CN113683669B

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

Info

Publication number: CN113683669B
Application number: CN202110318298.0A
Authority: CN
Inventors: 宋任涛; 冯阳; 马泽阳
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2023-05-16
Anticipated expiration: 2041-03-25
Also published as: CN113683669A

Abstract

Methods for accumulating a protein of interest in a plant based on interaction with a prolamin are disclosed. The invention verifies that target proteins NZP1, NZP2 and NZP3 respectively interact with prolamin 22-kD alpha-zein, and through experiments of expressing Zera-22 and target proteins NZP1, NZP2 or NZP3 in tobacco and expressing Zera and target proteins NZP1, NZP2 or NZP3 in tobacco, the results show that Zera-22 induces protein body formation in tobacco, and target proteins are positioned in protein bodies formed by Zera-22, and the target proteins NZP1, NZP2 and NZP3 can be accumulated in protein bodies formed by Zera-22 through interaction with 22-kD alpha-zein. Thus, accumulation of the target protein in plants can be achieved based on interaction with prolamin, and thus can be applied to protein production.

Description

Method for accumulating target protein in plant based on interaction with prolamin

Technical Field

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

Background

With the continued expansion of industrial and medical demand for recombinant proteins, transgenic plants are considered a safe, efficient and inexpensive means of production (Ma et al, 2003). Transgenic plants also have the potential to fold and assemble multimeric proteins correctly, without contamination by human pathogens, and to directly take unprocessed or partially processed plant material orally, as compared to traditional expression systems such as yeast, insect and mammalian cell cultures (Fischer et al 2004). Transgenic plants have shown wide application prospects as bioreactors in the production of vaccines, antibodies, and industrial enzymes, etc. over the past three decades, for example, plant platforms are used to produce various antibodies, vaccine antigens, protein allergens, enzymes and enzyme inhibitors, clotting factors, cytokines, and hormones, etc. (Giddings et al, 2000; ma et al, 2005). However, one major problem faced in the production of recombinant proteins using plant platforms is: the recombinant proteins accumulate at inadequate levels in plants (Doran, 2006). Expression of recombinant proteins in heterologous environments is naturally non-stationary and is also subject to the process of protein degradation that is prevalent in cells. Some recombinant proteins have no defined subcellular localization in cells and are likely to enter the intracellular protein degradation pathway (Faye et al 2005).

Studies of subcellular localization of recombinant proteins in plant expression systems have shown that subcellular localization of recombinant proteins in plant cells significantly affects the accumulation level of recombinant proteins in cells. Whereas the natural protein storage structure in plant seeds can provide a barrier to the storage of recombinant proteins (Conley et al, 2011). During the development of seeds of gramineae, a specific location of the endoplasmic reticulum in endosperm cells naturally accumulates a large amount of protein, and the endoplasmic reticulum of these regions bulges outward, forming a specific endoplasmic reticulum-derived compartment called a protein body (Shewry et al, 1995;Herman and Larkins,1999). Among them, protein bodies in corn endosperm cells have been studied intensively. The protein body is an important protein storage cell device in corn endosperm cells, and the protein content in the protein body accounts for about 60% of the total protein content of the cells. And the protein body is highly stable, and even in mature grains and grains stored for many years, the protein stored in the protein body is highly intact. Therefore, the corn protein body is an ideal place for high expression and accumulation of the exogenous protein. In the protein bodies of corn endosperm cells, prolamin is the predominant storage protein, accounting for about 80% of the total storage protein (Shewry et al, 1995;Herman and Larkins,1999;Fuji et al, 2007). Zein is mainly composed of four classes of alpha (19-kD, 22-kD), beta (14-kD), gamma (16-kD, 27-kD and 50-kD) and delta (10-kD, 18-kD), with the highest content of the alpha (19-kD, 22-kD) class of zein (Esen, 1987;Coleman andLarkins,1999;Song andMessing,2002).

In studies of protein formation, 27-kD gamma-zein was found to play an important role in the development of protein bodies (Wu and Messaging, 2010; guo et al, 2013). Inhibition of 27-kD gamma-zein expression in corn endosperm by RNAi technology results in a significant reduction in the number of proteosomes in the cell. It is shown that 27-kD gamma-zein has the effect of promoting proteosome formation. Further studies have found that 27-kD gamma-zein can induce protein body formation not only in corn 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 showed that the 27-kD gamma-zein contains four regions: a. a signal peptide comprising 19 amino acids, b.a region consisting of 8 PPVHL hexapeptide repeats (53 aa), c.a pro-x domain rich in proline (29 aa), d.a hydrophobic C-terminal domain rich in cysteine (111 a). Through investigation of the residence mechanism of 27-kD gamma-zein, it was found that there is no endoplasmic reticulum residence signal consisting of HDEL in the 27-kD gamma-zein sequence, and that both the residence in the endoplasmic reticulum and the induction of protein body formation depend on a structure consisting of regions a, b, c, which in turn is called Zera domain (Torrent et al 2009, llop-Tous et al 2010;Mainieri et al, 2014). Further studies showed that two cysteine residues at the N-terminus of 27-kD gamma-zein are critical for oligomerization of 27-kD gamma-zein, which is considered the first step in protein formation.

In addition to the 27-kD gamma-zein, another important class of prolamin in the protein body is the alpha class of prolamin (19-kD, 22-kD), which is responsible for the filling of the protein body (Holding et al, 2007), the highest content of prolamin in the protein body. By studying alpha-zein, it was found that alpha-zein itself does not have an endoplasmic reticulum retention signal, and that expression of alpha-zein alone in vitro does not induce protein body formation, indicating that alpha-zein does not have the ability to induce protein body formation. Kim et al have found through a series of prolamin interaction studies that 22-kD alpha-zein is capable of interacting with other types of prolamin (Kim et al, 2002). Coleman et al expressed both 27-kD gamma-zein and 22-kD alpha-zein in tobacco, and 22-kD alpha-zein was able to enter the protein bodies formed by 27-kD gamma-zein (Coleman et al, 1996).

In addition to prolamin, there are also significant amounts of non-prolamin (NZP) in the protein bodies of corn endosperm. Over 2000 non-prolamines were identified by mass spectrometry analysis of isolated and purified corn endosperm protein bodies, and by further analysis, these non-prolamines were found to belong to different metabolic pathways and carry signal peptides targeting other organelles (Wang et al, 2016). Studies of the non-prolamin NZP1 found that NZP1 itself carries a mitochondrial localized signal peptide, and expression of NZP1 in tobacco leaves was found to be localized in mitochondria. In corn endosperm cells, NZP1 is localized in both mitochondria and proteosomes. In addition, a number of non-prolamin proteins were identified that could accumulate in the protein body through interaction with the 22-kD. The mechanism of this prolamin-mediated accumulation of non-prolamin in the protein body has not been previously explicitly reported. And the research on the accumulation mechanism of the non-prolamin in the protein body provides useful information for the efficient accumulation of the recombinant protein in the corn endosperm or tobacco protein body.

Non-prolamin proteins are important for maintaining normal morphology of the protein body and improving protein quality in the protein body. In the past studies, O1, FL1 and O10 all involved in the development of protein bodies, regulated the distribution of prolamin in protein bodies, and maintained the normal morphology of protein bodies (Holding et al, 2007; wang et al, 2016; yao et al, 2016). Normal protein bodies are important to maintain the hardness of the grain. When NZP1 is mutated, grain development is affected, and simultaneously, protein body development is retarded, so that accumulation of 22-kD alpha-zein in protein bodies is affected. In the protein body, prolamin is the main storage protein, and in prolamin, the content of proline and glutamic acid is higher, but the content of tryptophan and serine is lower, so that the protein quality is not high. Serine and tryptophan in the NZP1, NZP2 and NZP3 proteins are higher than prolamin, and the protein storage proteins maintain the balance of amino acids in the seeds in protein bodies, so that the quality of the seed protein is improved. In addition, the protein body can be used as a bioreactor and can be used as an exogenous recombinant protein production platform. After the exogenous protein is recombined with NZP1, NZP2 or NZP3, the exogenous protein can be effectively accumulated in the protein body by utilizing the characteristic that the NZP1-3 can be accumulated in the protein body. In rice, the sakura allergen protein Cryj1 is transformed and expressed in rice, so that allergen proteins stably accumulated in protein bodies can be obtained, and meanwhile, the allergen proteins have better activity and can be used for desensitization reaction of people (Okada et al, 2003).

Disclosure of Invention

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

In order to solve the above technical problems, the present invention provides a method for accumulating a target protein in a plant based on interaction with prolamin, comprising the steps of:

s1, expressing the prolamin in a receptor plant;

s2, expressing the target protein interacted with the prolamin in a receptor plant;

the prolamin is a protein of A1, A2 or A3 as follows:

a1, the amino acid sequence is a protein shown as the amino acid sequence of a sequence 8 in a sequence table;

a2, carrying out substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid sequence shown as a sequence 8 in a sequence table to obtain a protein which has more than 80% of the identity with the protein shown in the A1) and is related to protein filling;

a3, N-terminal or/and C-terminal of A1) or A2).

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

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

b1 and the amino acid sequence are proteins shown in any one of the amino acid sequences of the sequence 2, the sequence 4 and the sequence 6 in the sequence table;

B2, the 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 any one of the sequence 2, the sequence 4 and the sequence 6 in the sequence table, has more than 80 percent of identity with the protein shown in the B1) and interacts with any one of the protein A1, the protein A2 and the protein A3;

b3, fusion protein to which B1 or B2 is linked;

b4, or a fusion protein obtained by ligating protein tags to the N-terminal or/and C-terminal of any one of the proteins described in B1, B2 and B3.

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

In the above method, the method may further comprise expressing in the recipient plant a protein associated with the formation of a protein of C1, C2 or C3 as follows:

the amino acid sequence of C1 is protein shown as the amino acid sequence of sequence 10 in a sequence table;

c2, carrying out substitution and/or deletion and/or addition on the amino acid sequence shown in the sequence 10 in the sequence table by one or more amino acid residues to obtain a protein which has more than 80% of the identity with the protein shown in the C1) and is related to protein formation;

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

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

In the above method, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

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

In the above method, the accumulation of the target protein in the plant may be accumulation of the target protein in a plant protein body.

In the above method, the expression of the prolamin may be achieved by introducing a gene encoding the prolamin into the recipient plant; the gene encoding the prolamin is a gene as shown in the following D1 or D2:

D1, the coding sequence (ORF) of the coding chain is a DNA molecule of sequence 7 of the sequence table;

d2 and nucleotide sequences are DNA molecules shown in sequence 7 in the sequence table.

In the above method, the expression of the target protein interacting with the prolamin may be achieved by introducing a gene encoding the target protein into the recipient plant; the gene encoding the target protein is a gene shown as the following E1, E2 or E3:

the coding sequence (ORF) of the E1 and coding chain is a DNA molecule of sequence 1 or sequence 3 or sequence 5 in the sequence table;

e2 and the nucleotide sequence are DNA molecules shown in sequence 1 or sequence 3 or sequence 5 of the sequence table;

e3, a DNA molecule comprising E1 or E2.

In the above method, the expression of the protein associated with the formation of a protein may be achieved by introducing a gene encoding the protein associated with the formation of a protein into the recipient plant; the gene encoding the protein related to protein formation is a gene represented by F1 or F2:

f1, the coding sequence (ORF) of the coding strand is a DNA molecule of sequence 9 of the sequence table;

f2 and nucleotide sequences are DNA molecules shown in sequence 9 in the sequence table.

In the above method, the gene encoding the protein related to the formation of a protein is preferably introduced into the recipient plant after the gene encoding the protein related to the formation of a protein is linked to the gene encoding the prolamin.

In the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant; further, the monocotyledonous plant is a gramineous plant, and the dicotyledonous plant is a plant of the family Solanaceae; still further, the gramineous plant is corn and the dicotyledonous plant is tobacco.

It is a second object of the present invention to provide the use of the above method for accumulating a protein of interest in a plant based on interaction with a prolamin in the production of the protein,

or, the use of the prolamin, the protein related to protein formation and the target protein for accumulating the target protein in a plant.

A third object of the present invention is to provide a protein set comprising the above prolamin, the above protein related to protein formation, and the above target protein.

The invention verifies that the target proteins NZP1, NZP2 and NZP3 respectively interact with the prolamin 22-kD alpha-zein through a yeast two-hybrid system or an LCI system. Through the immunoelectron microscope detection of the corn kernel protein body, the accumulation of NZP1 in the protein body is proved, and the accumulation of NZP1 in the 22kD prolamin-deficient mutant material is obviously reduced. Further, by expressing Zera-22 (a fusion protein of Zera and 22-kD. Alpha. -zein) and the target proteins NZP1, NZP2 or NZP3 in tobacco, and by experiments in which Zera and the target proteins NZP1, NZP2 or NZP3 were expressed in tobacco, it was revealed that Zera-22 induced protein body formation in tobacco, and the target proteins were localized in the protein body formed by Zera-22, it was demonstrated that the target proteins NZP1, NZP2 and NZP3 could accumulate in the protein body formed by Zera-22 through interaction with 22-kD. Alpha. -zein. Thus, the method of the present invention can be applied to protein production by achieving accumulation of a target protein in a plant based on interaction with a prolamin.

Drawings

FIG. 1 is a graph showing the interaction result of NZP1 and 22-kD alpha-zein in the present invention; wherein FIG. 1A is a graph of the results of example 1 interaction in yeast and FIG. 1B is a graph of the results of example 2 interaction in LCI system.

FIG. 2 is a graph showing the interaction results of NZP2 and NZP3 with 22-kD. Alpha. -zein, respectively, according to the present invention; wherein FIG. 2A is a graph of the results of example 1 interaction in yeast and FIG. 2B is a graph of the results of example 2 interaction in LCI system.

FIG. 3 is a graph showing the results of measuring the content of NZP1 in different mutant materials in example 3 of the present invention.

FIG. 4 is a graph showing the confirmation of NZP1 cell localization in example 4 of the present invention. Wherein, panel A of FIG. 4 shows density gradient centrifugation of protein bodies, and panel B of FIG. 4 shows immunoblotting to detect accumulation of NZP1 in protein bodies.

FIG. 5 shows the immunoelectron microscope observation of the localization of NZP1 in a protein body according to example 4 of the present invention.

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

FIG. 7 shows the observation of fluorescence signals after Zera-22-mChery transformation of tobacco in example 5 of the present invention.

FIG. 8 shows the observation of fluorescence signals of NZP1 co-expressed with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, FIG. 8A shows the observation result of fluorescence signals after the co-transformation of tobacco by Zera-22-mCherry and NZP 1-GFP; panel B of FIG. 8 shows the observation of fluorescence signals after co-transformation of tobacco with Zera-mCherry and NZP 1-GFP.

FIG. 9 shows the detection results of Western blotting of the co-expression of NZP1 with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein Panel A of FIG. 9 shows the results of Zera-22-mCherry and NZP1 protein content detection by total transgenic tobacco Western blotting with Zera-22-mCherry and NZP1 co-expression. Panel B of FIG. 9 shows the results of Zera-mCherry and NZP1 protein content detection by total transgenic tobacco protein immunoblotting for Zera-mCherry and NZP1 co-expression.

FIG. 10 shows the observation of fluorescence signals of NZP2 co-expressed with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, FIG. 10A shows the observation result of fluorescence signals after the co-transformation of tobacco by Zera-22-mCherry and NZP 2-GFP; panel B of FIG. 10 shows the observation of fluorescence signals after co-transformation of tobacco with Zera-mCherry and NZP 2-GFP.

FIG. 11 shows the observation of fluorescence signals of NZP3 co-expressed with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, FIG. 11A shows the observation result of fluorescence signals after the co-transformation of tobacco by Zera-22-mCherry and NZP 3-GFP; panel B of FIG. 11 shows the observation of fluorescence signals after co-transformation of tobacco with Zera-mCherry and NZP 3-GFP.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples described below are all conventional biochemical reagents, unless otherwise specified, and are commercially available.

1. Plant lines and strains

In the examples below, an opaque1 mutant (hereinafter abbreviated as o 1), an opaque2 mutant (hereinafter abbreviated as o 2), an opaque7 mutant (hereinafter abbreviated as o 7), a floury1 mutant (hereinafter abbreviated as fl 1) are described in non-patent documents "Yao D, qi W, li X, et al, maize opaque10 encodes a essential-specific protein essential for the proper distribution of zeins in endosperm protein bodies [ J ]. Plos Genetics,2016,12 (8): e1006270 ]. Fl1, o1, o2 and o7 were all generated by single gene mutation (Fl 1 mutant gene was GRMZM2G094532, o1 mutant gene was GRMZM2G449909, o2 mutant gene was GRMZM2G016150, and o7 mutant gene was GRMZM2G 074759), the mutant grain was developed normally. After the seeds are mature, the mutant seeds are opaque in powder quality, but the seeds can germinate normally. The wild type materials corresponding to the o1 mutant, the o2 mutant and the o7 mutant are maize inbred line W22, and the wild type material corresponding to the fl1 mutant is maize B73 inbred line. The mutant materials described above are publicly available from the national university of agriculture to repeat the experiments of this application and are not available for other uses.

GV3101 agrobacterium in the following examples is a Shanghai unique biological product, cat No. AC1001S.

2. Carrier body

pGBKT7 and pGADT7 in the examples described below are from the Matchmarker Gold Yeast-Two hybrid system yeast two-hybrid kit, product of Clontech under the trade designation 630442.

The P19 vectors described in the examples below are described in the non-patent documents GoldenBrand 2.0:a comprehensive DNA assembly framework for plant synthetic biology.Sarrion-Perdigonnes A, vazquez-Vilar M, palai J, castelijns B, force J, ziarsolo P, blanca J, granell A, orzaez D.plant Physiol.2013Jul;162 (3) 1618-31.10.1104/pp.113.217661 under the name pDCB3alpha.2-35S: P19: tnos. The public is available from the national university of agriculture to repeat the experiments of this application and is not available for other uses.

The CLUC and NLUC vectors in the examples described below are described in non-patent documents "Zhang Z, yang J, wu Y.Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque, prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins [ J ]. Plant Cell,2015,27 (4): 1162', available to the public from the university of agricultural in China, for repeated experiments of the present application, and are not useful for other applications.

pHB in The examples below is described in The non-patent documents "Mao J, zhang YC, sang Y, li QH, yang HQ.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 "SupportingMethods andMaterials", available to the public from the national university of agriculture, for repeated experiments of this application, are not useful for other purposes.

3. Antibodies and enzymes

The 27-kD gamma-zein antibodies, 22-kD alpha-zein antibodies, 16-kD gamma-zein antibodies and 19-kD alpha-zein antibodies in the examples described below are described in the non-patent literature, "Yao D, qi W, li X, et al, maize opaque10 Encoles a Cereal-Specific Protein That Is Essential for the Proper Distribution of Zeins in Endosperm Protein Bodies [ J ]. Plos Genetics,2016,12 (8): e 1006270", available from the university of agricultural China, for repeated experiments of this application, and are not useful for other applications.

The colloidal gold labeled goat anti-rabbit IgG (D17537) is a product of Beijing Walker biotechnology Co., ltd, and the NZP1 antibody is manufactured by ABclonal corporation under the project number WG-01036D.

The polymerase used in the following examples was Phanta high fidelity DNA polymerase, a product of Nanjinouzan Biotechnology, inc., under the designation P511-01.

4. Reagent(s)

The total RNA extraction kit of the polysaccharide of the Tiangen in the following examples is a product of Tiangen biotechnology company, and the product number is DP360.

The seamless cloning kit in the following examples is available from ABclonal corporation under the designation RK21020.

The yeast-Leu/-Trp solid medium (accession number 630317), the yeast-Leu/-Trp liquid medium (accession number 630316), and the yeast-Ade/-His/-Leu/-Trp solid medium (accession number 630323) in the following examples are all Clontech products.

The LUC luciferin substrate in the following examples is manufactured by Promega corporation under the designation P1041.

The ABclonal multi-fragment recombination kit in the examples below was purchased from Wuhan Aibotac Biotechnology Inc. under the designation RK21020.

In the following examples, unless otherwise specified, the 1 st 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 set up in triplicate, and the results averaged to represent a significance analysis result of P < 0.05.

Example 1, interaction verification of NZP1, NZP2 and NZP3 with 22-kD alpha-zein in Yeast System

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

Extracting total RNA of the corn B73 inbred line immature grain by using a total RNA extraction kit of the tencel polyphenol polysaccharide, and carrying out reverse transcription to obtain cDNA of the corn B73 inbred line immature grain.

1.1 construction of pGBKT7-NZP1 vector

The coding sequence of the NZP1 gene is shown as sequence 1 in a sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table). Designing 5 'end primers NZP1-F and 3' end primers NZP1-R according to the coding sequence of the NZP1 gene:

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

NZP1-R：5’-ATGCGGCCGCTGCAGGTCGACGGATCCTCACTTCTCAGCCGGCGCGGCCG-3’。

a DNA fragment encoding the NZP1 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP1-F and NZP 1-R.

And replacing the fragment (including the recognition site of BamHI and the small fragment of EcoRI) between restriction endonuclease BamHI and EcoRI cleavage sites of the pGBKT7 vector by using a seamless cloning kit, and keeping other sequences of the pGBKT7 vector unchanged to obtain a recombinant expression vector of NZP1 protein, which is named pGBKT7-NZP1.

1.2 construction of pGBKT7-NZP2 vector

The coding sequence of the NZP2 gene is shown as sequence 3 in the sequence table, and the coded protein is NZP2 (sequence 4 in the sequence table). Designing a 5 'end primer NZP2-F and a 3' end primer NZP2-R according to the coding sequence of the NZP2 gene:

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

NZP2-R：5’-ATGCGGCCGCTGCAGGTCGACGGATCCCTAGTCGCGGTTAATGAAGGCGA-3’。

a DNA fragment encoding the NZP2 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP2-F and NZP 2-R.

And replacing the fragment (including the recognition site of BamHI and the small fragment of EcoRI) between restriction endonuclease BamHI and EcoRI cleavage sites of the pGBKT7 vector by using a seamless cloning kit, and keeping other sequences of the pGBKT7 vector unchanged to obtain a recombinant expression vector of NZP2 protein, which is named pGBKT7-NZP2.

1.3 construction of pGBKT7-NZP3 vector

The coding sequence of the NZP3 gene is shown as sequence 5 in the sequence table, and the coded protein is NZP3 (sequence 6 in the sequence table). Designing a 5 'end primer NZP3-F and a 3' end primer NZP3-R according to the coding sequence of the NZP3 gene:

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

NZP3-R：5’-ATGCGGCCGCTGCAGGTCGACGGATCCTTAAACCCGAGACGTCCCCAGCT-3’。

a DNA fragment of the NZP3 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP3-F and NZP 3-R.

And replacing the fragment (including the recognition site of BamHI and the small fragment of EcoRI) between restriction endonuclease BamHI and EcoRI cleavage sites of the pGBKT7 vector by using a seamless cloning kit, and keeping other sequences of the pGBKT7 vector unchanged to obtain a recombinant expression vector of NZP3 protein, which is named pGBKT7-NZP3.

1.4 construction of pGADT7-22-kD alpha-zein vector

The coding sequence of the 22-kD alpha-zein gene is shown as sequence 7 in the sequence table, and the coded protein is 22-kD alpha-zein (sequence 8 in the sequence table). The 5 '-end primer 22-kD alpha-zein-F and the 3' -end primer 22-kD alpha-zein-R are designed according to the coding sequence of the 22-kD alpha-zein gene:

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

22-kDα-zein-R：5’-CATCTGCAGCTCGAGCTCGATGGATCCCTAAAAGATGGCACCTCCAACGATCG-3’。

A primer pair consisting of a 5 '-end primer 22-kD alpha-zein-F and a 3' -end primer 22-kD alpha-zein-R is used for amplifying the coding DNA fragment of the 22-kD alpha-zein gene from cDNA of the maize B73 inbred line immature grain.

The fragment obtained by amplification is used for replacing the fragment between restriction endonuclease BamHI and EcoRI enzyme cutting sites (including a small fragment of BamHI recognition site and EcoRI enzyme recognition site) of the pGADT7 vector by using a seamless cloning kit, other sequences of the pGADT7 vector are kept unchanged, and a recombinant expression vector of 22-kD alpha-zein protein is obtained and named pGADT7-22-kD alpha-zein.

2. Interaction verification of NZP1, NZP2 and NZP3 with 22-kD alpha-zein in Yeast systems

2.1 interaction verification of NZP1 and 22-kD alpha-zein in Yeast systems

Co-transforming pGBKT7-NZP1 constructed in the step 1.1 and pGADT7-22-kD alpha-zein constructed in the step 1.4 into a yeast competent AH109 strain, coating the strain on a yeast-Leu/-Trp solid culture medium, culturing for 3-5 days at 30 ℃, picking up a monoclonal and culturing in the yeast-Leu/-Trp liquid culture medium at 30 ℃ and 220rpm until OD is reached ₆₀₀ When the strain is=1.0, the strain is taken for gradient dilution, and is respectively coated on a yeast-Leu/-Trp solid culture medium and a yeast-Ade/-His/-Leu/-Trp solid culture medium to detect interaction. The results are shown in panel A of FIG. 1, indicating that NZP1 interacts with 22-kD alpha-zein in a yeast system.

2.2 interaction verification of NZP2 and 22-kD alpha-zein in Yeast systems

pGBKT7-NZP2 constructed in the step 1.2 is replaced by pGBKT7-NZP1 constructed in the step 2.1, the pGADT7-22-kD alpha-zein constructed in the step 1.4 is used for co-transforming the yeast competent AH109 strain, other conditions in the step 2.1 are kept unchanged, and interaction is detected, and the result is shown as a graph in FIG. 2, and shows that NZP2 and 22-kD alpha-zein interact in a yeast system.

2.3 interaction verification of NZP3 and 22-kD alpha-zein in Yeast systems

The pGBKT7-NZP3 constructed in the step 1.3 is replaced by pGBKT7-NZP1 constructed in the step 2.1, and pGADT7-22-kD alpha-zein constructed in the step 1.4 is used for co-transforming the yeast competent AH109 strain, other conditions in the step 2.1 are kept unchanged, and interaction is detected, and the result is shown as a graph in FIG. 2, and shows that NZP3 and 22-kD alpha-zein interact in a yeast system.

Example 2, NZP1, NZP2 and NZP3 interaction verification with 22-kD alpha-zein in LCI System

1. Construction of the CLUC-NZP1, CLUC-NZP2, CLUC-NZP3 and 22-kD alpha-zein-NLUC vectors

1.1 construction of the CLUC-NZP1 vector

The coding sequence of the NZP1 gene is shown as sequence 1 in a sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table). 5 '-end primers NZP1-F' and 3 '-end primers NZP1-R' are designed according to the coding sequence of the NZP1 gene:

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

NZP1-R’：5’-GGACGCGTACGAGATCTGGTCGACTCACTTCTCAGCCGGCGCGGCCG-3’。

A DNA fragment encoding the NZP1 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP1-F 'and NZP 1-R'.

And replacing the fragment (small fragment including the recognition site of BamHI and the recognition site of SalI) between restriction endonuclease BamHI and SalI enzyme cleavage sites of the CLUC vector by using a seamless cloning kit, and keeping other sequences of the CLUC vector unchanged to obtain a recombinant expression vector of the NZP1 protein, which is named as CLUC-NZP1.

1.2 construction of the CLUC-NZP2 vector

The coding sequence of the NZP2 gene is shown as sequence 3 in the sequence table, and the coded protein is NZP2 (sequence 4 in the sequence table). Designing 5 'end primers NZP2-F' and 3 'end primers NZP2-R' according to the coding sequence of the NZP2 gene:

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

NZP2-R’：5’-GGACGCGTACGAGATCTGGTCGACCTAGTCGCGGTTAATGAAGGCGA-3’。

a DNA fragment encoding the NZP2 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP2-F 'and NZP 2-R'.

And replacing the fragment (small fragment including the recognition site of BamHI and the recognition site of SalI) between restriction endonuclease BamHI and SalI enzyme cleavage sites of the CLUC vector by using a seamless cloning kit, and keeping other sequences of the CLUC vector unchanged to obtain a recombinant expression vector of the NZP2 protein, which is named as CLUC-NZP2.

1.3 construction of the CLUC-NZP3 vector

The coding sequence of the NZP3 gene is shown as sequence 5 in the sequence table, and the coded protein is NZP3 (sequence 6 in the sequence table). Designing 5 'end primers NZP3-F' and 3 'end primers NZP3-R' according to the coding sequence of the NZP3 gene:

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

NZP3-R’：5’-GGACGCGTACGAGATCTGGTCGACTTAAACCCGAGACGTCCCCAGCT-3’。

a DNA fragment encoding the NZP3 gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of NZP3-F 'and NZP 3-R'.

And replacing the fragment (small fragment including the recognition site of BamHI and the recognition site of SalI) between restriction endonuclease BamHI and SalI enzyme cleavage sites of the CLUC vector with the fragment obtained by amplification by using a seamless cloning kit, keeping other sequences of the CLUC vector unchanged, and obtaining a recombinant expression vector of the NZP3 protein, which is named as CLUC-NZP3.

1.4, 22-kD alpha-zein-NLUC vector construction

The coding sequence of the 22-kD alpha-zein gene is shown as sequence 7 in the sequence table, and the coded protein is 22-kD alpha-zein (sequence 8 in the sequence table). The 5 '-end primer 22-kD alpha-zein-F' and the 3 '-end primer 22-kD alpha-zein-R' are designed according to the coding sequence of the 22-kD alpha-zein gene:

22-kDα-zein-F’：5’-CTCGTACGCGTCCCGGGGCGGTACCATGGCTACCAAGATATTATCCC-3’；

22-kDα-zein-R’：5’-ACGAACGAAAGCTCTGCAGGTCGACAAAGATGGCACCTCCAACGATCG-3’。

and replacing a fragment (a small fragment including a KpnI recognition site and a SalI enzyme recognition site) between restriction endonuclease KpnI and SalI enzyme cleavage sites of the NLUC vector by using a seamless cloning kit, and keeping other sequences of the NLUC vector unchanged to obtain a recombinant expression vector of 22-kD alpha-zein protein, which is named 22-kD alpha-zein-NLUC.

2. Interaction verification of NZP1, NZP2 and NZP3 with 22-kD alpha-zein in LCI System

Tobacco seeds (Nicotiana benthamiana) were sterilized with 70% alcohol and planted in culture dishes containing MS+8g/L agar medium, and cultured in an incubator. The temperature is 25+/-1 ℃, the humidity is 70%, and 16 hours of illumination/8 hours of dark circulation illumination are carried out. After the seeds germinate, transplanting the germinated plants into nutrient soil, culturing for 3-4 weeks under unchanged conditions of temperature, humidity and illumination, and enabling the leaves to be used for instantaneous transformation when 3-4 true leaves grow.

2.1 interaction verification of NZP1 and 22-kD alpha-zein in LCI System

Transforming Agrobacterium GV3101 with p19 vector by heat shock to obtain GV3101 containing p19, designated as p19-GV3101, and culturing to obtain OD ₆₀₀ P19-GV3101 suspension=0.5.

Transforming Agrobacterium tumefaciens GV3101 with the CLUC-NZP1 constructed in step 1.1 by heat shock to obtain GV3101 containing CLUC-NZP1, named as CLUC-NZP1-GV3101, and culturing to obtain OD ₆₀₀ CLUC-NZP1-GV3101 suspension=0.5. Transforming the 22-kD alpha-zein-NLUC constructed in the step 1.4 into agrobacterium tumefaciens GV3101 by using a heat shock method to obtain GV3101 containing the 22-kD alpha-zein-NLUC, which is named as 22-kD alpha-zein-NLUC-GV 3101, and culturing to obtain OD ₆₀₀ 22-kD α -zein-NLUC-GV3101 suspension =0.5.

The p19-GV3101 suspension, the CLUC-NZP1-GV3101 suspension and the 22-kD alpha-zein-NLUC-GV 3101 suspension were mixed in a volume ratio of 0.5:1:1, and the mixture was injected into the leaf from the back of tobacco to perform infection by using a disposable syringe, and then cultured for 8 hours at 25.+ -. 1 ℃ under a humidity of 70%, followed by culturing for 24-48 hours under a circulating light of 16 hours light/8 hours darkness. LUC fluorescein substrate (Promega, 10 mM) was used for injection in the affected area and observations were made using a Tanon-5200image system. The results are shown in panel B of FIG. 1, indicating that NZP1 interacts with 22-kD alpha-zein in the LCI system.

2.2 interaction verification of NZP2 and 22-kD alpha-zein in LCI System

The CLUC-NZP2 constructed in the step 1.2 is replaced by the CLUC-NZP1 in the step 2.1, other conditions in the step 2.1 are kept unchanged, and interaction is detected, and the result is shown as a B diagram of FIG. 2, and shows that the NZP2 and the 22-kD alpha-zein interact in an LCI system.

2.3 interaction verification of NZP3 and 22-kD alpha-zein in LCI System

The CLUC-NZP3 constructed in the step 1.3 is replaced by the CLUC-NZP1 in the step 2.1, other conditions in the step 2.1 are kept unchanged, and interaction is detected, and the result is shown as a B diagram of FIG. 2, and shows that the NZP3 and the 22-kD alpha-zein interact in an LCI system.

Example 3 detection of the content of NZP1 in different mutants

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

And respectively extracting wild type kernels, fl1 mutants, o2 mutants and o7 mutant kernels endosperm total proteins, and performing immunoblotting detection by using a 22-kD alpha-zein antibody, a 16-kD gamma-zein antibody, a 19-kD alpha-zein antibody and a NZP1 antibody.

As a result, as shown in FIG. 3, in the o2 mutant, 19-kD. Alpha. -zein and 22-kD. Alpha. -zein were significantly reduced, and the content of NZP1 was also significantly reduced. In the o7 mutant, the content of the 19-kD alpha-zein and the 16-kD alpha-zein is obviously reduced, and the content of NZP1 is not obviously changed. It is stated that the content of NZP1 is related to the content of 22-kD. Alpha. -zein.

Example 4 confirmation of cell localization of NZP1

1. Density gradient centrifugation and immunoblotting detection confirmation

The endosperm part of the kernel after 20 days of selfing pollination of the maize inbred line B73 is taken and extracted with an extracting solution.

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

Sucrose discontinuous density gradient of centrifuge tube paved from bottom to top is: centrifugation at 0.6M,0.9M,1.2M,1.45M and 1.8M sucrose at 36900rpm at 4℃for 1h 30min gave L1 (0M/0.6M of the centrifuged product), L2 (0.6M/0.9M of the centrifuged product), L3 (0.9M/1.2M of the centrifuged product), L4 (1.2M/1.45M of the centrifuged product) and L5 (1.45M/1.8M of the centrifuged product) as a total of 5 cell components.

The content of proteosomes in each fraction was detected using a 22-kD alpha-zein antibody alert immunoblot.

Immunoblots were performed using NZP1 antibodies to detect the amount of NZP1 in each component.

The results are shown in FIG. 4B, where the NZP1 accumulated significantly in the protein body component (the protein body was located predominantly between the 1.45M/1.8M gradient, i.e., the L5 component).

2. Immune electron microscope confirmation

Obtaining immature grains of corn B73 after 15 days of self-pollination, preparing ultrathin sections, incubating NZP1 primary antibody (namely NZP1 antibody) at 4 ℃ for about 12-24 hours, incubating for 2 hours at room temperature, diluting colloidal gold-labeled rabbit anti-sheep IgG secondary antibody with PBS at a ratio of 1:10, and incubating for 20-30 minutes at room temperature.

As shown in fig. 5, NZP1 was confirmed to be distributed in the protein body by immunoelectron microscopy.

Example 5 Agrobacterium-mediated transient transformation and fluorescent observation of tobacco

The coding sequence of the NZP1 gene is shown as sequence 1 in a sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table) which is obtained on a MaizeGDB website. The coding sequence of the NZP2 gene is shown as sequence 3 in the sequence table, and the coded protein is NZP2 (sequence 4 in the sequence table). The coding sequence of the NZP3 gene is shown as sequence 5 in the sequence table, and the coded protein is NZP3 (sequence 6 in the sequence table). The coding sequence of the 22-kD alpha-zein gene is shown as sequence 7 in the sequence table, and the coded protein is 22-kD alpha-zein (sequence 8 in the sequence table). The coding sequence of the Zera gene is sequence 9 in a sequence table, and the coded protein sequence is Zera (sequence 10 in the sequence table). Similarly, the coding sequence of mCherry gene obtained from GenBank of NCBI website is shown in sequence table 11, and the amino acid sequence of the encoded protein is sequence table 12. The gene coding sequence of GFP is shown as sequence 13 in the sequence table, and the amino acid sequence of GFP is shown as sequence 14 in the sequence table.

Obtaining immature grains 15 days after pollination of the maize B73 inbred line, extracting total RNA by using a total RNA extraction kit of a tencel polyphenol polysaccharide sample, performing agarose gel electrophoresis, and detecting the total RNA extraction quality. The total RNA is subjected to reverse transcription by using a full gold One-step cDNA reverse transcription kit to obtain the cDNA of the maize B73 inbred line immature grain.

1. Construction of recombinant expression vectors

1.1 construction of pHB-mCHery vector

The primers were designed according to the coding sequence of the mCherry gene (sequence 11 in the sequence listing) as follows:

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

mCHerry-R：5’-TCTAGAGGATCAATTCGAGCTCCTTGTACAGCTCGTCCATG-3’。

a primer pair consisting of mCHery-F and mCHery-R is used for amplifying a coding DNA fragment of mCHery gene from cDNA of maize B73 inbred line immature grain.

Replacing the fragment of the restriction endonuclease SacI cleavage site (comprising the SacI enzyme recognition site) of the pHB vector with the fragment obtained by amplification (the map of the pHB vector is shown as A diagram of FIG. 6), and keeping other sequences of the pHB vector unchanged to obtain a recombinant expression vector of mCHerry, which is named pHB-mCHerry.

1.2 construction of pHB-GFP vector

Primers were designed based on the gene coding sequence of GFP (sequence 13 in the sequence listing) as follows:

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

GFP-R：5’-TCTAGAGGATCAATTCGAGCTCTTACTTGTACAGCTCGTCCA-3’。

the gene coding sequence of GFP was amplified from cDNA of maize B73 inbred immature grain with a primer pair consisting of GFP-F and GFP-R.

The fragment obtained by the amplification was used to replace the fragment of restriction endonuclease SacI enzyme (including SacI enzyme recognition site) of pHB vector (map of pHB vector is shown in A diagram of FIG. 6), and other sequences of pHB vector were kept unchanged, thus obtaining recombinant expression vector of GFP, named pHB-GFP.

1.3 construction of pHB-Zera-mCherry, pHB-Zera-22-mCherry vector

The primers were designed according to the coding sequence of the Zera gene (sequence 9 of the sequence table) as follows:

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

Zera-R：5’-CGCCCTTGCTCACCATGAGCTCCTGAGGCCGGGGCGG-3’。

the primer pair consisting of Zera-F and Zera-R is used for amplifying the encoding DNA fragment of the Zera gene from the cDNA of the maize B73 inbred line immature grain, and the encoding DNA fragment is used for constructing the pHB-Zera-mCherry vector.

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

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

a DNA fragment encoding the Zera gene was amplified from cDNA of maize B73 inbred line immature grain with a primer pair consisting of Zera (22) -F and Zera (22) -R, for constructing pHB-Zera-22-mCherry vector.

The primers were designed based on the coding sequence of the 22-kD alpha-zein gene (sequence 7 of the sequence listing) as follows:

22-kDα-zein-F”：5’-CCGCCCCGGCCTCAGCTTCTTGCGCTTTTTGCGA-3’；

22-kDα-zein-R”：5’-TCGCCCTTGCTCACCATGAGCTCAAAGATGGCACCTCCAACGATG-3’。

a DNA fragment encoding the 22-kD alpha-zein gene was amplified from cDNA of maize B73 inbred immature grain with a primer set consisting of 22-kD alpha-zein-F 'and 22-kD alpha-zein-R' for construction of pHB-Zera-22-mCherry vector. .

1.3.1 construction of pHB-Zera-mCherry vector

Zera was cloned into the pHB-mCHerry vector constructed in step 1.1 in the order of the first fusion gene from top to bottom as shown in panel B in FIG. 6: the coding DNA fragment of the amplified Zera gene is used for replacing a fragment between a BamHI enzyme cutting site and a SacI enzyme cutting site (a small fragment comprising a BamHI enzyme recognition site and a SacI enzyme recognition site) of a multi-fragment recombination kit of ABclonal, pHB-mCherry is taken as a framework, other sequences of the pHB-mCherry vector are kept unchanged, and the recombinant expression vector of the fusion protein of Zera-mCherry is obtained and named pHB-Zera-mCherry.

1.3.2 construction of pHB-Zera-22-mCherry vector

The DNA fragment encoding the above-amplified Zera gene and the DNA fragment encoding the above-amplified 22-kD. Alpha. -zein gene were ligated in vitro using fusion PCR in the order of the second fusion gene from top to bottom shown in panel B of FIG. 6 to obtain DNA fragments encoding the Zera and 22-kD. Alpha. -zein fusion protein genes, and cloned into the pHB-mCHerry vector constructed in step 1.1: the coding DNA fragment of the fusion protein gene obtained by amplification is used for replacing a fragment between a BamHI enzyme cutting site and a SacI enzyme cutting site (a small fragment comprising a BamHI enzyme recognition site and a SacI enzyme recognition site) of a pHB-mCherry vector by using an ABclonal multi-fragment recombination kit and taking the pHB-mCherry as a framework, other sequences of the pHB-mCherry vector are kept unchanged, and the recombinant expression vector of the fusion protein of Zera-22-mCherry is obtained and is named 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

The primers are designed according to the coding sequence (sequence 1 in the sequence table) of the NZP1 gene as follows:

NZP1-F”：5’-TCTCTCTCAAGCTGGATCCATGACGTCCGTGCGCAGCTGTGCCG-3’；

NZP1-R”：5’-CTCGCCCTTGCTCACGAGCTCCTTCTCAGCCGGCGCGGCCG-3’。

NZP1 was cloned into the pHB-GFP vector constructed in step 1.2 in the order of the third fusion gene from top to bottom as shown in panel B of FIG. 6: the amplified fragment was substituted for the fragment between the BamHI cleavage site and the SacI cleavage site of the restriction endonuclease of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using the ABclonal multi-fragment recombination kit using the pHB-GFP vector as a backbone, and designated pHB-NZP1-GFP.

1.4.2 construction of pHB-NZP2-GFP

The primers are designed according to the coding sequence (sequence 3 in the sequence table) of the NZP2 gene as follows:

NZP2-F”：5’-TCTCTCTCAAGCTGGATCCATGGTGGCCTCGCGGATCTTGCTCC-3’；

NZP2-R”：5’-CTCGCCCTTGCTCACGAGCTCGTCGCGGTTAATGAAGGCGA-3’。

NZP2 was cloned into the pHB-GFP vector constructed in step 1.2 in the order of the fourth fusion gene from top to bottom as shown in panel B in FIG. 6: the amplified fragment was substituted for the fragment between the BamHI cleavage site and the SacI cleavage site of the restriction endonuclease of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using the ABclonal multi-fragment recombination kit using the pHB-GFP vector as a backbone, and was named pHB-NZP2-GFP.

1.4.3 construction of pHB-NZP3-GFP

The primers were designed according to the coding sequence of the NZP3 gene (sequence 5 in the sequence table) as follows:

NZP3-F”：5’-TCTCTCTCAAGCTGGATCCATGGAGGGGCTGAGCTGGAGAACGG-3’；

NZP3-R”：5’-CTCGCCCTTGCTCACGAGCTCAACCCGAGACGTCCCCAGCT-3’。

NZP3 was cloned into the pHB-GFP vector constructed in step 1.2 in the order of the fifth fusion gene from top to bottom as shown in panel B in FIG. 6: the amplified fragment was substituted for the fragment between the BamHI cleavage site and the SacI cleavage site of the restriction endonuclease of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using the ABclonal multi-fragment recombination kit using the pHB-GFP vector as a backbone, and designated pHB-NZP3-GFP.

2. Gene expression in transiently transformed tobacco

2.1, NZP1, and Zera-22-mCherry and Zera-mCherry, respectively, transiently transforming tobacco genes

2.1.1 Agrobacterium-mediated transient transformation of tobacco

Transforming and transforming the pHB-Zera-mCherry vector constructed in the step 1 into agrobacterium GV3101 by using a heat shock method to obtain GV3101 containing pHB-Zera-mCherry, named pHB-Zera-mCherry-GV3101, and culturing to obtain OD ₆₀₀ pHB-Zera-mCherry-GV3101 suspension=0.5.

Transforming and transforming the pHB-Zera-22-mCherry vector constructed in the step 1 into agrobacterium GV3101 by using a heat shock method to obtain GV3101 containing pHB-Zera-22-mCherry, named pHB-Zera-22-mCherry-GV3101, and culturing to obtain OD ₆₀₀ pHB-Zera-22-mCherry-GV3101 suspension=0.5.

Transforming and transforming the pHB-NZP1-GFP vector constructed in the step 1 into agrobacterium GV3101 by using a heat shock method to obtain GV3101 containing pHB-NZP1-GFP, which is named pHB-NZP1-GFP-GV3101, and culturing to obtain OD ₆₀₀ pHB-NZP1-GFP-GV3101 suspension=0.5.

The p19-GV3101 suspension (labeled p 19), the pHB-Zera-mCherry-GV3101 suspension (labeled Zera-mCherry), the pHB-Zera-22-mCherry-GV3101 suspension (labeled Zera-22-mCherry) and the pHB-NZP1-GFP-GV3101 (labeled NZP 1-GFP) suspensions were mixed in the following combinations, the proportions shown being by volume:

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

combination 2: zera-22-mCherry+NZP1-GFP+p19, the mixing ratio of the suspension is 1:1:0.5;

combination 3: zera-mCherry+NZP1-GFP+p19, mixing ratios of suspensions were listed as 1:1:0.5.

The mixed solution of each combination is respectively injected into the leaf from the back of the tobacco by a disposable injector for infection, is dark-cultured for 8 hours at the temperature of 25+/-1 ℃ and the humidity of 70 percent, and is then cultured for 24-48 hours under 16 hours of light/8 hours of dark circulating light, so that the fresh transgenic tobacco sheets are obtained. Wherein, the transgenic tobacco sheets expressed by Zera-22-mCherry alone are obtained by infection of the combination 1; the combination 2 is infected to obtain a transgenic tobacco sheet which is co-expressed by Zera-mCherry and NZP 1; combination 3 infection resulted in Zera-22-mCherry and NZP1 co-expressed transgenic tobacco sheets.

2.1.2 fluorescent observations

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

As shown in FIG. 7, when Zera-22-mCherry was expressed alone (combination 1), a proteosome structure was able to be formed. As shown in FIG. 8, when Zera-mCherry and NZP1 are co-expressed (combination 3, see FIG. 8, panel B), NZP1 cannot accumulate in Zera-formed protein bodies. When Zera-22-mCherry and NZP1 are co-expressed (combination 2, see FIG. 8, panel A), NZP1 is able to accumulate in the Zera-formed protein bodies, and the fluorescent signals of both NZP1-GFP and Zera-22-mCherry are aligned, indicating that NZP1 is able to relocate in the Zera-22-formed protein bodies.

2.1.3 Western immunoblotting detection

And 2.1.1, extracting total protein from the transgenic tobacco sheets obtained in the step 2.1.1, quantifying an extracting solution containing the total protein by using a protein quantification kit, separating a protein sample by SDS-PAGE, transferring the protein onto a PVDF membrane by using a Western immunoblotting method (Western Blot), and detecting by using an NZP1 antibody and a 27-kD gamma-zein antibody.

T1-T4 are four different expression events, specifically respectively

As shown in Panel A of FIG. 9, there was a significant accumulation of NZP1-GFP when it was co-expressed with Zera-22-mCherry.

As shown in Panel B of FIG. 9, there was no significant accumulation of NZP1-GFP when it was co-expressed with Zera-mCherry.

2.2, NZP2 transient transformation of tobacco with Zera-22-mCherry and Zera-mCherry genes, respectively

And (3) replacing the pHB-NZP2-GFP vector constructed in the step (1) with the pHB-NZP1-GFP vector in the step (2.1.1), and keeping the other methods in the step (2.1.1) unchanged to obtain the transgenic tobacco sheet co-expressed by Zera-mCherry and NZP2 and the transgenic tobacco sheet co-expressed by Zera-22-mCherry and NZP 2. The obtained transgenic tobacco sheet was subjected to fluorescent observation using GFP expression.

As a result, when Zera-mCherry and NZP2 were co-expressed (see FIG. 10, panel B), NZP2 was unable to accumulate in the Zera-formed protein bodies, as shown in FIG. 10. When Zera-22-mCherry and NZP2 are co-expressed (see FIG. 10, panel A), the fluorescent signals of both NZP2-GFP and Zera-22-mCherry are aligned, indicating that NZP2 is capable of being relocated in the protein bodies formed by Zera-22.

2.3, NZP3, and Zera-22-mCherry and Zera-mCherry, respectively, transiently transforming tobacco genes

And (3) replacing the pHB-NZP3-GFP vector constructed in the step (1) with the pHB-NZP1-GFP vector in the step (2.1.1), and keeping the other methods in the step (2.1.1) unchanged to obtain the transgenic tobacco sheet co-expressed by Zera-mCherry and NZP3 and the transgenic tobacco sheet co-expressed by Zera-22-mCherry and NZP 3. The obtained transgenic tobacco sheet was subjected to fluorescent observation using GFP expression.

As a result, when Zera-mCherry and NZP3 were co-expressed (see FIG. 11, panel B), NZP3 was unable to accumulate in the Zera-formed protein bodies, as shown in FIG. 11. When Zera-22-mCherry and NZP3 were co-expressed (see FIG. 11, panel A), the fluorescent signals of both NZP3-GFP and Zera-22-mCherry were consistently located, indicating that NZP3 was able to be relocated in the protein bodies formed by Zera-22.

In conclusion, the invention verifies that the target proteins NZP1, NZP2 and NZP3 respectively interact with the prolamin 22-kD alpha-zein through a yeast two-hybrid system or an LCI system. Through the immunoelectron microscope detection of the corn kernel protein body, the accumulation of NZP1 in the protein body is proved, and the accumulation of NZP1 in the 22kD prolamin-deficient mutant material is obviously reduced. Further, by expressing Zera-22 (a fusion protein of Zera and 22-kD. Alpha. -zein) and the target proteins NZP1, NZP2 or NZP3 in tobacco, and by experiments in which Zera and the target proteins NZP1, NZP2 or NZP3 were expressed in tobacco, it was revealed that Zera-22 induced protein body formation in tobacco, and the target proteins were localized in the protein body formed by Zera-22, it was demonstrated that the target proteins NZP1, NZP2 and NZP3 could accumulate in the protein body formed by Zera-22 through interaction with 22-kD. Alpha. -zein.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Chinese university of agriculture

<120> method for accumulating target protein in plant based on interaction with prolamin

<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 agacccccgg 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 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 agggggatac 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 Val His Leu Pro Pro Pro Val His Leu

35 40 45

Pro Pro Pro Val His Leu 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 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 agaacacccc 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

1. A method for accumulating a protein of interest in a plant based on interaction with a prolamin, comprising the steps of:

s1, expressing the prolamin in a receptor plant;

the prolamin is a protein of A1 or A2 as follows:

a1, the amino acid sequence is protein of an amino acid sequence shown in a sequence 8 in a sequence table;

a2, connecting protein labels at the N terminal or/and the C terminal of A1) to obtain fusion proteins;

the target protein is the protein of the following B1 or B2:

b1 and the amino acid sequence are proteins with any one of the amino acid sequences shown in the sequence 2, the sequence 4 and the sequence 6 in the sequence table;

b2, a fusion protein obtained by linking protein tags to the N-terminal or/and the C-terminal of the protein described in B1;

the recipient plant is a monocot.

2. The method of claim 1, further comprising expressing in the recipient plant a protein associated with protein formation, the protein associated with protein formation being a C1 or C2 protein:

The amino acid sequence of C1 is protein of an amino acid sequence shown as a sequence 10 in a sequence table;

c2, N-terminal or/and C-terminal of C1) is linked to the protein tag.

3. The method according to any one of claims 1-2, wherein said expressing said prolamin is effected by introducing a gene encoding said prolamin into said recipient plant; the gene encoding the prolamin is a gene as shown in the following D1 or D2:

d1, the coding sequence of the coding chain is a DNA molecule of a sequence 7 in a sequence table;

4. The method of claim 3, wherein said expressing said protein of interest that interacts with said prolamin is accomplished by introducing a gene encoding said protein of interest into said recipient plant; the gene encoding the target protein is a gene shown as the following E1 or E2:

the coding sequence of the E1 and coding chain is a DNA molecule of sequence 1 or sequence 3 or sequence 5 in the sequence table;

e2 and the nucleotide sequence are DNA molecules shown in sequence 1 or sequence 3 or sequence 5 of the sequence table.

5. The method according to claim 2, wherein said expressing a protein associated with the formation of a protein is effected by introducing a gene encoding said protein associated with the formation of a protein into said recipient plant; wherein the gene encoding the protein related to protein formation is a gene represented by F1 or F2 as follows:

F1, the coding sequence of the coding chain is a DNA molecule of a sequence 9 in a sequence table;

6. The method according to claim 5, wherein the gene encoding the protein related to the formation of a protein is introduced into a recipient plant after the gene encoding the protein related to the formation of a protein is linked with the gene encoding the prolamin.

7. Use of the method according to any one of claims 1-6 for accumulating a protein of interest in a plant based on interaction with a prolamin for the production of the protein.