial use, such as the use of plant platforms to produce various antibodies, vaccine antigens, protein allergens, enzymes, and enzymes Inhibitors, coagulation factors, cytokines and hormones, etc. (Giddings et al., 2000; Ma et al., 2005). However, a major problem facing the production of recombinant proteins using plant platforms is that recombinant proteins do not accumulate at sufficient levels in plants (Doran, 2006). The expression of recombinant proteins in a heterologous environment is naturally unstable, and it also faces the ubiquitous protein degradation process in cells. Certain recombinant proteins do not have a clear subcellular localization in cells, and are likely to enter the intracellular protein degradation pathway (Faye et al., 2005).

Studies on the subcellular localization of recombinant proteins in plant expression systems have shown that the subcellular localization of recombinant proteins in plant cells significantly affects the accumulation level of recombinant proteins in cells. The natural protein storage structure in plant seeds can provide a barrier for the storage of recombinant proteins (Conley et al., 2011). During grass seed development, a large amount of protein naturally accumulates in specific locations of the endoplasmic reticulum in endosperm cells, and the ER in these regions bulges outward, forming specialized ER-derived compartments called protein bodies (Shewry et al., 1995; Herman and Larkins, 1999). Among them, the research on protein bodies in maize endosperm cells is more in-depth. Protein bodies are important protein storage organelles in maize endosperm cells, and the protein content in protein bodies accounts for about 60% of the total protein content in cells. Moreover, the protein body is highly stable, even in mature grains and grains stored for many years, the protein stored in the protein body is highly complete. Therefore, the zein body is an ideal place for high expression and high accumulation of exogenous proteins. In the protein body of maize endosperm cells, gliadin is the most important storage protein, accounting for about 80% of the total storage protein (Shewry et al., 1995; Herman and Larkins, 1999; Fuji et al., 2007). Zein mainly includes α (19-kD, 22-kD), β (14-kD), γ (16-kD, 27-kD and 50-kD) and δ (10-kD, 18-kD) four Among them, α(19-kD, 22-kD) prolamins have the highest content (Esen, 1987; Coleman and Larkins, 1999; Song and Messing, 2002).

In the study of protein body formation, it was found that 27-kDγ-zein plays an important role in the development of protein body (Wu and Messing, 2010; Guo et al., 2013). Inhibition of 27-kDγ-zein expression in maize endosperm by RNAi technology resulted in a significant decrease in the number of protein bodies in the cells. It shows that 27-kDγ-zein can promote the formation of protein bodies. Further studies have found that 27-kDγ-zein can induce the formation of protein bodies not only in maize kernels, but also in tobacco seeds, leaves and Arabidopsis leaves (Torrent et al., 2009; Llop- Tous et al., 2010; Mainieri et al., 2014). Sequence analysis shows that 27-kDγ-zein contains four regions: a. a signal peptide containing 19 amino acids, b. a region consisting of 8 PPVHL hexapeptide repeats (53aa), c. rich in proline pro-x domain (29aa), d. hydrophobic cysteine-rich C-terminal domain (111a). Through the study of the residence mechanism of 27-kDγ-zein, it is found that there is no endoplasmic reticulum residence signal composed of HDEL in the 27-kDγ-zein sequence, and the residence and induction of 27-kDγ-zein in the endoplasmic reticulum The formation of protein bodies depends on the structure composed of regions a, b, and c, which is also called Zera domain (Torrent et al., 2009; Llop-Tous et al., 2010; Mainieri et al., 2014). Further studies revealed that the two cysteine residues at the N-terminus of 27-kDγ-zein are critical for the oligomerization of 27-kDγ-zein, which is thought to be a protein body The first step in formation.

In addition to 27-kDγ-zein, another important class of gliadins in protein bodies is α-class gliadins (19-kD, 22-kD), and α-zein is responsible for the filling of protein bodies (Holding et al., 2007) , is the most abundant gliadin type in protein bodies. Through the study of α-zein, it was found that α-zein itself does not have an endoplasmic reticulum resident signal, and expression of α-zein alone in vitro cannot induce the formation of protein bodies, indicating that α-zein does not have the ability to induce protein body formation . Kim et al. found through a series of gliadin interaction studies that 22-kDα-zein can interact with other types of gliadins (Kim et al., 2002). Coleman et al. expressed both 27-kDγ-zein and 22-kDα-zein in tobacco, and 22-kDα-zein can enter the protein body formed by 27-kDγ-zein (Coleman et al., 1996).

In addition to prolamins, there are also a large number of non-prolamins (nonzeinprotein, NZP) in the protein body of corn endosperm. Wang et al. identified more than 2,000 non-gliadins through mass spectrometry analysis of the isolated and purified corn endosperm protein bodies. Further analysis found that these non-gliadins belonged to different metabolic pathways and had signals targeting other organelles. Peptides (Wang et al., 2016). The study of non-glamin NZP1 found that NZP1 itself has a signal peptide localized to mitochondria, and expressed NZP1 in tobacco leaves and found that it is located in mitochondria. In maize endosperm cells, NZP1 is localized in both mitochondria and protein bodies. In addition, multiple non-prolamins that accumulate in protein bodies through interactions with 22-kD were identified. This mechanism of gliadin-mediated accumulation of non-gliadins in protein bodies has not been clearly reported before. The study on the accumulation mechanism of non-prolamin in protein body provides useful information for the efficient accumulation of recombinant protein in maize endosperm or tobacco protein body.

Non-glamin is of great significance for maintaining the normal shape of protein bodies and improving the quality of proteins in protein bodies. In previous studies, O1, FL1 and O10 are all involved in the development of protein bodies, regulating the distribution of gliadin in protein bodies, and maintaining the normal shape of protein bodies (Holding et al., 2007; Wang et al., 2016; Yao et al., 2016;). A normal protein body is important to maintain kernel firmness. When NZP1 was mutated, grain development was affected, and protein body development was delayed, which affected the accumulation of 22-kDα-zein in protein bodies. In the protein body, gliadin is the main storage protein. In gliadin, the content of proline and glutamic acid is high, but the content of tryptophan and serine is low, so the protein quality is not high. Serine and tryptophan in NZP1, NZP2 and NZP3 proteins are higher than those in gliadin. These protein storage proteins maintain the balance of amino acids in the grain and help to improve the quality of grain protein. In addition, protein bodies, as bioreactors, can serve as platforms for exogenous recombinant protein production. After the exogenous protein is recombined with NZP1, NZP2 or NZP3, the feature that NZP1-3 can accumulate in the protein body is used to effectively accumulate the exogenous protein in the protein body. In rice, the cherry blossom allergen protein Cryj1 is modified and expressed in rice, and the allergen protein can be stably accumulated in the protein body. At the same time, the allergen protein has good activity and can be used for human desensitization. (Okada et al., 2003).

Contents of the invention

The technical problem to be solved by the invention is how to accumulate the target protein in the plant protein body.

In order to solve the above technical problems, the present invention provides a method for accumulating target proteins in plants based on the interaction with gliadin, comprising the following steps:

S1. Expressing the gliadin in recipient plants;

S2. Expressing the target protein that interacts with the prolamin in the recipient plant;

The prolamin is a protein of the following A1, A2 or A3:

A1, the amino acid sequence is the protein shown in the sequence 8 amino acid sequence in the sequence listing;

A2. Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in Sequence 8 in the sequence listing has more than 80% identity with the protein shown in A1) and is identical to the protein Body filling related proteins;

A3. A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2).

In the above method, the sequence 8 in the sequence listing consists of 266 amino acid residues.

In the above method, the target protein may be the following B1, B2, B3 or B4 protein:

B1. The amino acid sequence is the protein shown in any one of the amino acid sequences of Sequence 2, Sequence 4, and Sequence 6 in the sequence listing;

B2, the amino acid sequence shown in any one of Sequence 2, Sequence 4, and Sequence 6 in the sequence listing is obtained through substitution and/or deletion and/or addition of one or several amino acid residues and the protein shown in B1) A protein that has more than 80% identity and interacts with any of the proteins described in A1, A2 and A3;

B3, a fusion protein linked with B1 or B2;

B4. A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of any of the proteins described in B1, B2 and B3.

In the above method, sequence 2 in the sequence listing consists of 196 amino acid residues; sequence 4 in the sequence listing consists of 346 amino acid residues; sequence 6 in the sequence listing consists of 213 amino acid residues.

In the above method, it may also include expressing proteins related to protein body formation in recipient plants, and the proteins related to protein body formation are the following C1, C2 or C3 proteins:

C1, the amino acid sequence is the protein shown in the sequence 10 amino acid sequence in the sequence listing;

C2. Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in Sequence 10 in the sequence listing has more than 80% identity with the protein shown in C1) and is identical to the protein Proteins associated with body formation;

C3, the fusion protein obtained by linking protein tags at the N-terminal or/and C-terminal of C1) or C2).

In the above method, the sequence 10 in the sequence listing consists of 93 amino acid residues.

In the above methods, identity refers to the identity of amino acid sequences. Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (the default value) and search for the identity of a pair of amino acid sequences for calculation, and then the value (%) of the identity can be obtained.

In the above method, the above 80% identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above method, accumulating the target protein in the plant may be accumulating the target protein in plant protein bodies.

In the above method, the expression of the prolamin can be realized by introducing the gene encoding the prolamin into the recipient plant; the gene encoding the prolamin is as shown in D1 or D2 below Genes:

D1, the coding sequence (ORF) of the coding strand is the DNA molecule of sequence listing sequence 7;

D2. The nucleotide sequence is the DNA molecule shown in sequence 7 in the sequence listing.

In the above method, the expression of the target protein that interacts with the gliadin can be achieved by introducing a gene encoding the target protein into the recipient plant; the gene encoding the target protein is A gene as indicated by E1, E2 or E3 below:

E1, the coding sequence (ORF) of the coding strand is a DNA molecule of sequence 1 or sequence 3 or sequence 5;

E2. The nucleotide sequence is the DNA molecule shown in Sequence 1 or Sequence 3 or Sequence 5 in the sequence table;

E3. A DNA molecule comprising El or E2.

In the above method, the expression of the protein associated with protein body formation can be achieved by introducing into the recipient plant a gene encoding the protein associated with protein body formation; the encoding of the protein associated with protein body formation The gene of the protein is the gene shown in F1 or F2 as follows:

F1, the coding sequence (ORF) of the coding strand is the DNA molecule of sequence number 9;

F2, the nucleotide sequence is the DNA molecule shown in sequence 9 in the sequence table.

In the above method, the gene encoding the protein related to protein body formation is introduced into the recipient plant, preferably the gene encoding the protein related to protein body formation is linked to the gene encoding the prolamin Introduced into recipient plants.

In the above method, the recipient plant is a monocotyledon or a dicotyledon; further, the monocot is a gramineous plant, and the dicotyledon is a solanaceous plant; further, the gramineous plant is corn, and the dicot is tobacco.

The second object of the present invention is to provide the application of the method for accumulating target protein in plants based on the interaction with gliadin in the production of protein,

Or, the application of the above-mentioned prolamin, the above-mentioned protein related to protein body formation and the above-mentioned target protein in accumulating the target protein in plants.

The third object of the present invention is to provide a protein set, which is composed of the above-mentioned prolamin, the above-mentioned protein related to protein body formation and the above-mentioned target protein.

The present invention verifies that target proteins NZP1, NZP2 and NZP3 interact with gliadin 22-kDα-zein respectively through yeast two-hybrid system or LCI system. The accumulation of NZP1 in protein bodies was demonstrated by immunoelectron microscopy of maize kernel protein bodies, and the accumulation of NZP1 was significantly decreased in the 22kD gliadin-deficient mutant material. Further, by expressing Zera-22 (the fusion protein of Zera and 22-kDa α-zein) and target protein NZP1, NZP2 or NZP3 in tobacco, and the experiment of expressing Zera and target protein NZP1, NZP2 or NZP3 in tobacco, the result It shows that Zera-22 induces protein body formation in tobacco, and the target protein is localized in the protein body formed by Zera-22, which proves that the target proteins NZP1, NZP2 and NZP3 can accumulate in Zera-22 through the interaction with 22-kDα-zein in the formed protein bodies. Therefore, the method of the present invention can realize the accumulation of the target protein in plants based on the interaction with gliadin, and then be applied to protein production.

Description of drawings

Fig. 1 is the result diagram of the interaction between NZP1 and 22-kDα-zein in the present invention; wherein Fig. A of Fig. 1 is the result diagram of the interaction of Example 1 in yeast, and Fig. B of Fig. 1 is the diagram of the interaction of Example 2 in the LCI system The resulting graph of the interaction.

Fig. 2 is the result diagram of the interaction between NZP2 and NZP3 and 22-kDα-zein respectively in the present invention; wherein, Fig. A of Fig. 2 is the result diagram of the interaction of Example 1 in yeast, and Fig. B of Fig. 2 is the result of the interaction of Example 2 Graph of the results of the interaction in the LCI system.

Fig. 3 is a graph showing the detection results of NZP1 content in different mutant materials in Example 3 of the present invention.

Fig. 4 is the confirmation of NZP1 cell localization in Example 4 of the present invention. Among them, Figure A of Figure 4 is the separation of protein bodies by density gradient centrifugation, and Figure B of Figure 4 is the accumulation of NZP1 in protein bodies detected by Western blot.

Fig. 5 shows the localization of NZP1 in protein bodies observed by immunoelectron microscopy in Example 4 of the present invention.

Figure 6 is a vector construction diagram of Example 5 of the present invention, wherein Figure A in Figure 6 is a map of the pHB vector, and Figure B in Figure 6 is a schematic diagram of fusion gene construction.

Fig. 7 is the observation result of fluorescence signal after Zera-22-mChery transformed tobacco in Example 5 of the present invention.

Fig. 8 is the observation result of fluorescent signals co-expressed with Zera-22-mCherry and Zera-mCherry respectively in Example 5 of the present invention. Among them, Figure A of Figure 8 is the observation result of fluorescence signal after Zera-22-mCherry and NZP1-GFP co-transform tobacco; Figure B of Figure 8 is the observation result of fluorescence signal after Zera-mCherry and NZP1-GFP co-transform tobacco.

Fig. 9 is the result of Western blot detection of co-expression of NZP1 with Zera-22-mCherry and Zera-mCherry respectively in Example 5 of the present invention. 9 shows the result of detecting Zera-22-mCherry and NZP1 protein content by western blotting of the total protein of transgenic tobacco co-expressed with Zera-22-mCherry and NZP1. Panel B of Figure 9 shows the results of detecting the protein content of Zera-mCherry and NZP1 by Western blotting of the total protein of transgenic tobacco co-expressed with Zera-mCherry and NZP1.

Fig. 10 is the observation result of fluorescent signals co-expressed with Zera-22-mCherry and Zera-mCherry respectively in Example 5 of the present invention. Among them, Figure A of Figure 10 is the observation result of fluorescence signal after Zera-22-mCherry and NZP2-GFP co-transform tobacco; Figure B of Figure 10 is the observation result of fluorescence signal after Zera-mCherry and NZP2-GFP co-transform tobacco.

Fig. 11 is the observation result of fluorescent signals co-expressed with Zera-22-mCherry and Zera-mCherry respectively in Example 5 of the present invention. Among them, Figure A of Figure 11 is the observation result of fluorescence signal after Zera-22-mCherry and NZP3-GFP co-transform tobacco; Figure B of Figure 11 is the observation result of fluorescence signal after Zera-mCherry and NZP3-GFP co-transform tobacco.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples, unless otherwise specified, are conventional biochemical reagents, which can be obtained from commercial sources.

1. Plant strains and strains

In the following examples, opaque1 mutant (hereinafter abbreviated as o1), opaque2 mutant (hereinafter abbreviated as o2), opaque7 mutant (hereinafter abbreviated as o7), and floury1 mutant (hereinafter abbreviated as fl1) are described in the non-patent literature " YaoD, Qi W, Li X, et al.Maize opaque10 encodes a cereal-specific protein essential for the proper distribution of zeins in endosperm protein bodies[J].PlosGenetics,2016,12(8):e1006270.". fl1, o1, o2 and o7 were all produced by single gene mutation (fl1 mutant gene is GRMZM2G094532, o1 mutant gene is GRMZM2G449909, o2 mutant gene is GRMZM2G016150, o7 mutant gene is GRMZM2G074759), and the mutants have normal grain development. After the grains matured, the mutant grains appeared silty and opaque, but the grains could germinate normally. The wild-type materials corresponding to the o1 mutant, o2 mutant and o7 mutant are maize inbred line W22, and the wild-type materials corresponding to the fl1 mutant are maize inbred line B73. The above-mentioned mutant materials can be obtained from China Agricultural University by the public to repeat the experiment of this application, and cannot be used for other purposes.

The Agrobacterium agrobacterium GV3101 in the following examples is a product of Shanghai Weidi Biology, and the article number is AC1001S.

2. Carrier

The pGBKT7 and pGADT7 in the following examples come from the Matchmarker Gold Yeast-Two hybridsystem Yeast Two-Hybrid Kit, which is a product of Clontech Company, the catalog number is 630442.

The p19 vector in the following examples is described in the non-patent literature GoldenBraid 2.0: a comprehensiveDNA assembly framework for plant synthetic biology. Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. Plant Physiol. 2013 Jul;162(3):1618-31.10.1104/pp.113.217661, named pDGB3alpha2_35S:P19:Tnos. The public can obtain it from China Agricultural University to repeat the experiment of this application, and it cannot be used for other purposes.

The CLUC and NLUC carriers in the following examples are described in the non-patent literature "Zhang Z, Yang J, WuY. Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque2, Prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins[ J].Plant Cell,2015,27(4):1162.", the public can obtain it from China Agricultural University to repeat the experiment of this application, and it cannot be used for other purposes.

In the following examples, pHB is recorded in the non-patent literature "Mao J, Zhang YC, Sang Y, Li QH, YangHQ. From The Cover: A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening. Proc Natl Acad Sci U S A .2005;102(34):12270-12275.doi:10.1073/pnas.0501011102 "Supporting Methods and Materials", the public can obtain from China Agricultural University to repeat the experiment of this application, and cannot be used for other purposes.

3. Antibodies and enzymes

The 27-kDγ-zein antibody, 22-kDα-zein antibody, 16-kDγ-zein antibody and 19-kDα-zein antibody in the following examples are described in the non-patent literature "Yao D, Qi W, Li X, et al .Maize opaque10 Encodes a Cereal-Specific Protein That Is Essential for the Proper Distribution of Zeins in Endosperm Protein Bodies[J].Plos Genetics,2016,12(8):e1006270.", available from China Agricultural University, to repeat the experiment for this application , not for other purposes.

Colloidal gold-labeled goat anti-rabbit IgG (D17537) is a product of Beijing Wokai Biotechnology Co., Ltd., and the NZP1 antibody is produced by ABclonal Company, item number WG-01036D.

The polymerase used in the following examples is Phanta high-fidelity DNA polymerase, which is a product of Nanjing Novizan Biotechnology Co., Ltd., and the article number is P511-01.

4. Reagents

The Tiangen polyphenol polysaccharide total RNA extraction kit in the following examples is a product of Tiangen Biotechnology Company, and the article number is DP360.

The seamless cloning kit in the following examples is a product of ABclonal Company, Cat. No. RK21020.

Yeast-Leu/-Trp solid medium (product number 630317), yeast-Leu/-Trp liquid medium (product number 630316), yeast-Ade/-His/-Leu/-Trp solid culture in the following examples The base (article number is 630323) is the product of Clontech Company.

The LUC fluorescein substrate in the following examples is a product of Promega Company, the product number is P1041.

In the following examples, the ABclonal multi-fragment recombination kit was purchased from Wuhan Aibotec Biotechnology Company, the product number is RK21020.

In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The quantitative tests in the following examples, unless otherwise specified, were repeated three times, and the results were averaged. * indicates that the significant analysis result is P<0.05.

Example 1, NZP1, NZP2 and NZP3 and 22-kDα-zein interaction verification in yeast system

1. Construction of pGBKT7-NZP1, pGBKT7-NZP2, pGBKT7-NZP3 and pGADT7-22-kDα-zein vectors

The total RNA of immature grains of maize B73 inbred line was extracted with Tiangen polyphenol polysaccharide total RNA extraction kit, and the cDNA of immature grains of maize inbred line B73 was obtained by reverse transcription.

1.1. Construction of pGBKT7-NZP1 vector

The coding sequence of the NZP1 gene is shown in sequence listing sequence 1, and its encoded protein is NZP1 (sequence listing sequence 2). The 5' end primer NZP1-F and the 3' end primer NZP1-R were designed according to the coding sequence of the NZP1 gene:

NZP1-F: 5'-CTGCATATGGCCATGGAGGCCGAATTCCTGGTCCGGCAGGAGGCACTGCGGC-3';

NZP1-R: 5'-ATGCGGCCGCTGCAGGTCGACGGATCCTCACTTCTCAGCCGGCGCGGCCG-3'.

A primer pair consisting of NZP1-F and NZP1-R was used to amplify the coding DNA fragment of NZP1 gene from cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the EcoRI restriction site of the pGBKT7 vector with the above-mentioned amplified fragment (including the BamHI recognition site and the EcoRI enzyme recognition site) small fragment), keeping other sequences of the pGBKT7 vector unchanged, a recombinant expression vector of the NZP1 protein was obtained, named pGBKT7-NZP1.

1.2. Construction of pGBKT7-NZP2 vector

The coding sequence of the NZP2 gene is shown in sequence listing sequence 3, and its encoded protein is NZP2 (sequence listing sequence 4). The 5' end primer NZP2-F and the 3' end primer NZP2-R were designed according to the coding sequence of the NZP2 gene:

NZP2-F: 5'-CTGCATATGGCCATGGAGGCCGAATTCTGTACCGGAGCGGCGGCGGCGAAGC-3';

NZP2-R: 5'-ATGCGGCCGCTGCAGGTCGACGGATCCCTAGTCGCGGTTAATGAAGGCGA-3'.

A primer pair consisting of NZP2-F and NZP2-R was used to amplify the coding DNA fragment of NZP2 gene from cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the EcoRI restriction site of the pGBKT7 vector with the above-mentioned amplified fragment (including the BamHI recognition site and the EcoRI enzyme recognition site) small fragment) and keep other sequences of the pGBKT7 vector unchanged to obtain a recombinant expression vector of the NZP2 protein, named pGBKT7-NZP2.

1.3. Construction of pGBKT7-NZP3 vector

The coding sequence of the NZP3 gene is shown in sequence listing sequence 5, and its encoded protein is NZP3 (sequence listing sequence 6). The 5' end primer NZP3-F and the 3' end primer NZP3-R were designed according to the coding sequence of the NZP3 gene:

NZP3-F: 5'-CTGCATATGGCCATGGAGGCCGAATTCTTGCGCCCGGCCGAGGGCATCCGCT-3';

NZP3-R: 5'-ATGCGGCCGCTGCAGGTCGACGGATCCTTAAACCCGAGACGTCCCCAGCT-3'.

A primer pair consisting of NZP3-F and NZP3-R was used to amplify the DNA fragment of NZP3 gene from cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the EcoRI restriction site of the pGBKT7 vector with the above-mentioned amplified fragment (including the BamHI recognition site and the EcoRI enzyme recognition site) small fragment) and keep other sequences of the pGBKT7 vector unchanged to obtain a recombinant expression vector of NZP3 protein, named pGBKT7-NZP3.

1.4. Construction of pGADT7-22-kDα-zein vector

The coding sequence of the 22-kDα-zein gene is shown in sequence listing sequence 7, and the encoded protein is 22-kDα-zein (sequence listing sequence 8). According to the coding sequence of the 22-kDα-zein gene, the 5' end primer 22-kDα-zein-F and the 3' end primer 22-kDα-zein-R were designed:

22-kDα-zein-F: 5'-CATATGGCCATGGAGGCCAGTGAATTCTTCATTATTCCACAATGCTCACTTGCTCCT-3';

22-kDa-zein-R: 5'-CATCTGCAGCTCGAGCTCGATGGATCCCTAAAAGATGGCACCTCCAACGATCG-3'.

A primer pair consisting of 22-kDα-zein-F at the 5' end and 22-kDα-zein-R at the 3' end was used to amplify the coding of 22-kDα-zein gene from cDNA of immature kernels of maize B73 inbred line DNA fragments.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and EcoRI restriction sites of the pGADT7 vector (including the BamHI recognition site and the EcoRI enzyme recognition site) with the above-mentioned amplified fragment small fragment), keeping other sequences of the pGADT7 vector unchanged, a recombinant expression vector of 22-kDα-zein protein was obtained, named pGADT7-22-kDα-zein.

2. Interaction between NZP1, NZP2 and NZP3 and 22-kDα-zein in yeast system

2.1. Verification of interaction between NZP1 and 22-kDα-zein in yeast system

Co-transform yeast-competent AH109 strain with pGBKT7-NZP1 constructed in step 1.1 and pGADT7-22-kDα-zein constructed in step 1.4, spread on yeast-Leu/-Trp solid medium, and culture at 30°C for 3-5 days , Pick a single clone and culture it in yeast-Leu/-Trp liquid medium. The culture condition is ₃₀ °C, 220rpm. On Trp solid medium and yeast-Ade/-His/-Leu/-Trp solid medium, the interaction was detected. The results are shown in panel A of Figure 1, indicating that NZP1 interacts with 22-kDα-zein in the yeast system.

2.2. Verification of interaction between NZP2 and 22-kDα-zein in yeast system

Replace pGBKT7-NZP1 in step 2.1 with pGBKT7-NZP2 constructed in step 1.2 and pGADT7-22-kDα-zein constructed in step 1.4 to co-transform yeast-competent AH109 strain, keep other conditions in step 2.1 unchanged, detect the interaction, and the result As shown in panel A of Figure 2, it was shown that NZP2 interacted with 22-kDα-zein in the yeast system.

2.3. Verification of interaction between NZP3 and 22-kDα-zein in yeast system

Replace pGBKT7-NZP1 in step 2.1 with pGBKT7-NZP3 constructed in step 1.3 and pGADT7-22-kDα-zein constructed in step 1.4 to co-transform yeast-competent AH109 strain, keep other conditions in step 2.1 unchanged, detect the interaction, and the result As shown in panel A of Figure 2, it was shown that NZP3 interacted with 22-kDα-zein in the yeast system.

Example 2, Verification of the interaction between NZP1, NZP2 and NZP3 and 22-kDα-zein in the LCI system

The total RNA of immature grains of maize B73 inbred line was extracted with Tiangen polyphenol polysaccharide total RNA extraction kit, and the cDNA of immature grains of maize inbred line B73 was obtained by reverse transcription.

1. Construction of CLUC-NZP1, CLUC-NZP2, CLUC-NZP3 and 22-kDα-zein-NLUC vectors

1.1. Construction of CLUC-NZP1 vector

The coding sequence of the NZP1 gene is shown in sequence listing sequence 1, and its encoded protein is NZP1 (sequence listing sequence 2). Design the 5' end primer NZP1-F' and the 3' end primer NZP1-R' according to the coding sequence of the NZP1 gene:

NZP1-F': 5'-ACGAGCTCGGTACCCGGGATCCATGACGTCCGTGCGCAGCTGTGCCG-3';

NZP1-R': 5'-GGACGCGTACGAGATCTGGTCGACTCACTTCTCAGCCGGCGCGGCCG-3'.

A primer pair consisting of NZP1-F' and NZP1-R' was used to amplify the coding DNA fragment of NZP1 gene from the cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the SalI restriction site of the CLUC vector with the above-mentioned amplified fragment (including the recognition site of BamHI and the SalI enzyme recognition site) small fragment), keeping the other sequences of the CLUC vector unchanged, the recombinant expression vector of NZP1 protein was obtained, which was named as CLUC-NZP1.

1.2. Construction of CLUC-NZP2 vector

The coding sequence of the NZP2 gene is shown in sequence listing sequence 3, and its encoded protein is NZP2 (sequence listing sequence 4). According to the coding sequence of the NZP2 gene, the 5' end primer NZP2-F' and the 3' end primer NZP2-R' were designed:

NZP2-F': 5'-ACGAGCTCGGTACCCGGGATCCATGGTGGCCTCGCGGATCTTGCTCC-3';

NZP2-R': 5'-GGACGCGTACGAGATCTGGTCGACCTAGTCGCGGTTAATGAAGGCGA-3'.

A primer pair consisting of NZP2-F' and NZP2-R' was used to amplify the coding DNA fragment of NZP2 gene from the cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the SalI restriction site of the CLUC vector with the above-mentioned amplified fragment (including the recognition site of BamHI and the SalI enzyme recognition site) small fragment), keeping other sequences of the CLUC vector unchanged, the recombinant expression vector of NZP2 protein was obtained, named CLUC-NZP2.

1.3. Construction of CLUC-NZP3 vector

The coding sequence of the NZP3 gene is shown in sequence listing sequence 5, and its encoded protein is NZP3 (sequence listing sequence 6). Design the 5' end primer NZP3-F' and the 3' end primer NZP3-R' according to the coding sequence of the NZP3 gene:

NZP3-F': 5'-ACGAGCTCGGTACCCGGGATCCATGGAGGGGCTGAGCTGGAGAACGG-3';

NZP3-R': 5'-GGACGCGTACGAGATCTGGTCGACTTAAACCCGAGACGTCCCCAGCT-3'.

A primer pair consisting of NZP3-F' and NZP3-R' was used to amplify the coding DNA fragment of NZP3 gene from the cDNA of immature kernels of maize B73 inbred line.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease BamHI and the SalI restriction site of the CLUC vector with the above-mentioned amplified fragment (including the recognition site of BamHI and the SalI enzyme recognition site) small fragment), keeping the other sequences of the CLUC vector unchanged, the recombinant expression vector of NZP3 protein was obtained, which was named as CLUC-NZP3.

1.4. Construction of 22-kDα-zein-NLUC vector

The coding sequence of the 22-kDα-zein gene is shown in sequence listing sequence 7, and the encoded protein is 22-kDα-zein (sequence listing sequence 8). Design the 5' primer 22-kDα-zein-F' and the 3' primer 22-kDα-zein-R' according to the coding sequence of the 22-kDα-zein gene:

22-kDα-zein-F': 5'-CTCGTACGCGTCCCGGGGCGGTACCATGGCTACCAAGATATTATTCCC-3';

22-kDα-zein-R': 5'-ACGAACGAAAGCTCTGCAGGTCGACAAAGATGGCACCTCCAACGATCG-3'.

Using the seamless cloning kit, replace the fragment between the restriction endonuclease KpnI and the SalI restriction site of the NLUC vector with the above-mentioned amplified fragment (including the recognition site of KpnI and the SalI enzyme recognition site) small fragment), keeping other sequences of the NLUC vector unchanged, a recombinant expression vector of 22-kDα-zein protein was obtained, which was named 22-kDα-zein-NLUC.

2. NZP1, NZP2 and NZP3 interact with 22-kDα-zein in the LCI system for verification

Tobacco seeds (Nicotiana benthamiana) were sterilized with 70% alcohol, planted in a petri dish containing MS+8g/L agar medium, and cultivated in an incubator. The temperature is 25±1°C, the humidity is 70%, and the light cycle is 16 hours light/8 hours dark. After the seeds germinate, transplant the germinated plants into nutrient soil, keep the cultivation temperature, humidity and light conditions unchanged, and after 3-4 weeks of cultivation, when the leaves grow to 3-4 true leaves, the leaves can be used for cultivation. Instant transformation.

2.1, NZP1 and 22-kDα-zein interaction verification in the LCI system

The p19 vector was transformed into Agrobacterium GV3101 by heat shock method to obtain GV3101 containing p19, which was named p19-GV3101, and cultured to obtain p19-GV3101 suspension with OD ₆₀₀ =0.5.

The CLUC-NZP1 constructed in step 1.1 was transformed into Agrobacterium agrobacterium GV3101 by heat shock method to obtain GV3101 containing CLUC-NZP1, which was named CLUC-NZP1-GV3101, and cultured to obtain a CLUC-NZP1-GV3101 suspension with OD ₆₀₀ =0.5. The 22-kDα-zein-NLUC constructed in step 1.4 was transformed into Agrobacterium agrobacterium GV3101 by the heat shock method to obtain GV3101 containing 22-kDα-zein-NLUC, named 22-kDα-zein-NLUC-GV3101, and cultured to obtain OD ₆₀₀ = 0.5 of the 22-kDa-zein-NLUC-GV3101 suspension.

Mix the p19-GV3101 suspension, CLUC-NZP1-GV3101 suspension, and 22-kDα-zein-NLUC-GV3101 suspension at a volume ratio of 0.5:1:1, and inject them into the leaves from the back of the tobacco using a disposable syringe for infection , 25±1° C., 70% humidity and dark culture for 8 hours, and then cultured for 24-48 hours under a cycle of light of 16 hours of light/8 hours of darkness. LUC fluorescein substrate (Promega, 10 mM) was injected into the infected area, and Tanon-5200 image system was used for observation. The results are shown in panel B of Fig. 1, indicating that NZP1 interacts with 22-kDα-zein in the LCI system.

2.2. Verification of the interaction between NZP2 and 22-kDα-zein in the LCI system

Replace the CLUC-NZP1 in step 2.1 with the CLUC-NZP2 constructed in step 1.2, keep the other conditions of step 2.1 unchanged, and detect the interaction. interact in the system.

2.3. Verification of the interaction between NZP3 and 22-kDα-zein in the LCI system

Replace the CLUC-NZP1 in step 2.1 with the CLUC-NZP3 constructed in step 1.3, keep the other conditions in step 2.1 unchanged, and detect the interaction. interact in the system.

Embodiment 3 detects the content of NZP1 of different mutants

The o1 mutant, fl1 mutant, o2 mutant, o7 mutant and corresponding wild type were used as experimental materials.

The total protein of wild-type grain, fl1 mutant, o2 mutant, o7 mutant grain endosperm was extracted respectively, and detected by western blot using 22-kDα-zein antibody, 16-kDγ-zein antibody, 19-kDα-zein antibody and NZP1 antibody .

The results are shown in Figure 3, in the o2 mutant, 19-kDα-zein and 22-kDα-zein decreased significantly, and the content of NZP1 also decreased significantly. In the o7 mutant, the contents of 19-kDα-zein and 16-kDα-zein decreased significantly, while the content of NZP1 did not change significantly. It shows that the content of NZP1 is related to the content of 22-kDaα-zein.

Example 4 confirms the cellular localization of NZP1

1. Density gradient centrifugation and immunoblotting confirmation

The endosperm part of the grain of the corn inbred line B73 20 days after self-pollination was taken and extracted with the extract.

Extract solution: 10 mM Tris-cl, 10 mM MgCl ₂ , 5 mM KCl, 10 mM PMSF, 1 mM DTT, 7.2% sucrose.

The discontinuous density gradient of sucrose laid from the bottom to the top of the centrifuge tube is: 0.6M, 0.9M, 1.2M, 1.45M and 1.8M sucrose, centrifuged at 36900rpm, 4°C, 1h 30min, to separate different cell components (Figure 4 A figure), obtain L1 (the centrifugal product of 0M/0.6M), L2 (the centrifugal product of 0.6M/0.9M), L3 (the centrifugal product of 0.9M/1.2M), L4 (the centrifugal product of 1.2M/1.45M ), L5 (centrifuged product of 1.45M/1.8M), a total of 5 cell fractions.

The content of protein bodies in each fraction was detected by 22-kDα-zein antibody alert immunoblotting.

NZP1 antibody was used to detect the content of NZP1 in each fraction by immunoblotting.

The results are shown in B of FIG. 4 , in the fraction of the protein body (the protein body is mainly located between the 1.45M/1.8M gradient, ie the L5 fraction), NZP1 was significantly accumulated.

2. Immunoelectron microscope confirmation

Obtain immature grains of corn B73 15 days after self-pollination, make ultrathin sections, incubate with NZP1 primary antibody (ie NZP1 antibody) at 4°C for about 12-24 hours, then incubate at room temperature for 2 hours, colloidal gold-labeled rabbit anti-goat IgG secondary antibody Dilute with PBS at a ratio of 1:10 and incubate at room temperature for 20-30min.

As shown in Figure 5, it was confirmed that NZP1 was distributed in protein bodies by immunoelectron microscopy.

Example 5 Agrobacterium-mediated tobacco transient transformation and fluorescence observation

Firstly, the coding sequence of the NZP1 gene was respectively obtained from the website of MaizeGDB, as shown in Sequence Listing 1, and its encoded protein is NZP1 (Sequence Listing 2). The coding sequence of the NZP2 gene is shown in sequence listing sequence 3, and its encoded protein is NZP2 (sequence listing sequence 4). The coding sequence of the NZP3 gene is shown in sequence listing sequence 5, and its encoded protein is NZP3 (sequence listing sequence 6). The coding sequence of the 22-kDα-zein gene is shown in sequence listing sequence 7, and the encoded protein is 22-kDα-zein (sequence listing sequence 8). The coding sequence of the Zera gene is sequence listing sequence 9, and its encoded protein sequence is Zera (sequence listing sequence 10). Similarly, the coding sequence of the mCherry gene obtained from GenBank of the NCBI website is shown in Sequence Listing No. 11, and the amino acid sequence of the encoded protein is shown in Sequence Listing No. 12. The gene coding sequence of GFP is shown in sequence number 13 of the sequence listing, and the amino acid sequence of GFP protein is shown in sequence number 14 of the sequence listing.

The immature grains of the corn B73 inbred line were obtained 15 days after pollination, and the total RNA was extracted using the Tiangen polyphenol polysaccharide sample total RNA extraction kit, and the quality of the total RNA extraction was detected by agarose gel electrophoresis. The total RNA was reverse-transcribed using the Full Type Gold One-step cDNA Reverse Transcription Kit to obtain the cDNA of the immature kernels of the maize B73 inbred line.

1. Construction of recombinant expression vector

1.1. Construction of pHB-mCHerry vector

According to the coding sequence (sequence 11 of the sequence listing) of mCherry gene, primers are designed as follows:

mCHerry-F: 5'-TCGAGCTGCAGGAGCTCATGGTGAGCA AGGGCGAGGAGGAT-3';

mCHerry-R: 5'-TCTAGAGGATCAATTCGAGCTCCTTGTACAGCTCGTCCATG-3'.

A primer pair consisting of mCHerry-F and mCHerry-R was used to amplify the coding DNA fragment of mCHerry gene from the cDNA of immature kernels of maize B73 inbred line.

Replace the fragment (including the SacI enzyme recognition site) of the restriction endonuclease SacI restriction endonuclease SacI restriction site of the pHB carrier (the graph of the pHB carrier is shown in Fig. 6 A figure) with the fragment obtained by the above-mentioned amplification, and keep the pHB carrier The other sequences of the mCHerry were kept unchanged, and the recombinant expression vector of mCHerry was obtained, which was named pHB-mCHerry.

1.2. Construction of pHB-GFP vector

According to the gene coding sequence (sequence listing sequence 13) of GFP, primers are designed as follows:

GFP-F: 5'-TCGAGCTGCAGGAGCTCGTGAGCAAGGGCGAGGAGC-3';

GFP-R: 5'-TCTAGAGGATCAATTCGAGCTCTTACTTGTACAGCTCGTCCA-3'.

A primer pair consisting of GFP-F and GFP-R was used to amplify the coding sequence of GFP gene from cDNA of immature kernels of maize B73 inbred line.

Replace the fragment (including the SacI enzyme recognition site) of the restriction endonuclease SacI enzyme of the pHB carrier (the collection of graphs of the pHB carrier as shown in Fig. 6 A figure) with the fragment obtained by the above-mentioned amplification, keep the pHB carrier Other sequences remained unchanged, and the recombinant expression vector of GFP was obtained, which was named pHB-GFP.

1.3. Construction of pHB-Zera-mCherry and pHB-Zera-22-mCherry vectors

According to the coding sequence (sequence listing sequence 9) of Zera gene, primers are designed as follows:

Zera-F: 5'-TCTCTCTCAAGCTGGATCCATGAGGGTGTTGCTCGTTGCC-3';

Zera-R: 5'-CGCCCTTGCTCACCATGAGCTCCTGAGGCCGGGGCGG-3'.

A primer pair composed of Zera-F and Zera-R was used to amplify the coding DNA fragment of Zera gene from the cDNA of immature kernels of maize B73 inbred line, and used to construct the pHB-Zera-mCherry vector.

According to the coding sequence (sequence listing sequence 9) of Zera gene, primers are designed as follows:

Zera(22)-F: 5'-TCTCTCTCAAGCTGGATCCATGAGGGTGTTGCTCGTTGCC-3';

Zera(22)-R: 5'-TCGCAAAAAGCGCAAGAAGCTGAGGCCGGGGCGG-3';

A primer pair consisting of Zera(22)-F and Zera(22)-R was used to amplify the coding DNA fragment of the Zera gene from the cDNA of the immature kernel of the maize B73 inbred line for the construction of the pHB-Zera-22-mCherry vector .

According to the coding sequence (sequence listing sequence 7) of the 22-kDa-zein gene, the primers are designed as follows:

22-kDα-zein-F": 5'-CCGCCCCGGCCTCAGCTTCTTGCGCTTTTTGCGA-3';

22-kDα-zein-R": 5'-TCGCCCTTGCTCACCATGAGCTCAAAGATGGCACCTCCAACGATG-3'.

A primer pair consisting of 22-kDα-zein-F” and 22-kDα-zein-R” was used to amplify the coding DNA fragment of the 22-kDα-zein gene from the cDNA of immature kernels of the maize B73 inbred line for the construction of pHB-Zera-22-mCherry vector. .

1.3.1. Construction of pHB-Zera-mCherry vector

According to the order of the first fusion gene counted from top to bottom shown in Figure B in Figure 6, clone Zera onto the pHB-mCHerry vector constructed in step 1.1: use the ABclonal multi-fragment recombination kit to pHB-mCherry As a backbone, replace the fragment between the multiple cloning site BamHI restriction site and the SacI restriction site of the pHB-mCherry vector with the coding DNA fragment of the Zera gene amplified above (including the recognition site of the BamHI enzyme and SacI A small fragment including the enzyme recognition site), keep other sequences of the pHB-mCherry vector unchanged, obtain the recombinant expression vector of the fusion protein of Zera-mCherry, and name it as pHB-Zera-mCherry.

1.3.2. Construction of pHB-Zera-22-mCherry vector

According to the sequence of the second fusion gene counted from top to bottom shown in Figure B in Figure 6, use fusion PCR to connect the coding DNA fragment of the Zera gene obtained by the above amplification and the 22- The coding DNA fragment of the kDα-zein gene was obtained to obtain the coding DNA fragment of the Zera and 22-kDα-zein fusion protein gene, and clone it into the pHB-mCHerry vector constructed in step 1.1: use the ABclonal multi-fragment recombination kit to pHB-mCherry is the backbone, and the DNA fragment encoding the fusion protein gene amplified above is replaced with the fragment between the multi-cloning site BamHI restriction site and the SacI restriction site of the pHB-mCherry vector (including recognition of the BamHI enzyme site and SacI enzyme recognition site), keep the other sequences of the pHB-mCherry carrier unchanged, obtain the recombinant expression vector of the fusion protein of Zera-22-mCherry, and name it as pHB-Zera-22-mCherry .

1.4. Construction of pHB-NZP1-GFP, pHB-NZP2-GFP and pHB-NZP3-GFP vectors

1.4.1. Construction of pHB-NZP1-GFP

According to the coding sequence (sequence listing sequence 1) of NZP1 gene, primers are designed as follows:

NZP1-F": 5'-TCTCTCTCAAGCTGGATCCATGACGTCCGTGCGCAGCTGTGCCG-3';

NZP1-R": 5'-CTCGCCCTTGCTCACGAGCTCCTTCTCAGCCGGCGCGGCCG-3'.

A primer pair consisting of NZP1-F" and NZP1-R" was used to amplify the coding DNA fragment of NZP1 gene from cDNA of immature kernels of maize B73 inbred line.

According to the order of the third fusion gene from top to bottom shown in Figure 6, B, clone NZP1 onto the pHB-GFP vector constructed in step 1.2: use the ABclonal multi-fragment recombination kit to pHB-GFP The carrier is the backbone, and the above-mentioned amplified fragment is replaced with the fragment between the restriction endonuclease BamHI restriction site and the SacI restriction site of the pHB-GFP carrier (comprising the recognition site of the BamHI enzyme and the SacI enzyme recognition site A small fragment within), and named pHB-NZP1-GFP.

1.4.2. Construction of pHB-NZP2-GFP

According to the coding sequence (sequence listing sequence 3) of NZP2 gene, primers are designed as follows:

NZP2-F": 5'-TCTCTCTCAAGCTGGATCCATGGTGGCCTCGCGGATCTTGCTCC-3';

NZP2-R": 5'-CTCGCCCTTGCTCACGAGCTCGTCGCGGTTAATGAAGGCGA-3'.

A primer pair consisting of NZP2-F" and NZP2-R" was used to amplify the coding DNA fragment of NZP2 gene from the cDNA of immature kernels of maize B73 inbred line.

According to the sequence of the fourth fusion gene from top to bottom shown in Figure 6, B, clone NZP2 into the pHB-GFP vector constructed in step 1.2: use the ABclonal multi-fragment recombination kit to pHB-GFP The carrier is the backbone, and the above-mentioned amplified fragment is replaced with the fragment between the restriction endonuclease BamHI restriction site and the SacI restriction site of the pHB-GFP carrier (comprising the recognition site of the BamHI enzyme and the SacI enzyme recognition site A small fragment within), and named pHB-NZP2-GFP.

1.4.3. Construction of pHB-NZP3-GFP

According to the coding sequence of NZP3 gene (sequence listing sequence 5) design primer is as follows:

NZP3-F": 5'-TCTCTCTCAAGCTGGATCCATGGAGGGGCTGAGCTGGAGAACGG-3';

NZP3-R": 5'-CTCGCCCTTGCTCACGAGCTCAACCCGAGACGTCCCCAGCT-3'.

A primer pair consisting of NZP3-F" and NZP3-R" was used to amplify the coding DNA fragment of NZP3 gene from the cDNA of immature kernels of maize B73 inbred line.

According to the sequence of the fifth fusion gene from top to bottom shown in Figure 6, B, clone NZP3 onto the pHB-GFP vector constructed in step 1.2: use the ABclonal multi-fragment recombination kit to pHB-GFP The carrier is the backbone, and the above-mentioned amplified fragment is replaced with the fragment between the restriction endonuclease BamHI restriction site and the SacI restriction site of the pHB-GFP carrier (comprising the recognition site of the BamHI enzyme and the SacI enzyme recognition site A small fragment within), and named pHB-NZP3-GFP.

2. Gene expression of transiently transformed tobacco

Tobacco seeds (Nicotiana benthamiana) were sterilized with 70% alcohol, planted in a petri dish containing MS+8g/L agar medium, and cultivated in an incubator. The temperature is 25±1°C, the humidity is 70%, and the light cycle is 16 hours light/8 hours dark. After the seeds germinate, transplant the germinated plants into nutrient soil, keep the cultivation temperature, humidity and light conditions unchanged, and after 3-4 weeks of cultivation, when the leaves grow to 3-4 true leaves, the leaves can be used for cultivation. Instant transformation.

2.1. NZP1 and Zera-22-mCherry and Zera-mCherry transiently transform tobacco genes respectively

2.1.1 Agrobacterium-mediated transient transformation of tobacco

The p19 vector was transformed into Agrobacterium GV3101 by heat shock method to obtain GV3101 containing p19, which was named p19-GV3101, and cultured to obtain p19-GV3101 suspension with OD ₆₀₀ =0.5.

The pHB-Zera-mCherry vector constructed in step 1 was transformed into Agrobacterium GV3101 using the heat shock method to obtain GV3101 containing pHB-Zera-mCherry, which was named pHB-Zera-mCherry-GV3101, and cultured to obtain _pHB- Zera-mCherry-GV3101 suspension.

The pHB-Zera-22-mCherry vector constructed in step 1 was transformed into Agrobacterium GV3101 by heat shock method to obtain GV3101 containing pHB-Zera-22-mCherry, which was named pHB-Zera-22-mCherry-GV3101, and cultivated to obtain OD ₆₀₀ =0.5 pHB-Zera-22-mCherry-GV3101 suspension.

The pHB-NZP1-GFP vector constructed in step 1 was transformed into Agrobacterium GV3101 using the heat shock method to obtain GV3101 containing pHB-NZP1-GFP, which was named pHB-NZP1-GFP-GV3101, and cultured to obtain _pHB- NZP1-GFP-GV3101 suspension.

The p19-GV3101 suspension (marked as p19), pHB-Zera-mCherry-GV3101 suspension (marked as Zera-mCherry), pHB-Zera-22-mCherry-GV3101 suspension (marked as Zera-22-mCherry) and The pHB-NZP1-GFP-GV3101 (marked as NZP1-GFP) suspension was mixed according to the following combinations, and the ratios shown are all volume ratios:

Combination 1: Zera-22-mCherry+p19, the suspension mixing ratio is 1:1;

Combination 2: Zera-22-mCherry+NZP1-GFP+p19, the suspension mixing ratio is 1:1:0.5;

Combination 3: Zera-mCherry+NZP1-GFP+p19, the suspension mixing ratio is listed as 1:1:0.5.

The mixed solution of each combination was injected into the leaves from the back of the tobacco using a disposable syringe to infect, and cultured in the dark at 25±1°C and 70% humidity for 8 hours, and then cultivated under the cycle light of 16 hours of light/8 hours of darkness for 24-48 hours. Hours, fresh transgenic tobacco sheets were obtained. Among them, the infection of combination 1 obtained transgenic tobacco sheets expressing Zera-22-mCherry alone; the infection of combination 2 obtained transgenic tobacco sheets co-expressing Zera-mCherry and NZP1; the infection of combination 3 obtained Zera-22 - Transgenic tobacco sheets co-expressing mCherry and NZP1.

2.1.2. Fluorescence observation

Fluorescence observation was performed on the transgenic tobacco slices obtained in step 2.1.1 using GFP expression.

As shown in Figure 7, when Zera-22-mCherry was expressed alone (combination 1), protein body structures could be formed. As shown in Figure 8, when Zera-mCherry and NZP1 were co-expressed (combination 3, see panel B of Figure 8), NZP1 could not accumulate in the protein bodies formed by Zera. When Zera-22-mCherry and NZP1 are co-expressed (combination 2, see Figure 8, panel A), NZP1 can accumulate in the protein bodies formed by Zera, and the fluorescent signals of NZP1-GFP and Zera-22-mCherry are located in the same position, It shows that NZP1 can relocate in the protein body formed by Zera-22.

2.1.3 Western blot detection

Extract the total protein from the transgenic tobacco sheet obtained in step 2.1.1, and quantify the extract containing the total protein using a protein quantification kit. After the protein sample is separated by SDS-PAGE, transfer the protein to PVDF membrane, and then detected with NZP1 antibody and 27-kDγ-zein antibody.

T1-T4 are four different expression events, specifically

As shown in panel A of Fig. 9, when NZP1-GFP was co-expressed with Zera-22-mCherry, there was obvious accumulation of NZP1-GFP.

As shown in panel B of Figure 9, when NZP1-GFP was co-expressed with Zera-mCherry, there was no obvious accumulation of NZP1-GFP.

2.2. NZP2 and Zera-22-mCherry and Zera-mCherry transiently transform tobacco genes respectively

Replace the pHB-NZP1-GFP carrier in step 2.1.1 with the pHB-NZP2-GFP carrier constructed in step 1, keep the other methods of step 2.1.1 unchanged, and obtain the transgenic tobacco sheet co-expressed with Zera-mCherry and NZP2, And Zera-22-mCherry and NZP2 co-express transgenic tobacco sheets. Fluorescence observation was performed on the obtained transgenic tobacco slices using GFP expression.

The results were shown in Figure 10, when Zera-mCherry and NZP2 were co-expressed (see panel B of Figure 10), NZP2 could not accumulate in the protein bodies formed by Zera. When Zera-22-mCherry and NZP2 were co-expressed (see panel A of Figure 10), the fluorescent signals of NZP2-GFP and Zera-22-mCherry were located in the same position, indicating that NZP2 could be relocated in the protein body formed by Zera-22.

2.3. NZP3 and Zera-22-mCherry and Zera-mCherry transiently transform tobacco genes respectively

The pHB-NZP3-GFP carrier constructed in step 1 was replaced with the pHB-NZP1-GFP carrier in step 2.1.1, and the other methods of step 2.1.1 were kept unchanged to obtain a transgenic tobacco sheet co-expressed with Zera-mCherry and NZP3, And Zera-22-mCherry and NZP3 co-express transgenic tobacco sheets. Fluorescence observation was performed on the obtained transgenic tobacco slices using GFP expression.

The results were shown in Figure 11, when Zera-mCherry and NZP3 were co-expressed (see panel B of Figure 11), NZP3 could not accumulate in the protein bodies formed by Zera. When Zera-22-mCherry and NZP3 are co-expressed (see Figure 11, A), the fluorescent signals of NZP3-GFP and Zera-22-mCherry are located in the same position, indicating that NZP3 can be relocated in the protein body formed by Zera-22.

In summary, the present invention verifies that the target proteins NZP1, NZP2 and NZP3 interact with gliadin 22-kDα-zein respectively through yeast two-hybrid system or LCI system. The accumulation of NZP1 in protein bodies was demonstrated by immunoelectron microscopy of maize kernel protein bodies, and the accumulation of NZP1 was significantly decreased in the 22kD gliadin-deficient mutant material. Further, by expressing Zera-22 (the fusion protein of Zera and 22-kDa α-zein) and target protein NZP1, NZP2 or NZP3 in tobacco, and the experiment of expressing Zera and target protein NZP1, NZP2 or NZP3 in tobacco, the result It shows that Zera-22 induces protein body formation in tobacco, and the target protein is localized in the protein body formed by Zera-22, which proves that the target proteins NZP1, NZP2 and NZP3 can accumulate in Zera-22 through the interaction with 22-kDα-zein in the formed protein bodies.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Sequence Listing

<110> China Agricultural University

<120> Method for Accumulating Target Proteins in Plants Based on Interaction with Glamins

<130> GNCSY210379

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 591

<212> DNA

<213> Corn (Zea mays)

<400> 1

atgacgtccg tgcgcagctg tgccgcgtcg ttaaccccgt cgatccgcgg gatgtcgggg 60

ctggtccggc aggaggcact gcggcgcgag ctggacgggt gccagctgct ggcgggcatc 120

tggtgccacg ggttcacggt ggcgcagctg cggagcatcc gcgcgtcgct gcccgacgcg 180

gcgcggctgg tggtggcgaa gaactcggac atggcggcgg cggtggcggg cacccggtgg 240

gaggcgctga ggccgtgcgc gcggggcatg aacgcgtggc tgttcgtgcg ctccgacgag 300

atcccgccgg cgctcaagcc ctaccgcgac ttccagaagg agtggaagct gcagctcaac 360

gacttcaccg gcgccgtcta cgagggacgg ctctacgggc ccgacgactt cgcgcagctc 420

gagaacatgc ccaccagggc gcagtcctac cagtacctcc tcggatgcct gcagatgccc 480

gccgtcaacg tcctcgccgt cctacgggcg cgtcaggagg cactgtccgc ggaggccgac 540

aagccgcccg ccgaggggga gggggaggcg gccgcgccgg ctgagaagtg a 591

<210> 2

<211> 196

<212> PRT

<213> Corn (Zea mays)

<400> 2

Met Thr Ser Val Arg Ser Cys Ala Ala Ser Leu Thr Pro Ser Ile Arg

1 5 10 15

Gly Met Ser Gly Leu Val Arg Gln Glu Ala Leu Arg Arg Glu Leu Asp

20 25 30

Gly Cys Gln Leu Leu Ala Gly Ile Trp Cys His Gly Phe Thr Val Ala

35 40 45

Gln Leu Arg Ser Ile Arg Ala Ser Leu Pro Asp Ala Ala Arg Leu Val

50 55 60

Val Ala Lys Asn Ser Asp Met Ala Ala Ala Val Ala Gly Thr Arg Trp

65 70 75 80

Glu Ala Leu Arg Pro Cys Ala Arg Gly Met Asn Ala Trp Leu Phe Val

85 90 95

Arg Ser Asp Glu Ile Pro Pro Ala Leu Lys Pro Tyr Arg Asp Phe Gln

100 105 110

Lys Glu Trp Lys Leu Gln Leu Asn Asp Phe Thr Gly Ala Val Tyr Glu

115 120 125

Gly Arg Leu Tyr Gly Pro Asp Asp Phe Ala Gln Leu Glu Asn Met Pro

130 135 140

Thr Arg Ala Gln Ser Tyr Gln Tyr Leu Leu Gly Cys Leu Gln Met Pro

145 150 155 160

Ala Val Asn Val Leu Ala Val Leu Arg Ala Arg Gln Glu Ala Leu Ser

165 170 175

Ala Glu Ala Asp Lys Pro Pro Ala Glu Gly Glu Gly Glu Ala Ala Ala

180 185 190

Pro Ala Glu Lys

195

<210> 3

<211> 1041

<212> DNA

<213> Corn (Zea mays)

<400> 3

atggtggcct cgcggatctt gctcccgctg ctgctctccc tcttcctatg ctcgcattgc 60

tgtaccggag cggcggcggc gaagcggcag ctgctgcagg cgcagtcgca ggtgaagttc 120

gacttctccc cgttcctgat cgagtacaag aacgggcgcg tgaagcggct gatgggcacc 180

aacgtggtgt ccgcgtcgtc ggacgcgctg acgggcgtca cctcccgcga cgtgaccatc 240

gacgcttcga cgggcgtcgc cgcgcggctc tacctcccga gcttccgcgc cagcgcccgg 300

gtgcccgtgc tcgtctactt ccacggcggc gcgttcgtgg tggagtcggc gttcacgccc 360

atctaccacg cctacctcaa cacgctggcc gccagggcgg gcgtggtggc cgtgtcggtg 420

aactaccggc tggcgccgga gcacccgctc ccggcggcgt acgacgactc ctgggcggcg 480

ctcaggtggg tgctggcgag cgcggccggg tcggacccgt ggctggccca gtacggcgac 540

ctgttccgcc tgttcctggc cggcgacagc gccggcggca acatcgcgca caacctggca 600

ctgcgcgcgg gggaggaagg cctggacggc ggcgcgcgga tcaagggcgt ggcgctgctg 660

gacccctact tccagggccg gagccccgtg ggcgccgagt ccgcggaccc ggcgtacctc 720

cagtccgcgg cgcgcacctg gagcttcatc tgcgcgggga ggtacccgat caaccacccc 780

tacgcggacc cgctcctgct gccggcctcc tcgtggcagc acctcggcgc ctcccgcgtg 840

ctggtcaccg tgtcggggca ggaccgcctc agcccctggc agcgcgggta ctacgccgcg 900

ctccagggca gcggctggcc cggcgaggcc gagctgtacg agaccccccgg cgagggccac 960

gtctacttcc tcaccaagct tggctcgccg caggcgctcg ccgagatggc caagctcgtc 1020

gccttcatta accgcgacta g 1041

<210> 4

<211> 346

<212> PRT

<213> Corn (Zea mays)

<400> 4

Met Val Ala Ser Arg Ile Leu Leu Pro Leu Leu Leu Ser Leu Phe Leu

1 5 10 15

Cys Ser His Cys Cys Thr Gly Ala Ala Ala Ala Ala Lys Arg Gln Leu Leu

20 25 30

Gln Ala Gln Ser Gln Val Lys Phe Asp Phe Ser Pro Phe Leu Ile Glu

35 40 45

Tyr Lys Asn Gly Arg Val Lys Arg Leu Met Gly Thr Asn Val Val Ser

50 55 60

Ala Ser Ser Asp Ala Leu Thr Gly Val Thr Ser Arg Asp Val Thr Ile

65 70 75 80

Asp Ala Ser Thr Gly Val Ala Ala Arg Leu Tyr Leu Pro Ser Phe Arg

85 90 95

Ala Ser Ala Arg Val Pro Val Leu Val Tyr Phe His Gly Gly Ala Phe

100 105 110

Val Val Glu Ser Ala Phe Thr Pro Ile Tyr His Ala Tyr Leu Asn Thr

115 120 125

Leu Ala Ala Arg Ala Gly Val Val Ala Val Ser Val Asn Tyr Arg Leu

130 135 140

Ala Pro Glu His Pro Leu Pro Ala Ala Tyr Asp Asp Ser Trp Ala Ala

145 150 155 160

Leu Arg Trp Val Leu Ala Ser Ala Ala Gly Ser Asp Pro Trp Leu Ala

165 170 175

Gln Tyr Gly Asp Leu Phe Arg Leu Phe Leu Ala Gly Asp Ser Ala Gly

180 185 190

Gly Asn Ile Ala His Asn Leu Ala Leu Arg Ala Gly Glu Glu Gly Leu

195 200 205

Asp Gly Gly Ala Arg Ile Lys Gly Val Ala Leu Leu Asp Pro Tyr Phe

210 215 220

Gln Gly Arg Ser Pro Val Gly Ala Glu Ser Ala Asp Pro Ala Tyr Leu

225 230 235 240

Gln Ser Ala Ala Arg Thr Trp Ser Phe Ile Cys Ala Gly Arg Tyr Pro

245 250 255

Ile Asn His Pro Tyr Ala Asp Pro Leu Leu Leu Pro Ala Ser Ser Trp

260 265 270

Gln His Leu Gly Ala Ser Arg Val Leu Val Thr Val Ser Gly Gln Asp

275 280 285

Arg Leu Ser Pro Trp Gln Arg Gly Tyr Tyr Ala Ala Leu Gln Gly Ser

290 295 300

Gly Trp Pro Gly Glu Ala Glu Leu Tyr Glu Thr Pro Gly Glu Gly His

305 310 315 320

Val Tyr Phe Leu Thr Lys Leu Gly Ser Pro Gln Ala Leu Ala Glu Met

325 330 335

Ala Lys Leu Val Ala Phe Ile Asn Arg Asp

340 345

<210> 5

<211> 642

<212> DNA

<213> Corn (Zea mays)

<400> 5

atggaggggc tgagctggag aacggtgtgt tgtttatcgg tgctctgtgc cgtgctgttc 60

ttgcgcccgg ccgagggcat ccgcttcgtg atcgataggg aagagtgctt ctcgcataac 120

gtggaatacg aggggatac tgtccatgta tccttcgtcg tcatcaaggc tgacaccccg 180

tggcattaca gcgaggaggg cgtcgatctt gtggttaagg atcctaatgg cgctcaagtc 240

cgtgattccc gagataagac tagtgacaag tttgagttca tagttcagaa gagaggcgtc 300

catcgcttct gcttcacgaa caaatcccca tatcacgaaa cgatagactt cgatgttcat 360

gttggccatt tttcatattt cgagcagcat gccaaagatg agcattttgc accacttttt 420

gagcaaatag gcaagttgga tgaggcactt tacaatattc agttcgaaca gcactggcta 480

gaggcccaga ctgaccgtca agcaatattg aacgagaaca tgagcaggag ggcagtccat 540

aaggcgctct tcgagtcagc ggcgctgatc gccgccagcg tcatccaagt ctacctcctg 600

cgccggctct tcgagcgcaa gctggggacg tctcgggttt aa 642

<210> 6

<211> 213

<212> PRT

<213> Corn (Zea mays)

<400> 6

Met Glu Gly Leu Ser Trp Arg Thr Val Cys Cys Leu Ser Val Leu Cys

1 5 10 15

Ala Val Leu Phe Leu Arg Pro Ala Glu Gly Ile Arg Phe Val Ile Asp

20 25 30

Arg Glu Glu Cys Phe Ser His Asn Val Glu Tyr Glu Gly Asp Thr Val

35 40 45

His Val Ser Phe Val Val Ile Lys Ala Asp Thr Pro Trp His Tyr Ser

50 55 60

Glu Glu Gly Val Asp Leu Val Val Lys Asp Pro Asn Gly Ala Gln Val

65 70 75 80

Arg Asp Ser Arg Asp Lys Thr Ser Asp Lys Phe Glu Phe Ile Val Gln

85 90 95

Lys Arg Gly Val His Arg Phe Cys Phe Thr Asn Lys Ser Pro Tyr His

100 105 110

Glu Thr Ile Asp Phe Asp Val His Val Gly His Phe Ser Tyr Phe Glu

115 120 125

Gln His Ala Lys Asp Glu His Phe Ala Pro Leu Phe Glu Gln Ile Gly

130 135 140

Lys Leu Asp Glu Ala Leu Tyr Asn Ile Gln Phe Glu Gln His Trp Leu

145 150 155 160

Glu Ala Gln Thr Asp Arg Gln Ala Ile Leu Asn Glu Asn Met Ser Arg

165 170 175

Arg Ala Val His Lys Ala Leu Phe Glu Ser Ala Ala Leu Ile Ala Ala

180 185 190

Ser Val Ile Gln Val Tyr Leu Leu Arg Arg Leu Phe Glu Arg Lys Leu

195 200 205

Gly Thr Ser Arg Val

210

<210> 7

<211> 801

<212> DNA

<213> Corn (Zea mays)

<400> 7

atggctacca agatattatc cctccttgcg cttcttgcgc tttttgcgag cgcaacaaat 60

gcgttcatta ttccacaatg ctcacttgct ccaagttcca ttattacaca gttcctccca 120

ccagttactt caatgggctt cgaacaccca gctgtgcaag cctataggct acaacaagca 180

attgcggcga gcgtcttaca acaaccaatt tcccagttgc aacaacaatc cttggcacat 240

ctaacaatac aaaccatcgc aacgcaacag caacaacaat tcctaccagc actgagccac 300

ctagccatgg tgaaccctgc cgcctacttg caacagcagt tgcttgcatc aaacccactt 360

gctctggcaa acgtagttgc aaaccagcca caacaacagc tgcaacagtt tctgccagcg 420

ctcagtcaac tagccatggt gaaccctgcc gcctacctac aacagcaaca actgctttca 480

tctagcccgc tcgctgtggc caatgcacct acatacctgc aacaacaatt gttgcaacag 540

attgtaccag ctctgactca gctagttgtg gcaaaccctg ctgcctactt gcaacagctg 600

cttccattca accaactgac tatgtcgaac tctgctgcgt acctacaaca gcgacaacag 660

ttacttaatc cactagcagt ggctaaccca ttggtcgctg ccttcctaca gcagcaacaa 720

ttgctgccat acaaccagtt ctctttgata aaccctgtct tgtcgaggca gcaacccatc 780

gttggaggtg ccatctttta g 801

<210> 8

<211> 266

<212> PRT

<213> Corn (Zea mays)

<400> 8

Met Ala Thr Lys Ile Leu Ser Leu Leu Ala Leu Leu Ala Leu Phe Ala

1 5 10 15

Ser Ala Thr Asn Ala Phe Ile Ile Pro Gln Cys Ser Leu Ala Pro Ser

20 25 30

Ser Ile Ile Thr Gln Phe Leu Pro Pro Val Thr Ser Met Gly Phe Glu

35 40 45

His Pro Ala Val Gln Ala Tyr Arg Leu Gln Gln Ala Ile Ala Ala Ser

50 55 60

Val Leu Gln Gln Pro Ile Ser Gln Leu Gln Gln Gln Ser Leu Ala His

65 70 75 80

Leu Thr Ile Gln Thr Ile Ala Thr Gln Gln Gln Gln Gln Phe Leu Pro

85 90 95

Ala Leu Ser His Leu Ala Met Val Asn Pro Ala Ala Tyr Leu Gln Gln

100 105 110

Gln Leu Leu Ala Ser Asn Pro Leu Ala Leu Ala Asn Val Val Ala Asn

115 120 125

Gln Pro Gln Gln Gln Leu Gln Gln Phe Leu Pro Ala Leu Ser Gln Leu

130 135 140

Ala Met Val Asn Pro Ala Ala Tyr Leu Gln Gln Gln Gln Leu Leu Ser

145 150 155 160

Ser Ser Pro Leu Ala Val Ala Asn Ala Pro Thr Tyr Leu Gln Gln Gln

165 170 175

Leu Leu Gln Gln Ile Val Pro Ala Leu Thr Gln Leu Val Val Ala Asn

180 185 190

Pro Ala Ala Tyr Leu Gln Gln Leu Leu Pro Phe Asn Gln Leu Thr Met

195 200 205

Ser Asn Ser Ala Ala Tyr Leu Gln Gln Arg Gln Gln Leu Leu Asn Pro

210 215 220

Leu Ala Val Ala Asn Pro Leu Val Ala Ala Phe Leu Gln Gln Gln Gln

225 230 235 240

Leu Leu Pro Tyr Asn Gln Phe Ser Leu Ile Asn Pro Val Leu Ser Arg

245 250 255

Gln Gln Pro Ile Val Gly Gly Ala Ile Phe

260 265

<210> 9

<211> 279

<212> DNA

<213> Corn (Zea mays)

<400> 9

atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg 60

catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120

catctgccac ctccggttca cctgccacct ccggtgcatc tcccaccgcc ggtccacctg 180

ccgccgccgg tccacctgcc accgccggtc catgtgccgc cgccggttca tctgccgccg 240

ccaccatgcc actaccctac tcaaccgccc cggcctcag 279

<210> 10

<211> 93

<212> PRT

<213> Corn (Zea mays)

<400> 10

Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser

1 5 10 15

Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro

20 25 30

Pro Val His Leu Pro Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu

35 40 45

Pro Pro Pro Val His Leu Pro Pro Pro Pro Val His Leu Pro Pro Pro Val

50 55 60

His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro

65 70 75 80

Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln

85 90

<210> 11

<211> 708

<212> DNA

<213> Corn (Zea mays)

<400> 11

atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660

cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaag 708

<210> 12

<211> 236

<212> PRT

<213> Corn (Zea mays)

<400> 12

Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

1 5 10 15

Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

100 105 110

Thr Gln Asp Ser Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 13

<211> 714

<212> DNA

<213> Corn (Zea mays)

<400> 13

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacaccccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caag 714

<210> 14

<211> 238

<212> PRT

<213> Corn (Zea mays)

<400> 14

Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

Claims (7)

1. the method for accumulating target protein in plant based on the interaction with prolamin, it is characterized in that, comprises the steps: S1. Expressing the gliadin in recipient plants; S2. Expressing the target protein that interacts with the prolamin in the recipient plant; The prolamin is a protein of the following A1 or A2: A1, the amino acid sequence is a protein with the amino acid sequence shown in Sequence 8 in the sequence listing; A2. A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1); The target protein is the following B1 or B2 protein: B1. The amino acid sequence is a protein with any amino acid sequence shown in Sequence 2, Sequence 4, or Sequence 6 in the sequence listing; B2. A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of the protein described in B1; The recipient plant is a monocotyledonous plant. 2. The method according to claim 1, further comprising expressing a protein associated with protein body formation in the recipient plant, the protein associated with protein body formation being the following C1 or C2 proteins: C1. The amino acid sequence is a protein with the amino acid sequence shown in Sequence 10 in the sequence listing; C2, the fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of C1). 3. The method according to any one of claims 1-2, characterized in that, the expression of the gliadin is achieved by introducing a gene encoding the gliadin in the recipient plant; The gene encoding the gliadin is the gene shown in D1 or D2 as follows: D1. The coding sequence of the coding strand is the DNA molecule of sequence 7 in the sequence listing; D2. The nucleotide sequence is the DNA molecule shown in sequence 7 in the sequence listing. 4. The method according to claim 3, wherein the expression of the target protein interacting with the prolamin is achieved by introducing a gene encoding the target protein into the recipient plant; The gene encoding the target protein is the gene shown in E1 or E2 as follows: E1. The coding sequence of the coding strand is a DNA molecule of sequence 1 or sequence 3 or sequence 5; E2. The nucleotide sequence is the DNA molecule shown in Sequence 1 or Sequence 3 or Sequence 5 in the sequence listing. 5. The method according to claim 2, wherein the expression of the protein associated with protein body formation is achieved by introducing a gene encoding the protein associated with protein body formation in the recipient plant; wherein , the gene encoding the protein associated with protein body formation is the gene shown in F1 or F2 as follows: F1, the coding sequence of the coding strand is the DNA molecule of sequence 9 in the sequence listing; F2, the nucleotide sequence is the DNA molecule shown in sequence 9 in the sequence table. 6. The method according to claim 5, characterized in that, in the recipient plant, introducing the gene encoding the protein associated with protein body formation is to combine the gene encoding the protein associated with protein body formation with The genes encoding the prolamin are introduced into the recipient plants together after connection. 7. The application of the method for accumulating target protein in plants based on the interaction with gliadin according to any one of claims 1-6 in the production of protein.

Images