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

文档序号：1871406 发布日期：2021-11-23 浏览：15次中文

阅读说明：本技术 基于与醇溶蛋白的互作在植物中积累目标蛋白的方法 (Method for accumulating target protein in plant based on interaction with prolamin ) 是由宋任涛冯阳马泽阳于 2021-03-25 设计创作，主要内容包括：本发明公开了基于与醇溶蛋白的互作在植物中积累目标蛋白的方法。本发明验证了目标蛋白NZP1、NZP2和NZP3分别与醇溶蛋白22-kDα-zein互作,并通过在烟草中表达Zera-22和目标蛋白NZP1、NZP2或NZP3,以及在烟草中表达Zera和目标蛋白NZP1、NZP2或NZP3的实验,结果表明Zera-22在烟草中诱导蛋白体形成,目标蛋白定位在Zera-22形成的蛋白体中,证明了目标蛋白NZP1、NZP2和NZP3能够通过与22-kDα-zein的互作积累在Zera-22形成的蛋白体中。由此,可基于与醇溶蛋白的互作实现目标蛋白在植物中的积累,进而应用于蛋白质生产中。(The invention discloses a method for accumulating target protein in plants based on interaction with prolamin. 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 formation in tobacco, the target proteins are positioned in a protein body formed by Zera-22, and the target proteins NZP1, NZP2 and NZP3 are proved to be capable of accumulating in the protein body formed by Zera-22 through the interaction with 22-kD alpha-zein. Therefore, the target protein can be accumulated in plants based on the interaction with the prolamin, and the method can be further applied to protein production.)

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

s1, expressing the prolamin in a recipient plant;

s2, expressing the target protein interacting with the prolamin in a recipient plant;

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

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

a2, a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 8 in the sequence table, has more than 80% of identity with the protein shown in A1) and is related to protein body filling;

a3, a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

2. The method of claim 1, wherein the target protein is a protein of B1, B2, B3, or B4:

b1, the amino acid sequence is protein shown by any one of amino acid sequences of sequence 2, sequence 4 and sequence 6 in the sequence table;

b2, a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of 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% of identity with the protein shown in B1) and interacts with the protein shown in any one of A1, A2 and A3;

b3, a fusion protein linked to B1 or B2;

b4, and a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of any one of the proteins B1, B2 and B3.

3. The method of claim 1 or 2, further comprising expressing in the recipient plant a protein associated with proteosome formation, the protein associated with proteosome formation being a protein of C1, C2, or C3 as follows:

c1, the amino acid sequence is the protein shown by the amino acid sequence of the sequence 10 in the sequence table;

c2, a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown as the sequence 10 in the sequence table, has more than 80% of identity with the protein shown as C1) and is related to the formation of proteosome;

c3, C1) or C2) at the N-terminus or/and the C-terminus.

4. The method of any of claims 1-3, wherein said expression of said prolamin protein is effected by introducing a gene encoding said prolamin protein into said recipient plant; the gene for coding the prolamin is a gene shown as D1 or D2:

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

d2, the nucleotide sequence is a DNA molecule shown in sequence 7 of the sequence table.

5. The method of any of claims 4, wherein said expressing said target protein that interacts with said prolamin protein is effected by introducing a gene encoding said target protein into said recipient plant; the gene encoding the target protein is a gene shown as E1, E2 or E3 as follows:

e1, wherein the coding sequence (ORF) of the coding strand is a DNA molecule of sequence 1 or sequence 3 or sequence 5 in the sequence table;

e2, the nucleotide sequence is a DNA molecule shown in sequence 1 or sequence 3 or sequence 5 of the sequence table;

e3, DNA molecule comprising E1 or E2.

6. The method according to claim 5, wherein said expressing a protein involved in proteosome formation is effected by introducing a gene encoding said protein involved in proteosome formation into said recipient plant; the gene for coding the protein related to proteosome formation is a gene shown as F1 or F2:

f1, wherein the coding sequence of the coding strand is a DNA molecule of sequence 9 in the sequence table;

f2, wherein the nucleotide sequence is a DNA molecule shown in sequence 9 of the sequence table.

7. The method according to claim 6, wherein the gene encoding the protein involved in protein body formation is introduced into the recipient plant, preferably after the gene encoding the protein involved in protein body formation is linked to the gene encoding the prolamin protein.

8. The method of any one of claims 1 to 7, wherein the recipient plant is a monocot or a dicot.

9. Use of a method according to any of claims 1 to 8 for accumulating a protein of interest in a plant based on interaction with a prolamin protein for the production of a protein,

or, the use of a prolamin, a protein associated with protein body formation and a protein of interest according to claims 1 to 8 for the accumulation of a protein of interest in a plant.

10. A protein set consisting of the prolamin protein of claims 1-8, the protein associated with protein body formation, and the protein of interest.

Technical Field

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

Background

With the continued expansion of the demand for recombinant proteins in industry and medicine, transgenic plants are considered a safe, efficient and inexpensive means of production (Ma et al, 2003). In contrast to traditional expression systems (such as yeast, insect and mammalian cell culture), transgenic plants also have the potential to be free of human pathogen contamination, to be able to correctly fold and assemble multimeric proteins, and to directly orally ingest raw or partially processed plant material (Fischer et al, 2004). During the past three decades, transgenic plants have shown broad application as bioreactors for the production of vaccines, antibodies and industrial enzymes, such as various antibodies, vaccine antigens, protein allergens, enzymes and enzyme inhibitors, blood clotting factors, cytokines and hormones, etc., using plant platforms (Giddings et al, 2000; Ma et al, 2005). However, one of the major problems facing the production of recombinant proteins using plant platforms is: recombinant proteins accumulate in plants at insufficient levels (Doran, 2006). The expression of recombinant proteins in heterologous environments is naturally not fixed and is also subject to the process of protein degradation that is ubiquitous in cells. Without a clear subcellular localization of certain recombinant proteins in cells, it is likely that intracellular protein degradation pathways will be entered (Faye et al, 2005).

Studies of 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 level of accumulation of recombinant proteins in the cells. While the native protein storage structures in plant seeds can provide a barrier to the storage of recombinant proteins (Conley et al, 2011). During the seed development of gramineous plants, large amounts of protein are naturally accumulated in specific locations of the endoplasmic reticulum in endosperm cells, and the endoplasmic reticulum in these regions bulges outward to form a special endoplasmic reticulum-derived compartment called proteosome (Shewry et al, 1995; Herman and Larkins, 1999). Among them, the study of proteosome in corn endosperm cell is more intensive. The protein body is an important protein storage organelle in corn endosperm cells, and the protein content in the protein body accounts for about 60 percent of the total protein content of the cells. And the protein bodies are highly stable, and the protein stored in the protein bodies is highly intact even in mature grains and grains stored for years. Therefore, the corn protein body is an ideal place for high expression and high accumulation of exogenous protein. Among the protein bodies of corn endosperm cells, prolamin is the most predominant storage protein, accounting for around 80% of the total amount of storage protein (Shewry et al, 1995; Herman and Larkins, 1999; Fuji et al, 2007). Zein mainly comprises four classes, namely alpha (19-kD, 22-kD), beta (14-kD), gamma (16-kD, 27-kD and 50-kD) and delta (10-kD, 18-kD), wherein the content of the alpha (19-kD, 22-kD) type zein is the highest (Esen, 1987; Coleman and Larkins, 1999; Song and Messing, 2002).

In studies on proteosome formation, it was found that 27-kD γ -zein plays an important role in the development of proteosomes (Wu and Messing, 2010; Guo et al, 2013). Inhibition of the expression of 27-kD gamma-zein in maize endosperm by RNAi technology results in a significant reduction in the number of proteosome in the cells. It is demonstrated that 27-kD gamma-zein has the function of promoting protein formation. Further studies found that 27-kD γ -zein can induce the formation of protein bodies not only in corn kernel, but also in tobacco seed, lamina and arabidopsis lamina (tortent et al, 2009; Llop-Tous et al, 2010; Mainieri et al, 2014). Sequence analysis showed that 27-kD γ -zein comprises four regions: a. a signal peptide comprising 19 amino acids, b.a region consisting of 8 PPVHL hexapeptide repeats (53aa), c.a pro-x domain rich in proline (29aa), d.a hydrophobic C-terminal domain rich in cysteine (111 a). Through studies on the retention mechanism of 27-kD gamma-zein, it was found that the endoplasmic reticulum retention signal composed of HDEL is absent in the 27-kD gamma-zein sequence, and that the retention of 27-kD gamma-zein in the endoplasmic reticulum and the formation of induced protein bodies both depend on a structure composed of regions a, b, c, which is in turn called Zera domain (Torrent et al, 2009; Llop-Tous et al, 2010; Mainieri et al, 2014). Further studies have shown that the two cysteine residues at the N-terminus of 27-kD γ -zenin are crucial for oligomerization of 27-kD γ -zenin, which is considered to be the first step in the formation of the proteosome.

In addition to 27-kD γ -zein, another important class of prolamins in protein bodies is the α -gliadins (19-kD, 22-kD), with α -zein being responsible for the filling of the protein body (Holding et al, 2007), the highest prolamin class in protein bodies. The research on the alpha-zein shows that the alpha-zein does not have an endoplasmic reticulum retention signal per se, and the expression of the alpha-zein alone in vitro can not induce the formation of protein bodies, which indicates that the alpha-zein does not have the capacity of inducing the formation of the protein bodies. Kim et al discovered, through a series of prolamin interaction studies, that 22-kD α -zein is capable of interacting with other types of prolamin proteins (Kim et al, 2002). Coleman et al expressed both 27-kD γ -zein and 22-kD α -zein in tobacco, 22-kD α -zein being able to enter the protein body formed by 27-kD γ -zein (Coleman et al, 1996).

In addition to prolamins, a number of non-prolamins (NZP) are also present in the protein body of maize endosperm. Wang et al identified over 2000 non-prolamins by mass spectrometry of the isolated and purified corn endosperm protein bodies, and further analyzed to find that these non-prolamins belong to different metabolic pathways and carry signal peptides that target other organelles (Wang et al, 2016). Studies on the non-prolamin NZP1 found that NZP1 itself carries a mitochondrial localization signal peptide, which was found to be localized in mitochondria when NZP1 was expressed in tobacco leaves. In corn endosperm cells, NZP1 is localized in both mitochondria and proteosome. In addition, a number of non-prolamin proteins have been identified which are capable of accumulating in the protein body by interaction with 22-kD. The mechanism of prolamin-mediated accumulation of non-prolamin in the protein body has not been reported explicitly. And the research on the non-alcohol soluble protein accumulation mechanism in the protein body provides useful information for the high-efficiency accumulation of the recombinant protein in corn endosperm or tobacco protein body.

The non-alcohol soluble protein has important significance for maintaining the normal shape of the protein body and improving the quality of the protein in the protein body. In previous researches, O1, FL1 and O10 are all involved in the development of a protein body, regulate the distribution of prolamin in the protein body and maintain the normal shape of the protein body (Holding et al, 2007; Wang et al, 2016; Yao et al, 2016;). Normal proteosomes are important to maintain kernel hardness. When NZP1 is mutated, the development of kernel is affected, and the proteosome development is retarded, so that the accumulation of 22-kD alpha-zein in the proteosome is affected. In the protein body, prolamin is the main storage protein, and the prolamin contains high content of proline and glutamic acid, but contains low content of tryptophan and serine, so that the protein quality is not high. Serine and tryptophan in the proteins NZP1, NZP2 and NZP3 are higher than prolamin, and the protein storage proteins maintain the balance of amino acids in grains in a protein body and contribute to improving the quality of the grains. In addition, the protein body serves as a bioreactor and can serve as a foreign recombinant protein production platform. After the exogenous protein is recombined with NZP1, NZP2 or NZP3, the characteristic that NZP1-3 can be accumulated in a protein body is utilized, so that the exogenous protein can be effectively accumulated in the protein body. In rice, the oriental cherry allergen protein Cryj1 is transformed and expressed in rice, so that the allergen protein stably accumulated in a protein body can be obtained, and the allergen protein also has better activity and can be used for desensitization of human (Okada et al, 2003).

Disclosure of Invention

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

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 a prolamin, comprising the steps of:

s1, expressing the prolamin in a recipient plant;

s2, expressing the target protein interacting with the prolamin in a recipient plant;

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

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

a3, a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

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

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

b1, the amino acid sequence is protein shown by any one of amino acid sequences of sequence 2, sequence 4 and sequence 6 in the sequence table;

b3, a fusion protein linked to B1 or B2;

b4, and a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of any one of the proteins 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; the sequence 6 in the sequence table consists of 213 amino acid residues.

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

c1, the amino acid sequence is the protein shown by the amino acid sequence of the sequence 10 in the sequence table;

c3, C1) or C2) at the N-terminus or/and the C-terminus.

In the above method, sequence 10 in the sequence table is composed of 93 amino acid residues.

In the above methods, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

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

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

In the above method, the expression of the prolamin protein can be achieved by introducing a gene encoding the prolamin protein into the recipient plant; the gene for coding the prolamin is a gene shown as D1 or D2:

d1, wherein the coding sequence (ORF) of the coding strand is a DNA molecule of sequence 7 in the sequence table;

d2, the nucleotide sequence is a DNA molecule shown in sequence 7 of the sequence table.

In the above method, the expression of the target protein interacting with the prolamin protein can 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 E1, E2 or E3 as follows:

e1, wherein the coding sequence (ORF) of the coding strand is a DNA molecule of sequence 1 or sequence 3 or sequence 5 in the sequence table;

e2, the nucleotide sequence is a DNA molecule shown in sequence 1 or sequence 3 or sequence 5 of the sequence table;

e3, DNA molecule comprising E1 or E2.

In the above method, the expression of the protein involved in the formation of protein bodies may be carried out by introducing a gene encoding the protein involved in the formation of protein bodies into the recipient plant; the gene for coding the protein related to proteosome formation is a gene shown as F1 or F2:

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

f2, wherein the nucleotide sequence is a DNA molecule shown in sequence 9 of the sequence table.

In the above method, the gene encoding the protein involved in protein body formation is introduced into the recipient plant, and preferably, the gene encoding the protein involved in protein body formation and the gene encoding the prolamin are introduced into the recipient plant together after being linked.

In the above method, the recipient plant is a monocotyledon or dicotyledon; further, the monocotyledon is a gramineae plant, and the dicotyledon is a solanaceae plant; 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 plants based on interaction with a prolamin protein for the production of proteins,

or the use of the prolamin, the protein involved in the formation of protein bodies, and the target protein for the accumulation of the target protein in plants.

The third object of the present invention is to provide a protein set comprising the prolamin, the protein involved in protein body formation, and the target protein.

The invention verifies that target proteins NZP1, NZP2 and NZP3 respectively interact with prolamin 22-kD alpha-zein by a yeast two-hybrid system or an LCI system. The detection of an immunoelectron microscope of a corn kernel protein body proves that NZP1 is accumulated in the protein body, and the accumulation amount of NZP1 in a 22kD gliadin-deficient mutant material is obviously reduced. Further, it was confirmed that the proteins NZP1, NZP2 and NZP3 of interest can be accumulated in a protein body formed by Zera-22 by the interaction with 22-kD α -zein, as a result of expressing Zera-22 (fusion protein of Zera and 22-kD α -zein) and the protein NZP1, NZP2 or NZP3 of interest in tobacco and experiments expressing Zera and the protein NZP1, NZP2 or NZP3 of interest in tobacco, showing that Zera-22 induces protein formation in tobacco and the protein of interest is localized in the protein body formed by Zera-22. Therefore, the method disclosed by the invention can realize the accumulation of the target protein in the plant based on the interaction with the prolamin, and further can be applied to the production of the protein.

Drawings

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

FIG. 2 is a graph showing the results of the interaction of NZP2 and NZP3 with 22-kD α -zein, respectively, in the present invention; wherein, the A diagram of FIG. 2 is the interaction result diagram of the example 1 in the yeast, and the B diagram of FIG. 2 is the interaction result diagram of the example 2 in the 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 confirmation of the cell localization of NZP1 in example 4 of the present invention. Wherein, the A picture of figure 4 is density gradient centrifugation protein body, the B picture of figure 4 is immunoblotting detection NZP1 accumulation in protein body.

FIG. 5 shows the localization of NZP1 in proteosome by immunoelectron microscopy in example 4 of the present invention.

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

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

FIG. 8 shows the observation results of fluorescent signals co-expressed by NZP1 with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, the A picture of FIG. 8 is the observation result of the fluorescence signal of tobacco co-transformed by Zera-22-mCherry and NZP 1-GFP; FIG. 8, panel B, is an observation of fluorescence signals from tobacco co-transformed with Zera-mCherry and NZP 1-GFP.

FIG. 9 shows the results of immunoblotting detection of the co-expression of NZP1 with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, the A picture of figure 9 is the result of detecting the content of the proteins of Zera-22-mCherry and NZP1 by the immunoblotting method of the total protein of the transgenic tobacco co-expressed by Zera-22-mCherry and NZP 1. FIG. 9B is the result of immunoblotting of transgenic tobacco total protein co-expressed by Zera-mCherry and NZP1 to detect the content of Zera-mCherry and NZP1 proteins.

FIG. 10 shows the observation results of fluorescent signals co-expressed by NZP2 with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, the A picture of FIG. 10 is the observation result of the fluorescence signal of tobacco co-transformed by Zera-22-mCherry and NZP 2-GFP; FIG. 10, panel B, is an observation of fluorescence signals from tobacco co-transformed with Zera-mCherry and NZP 2-GFP.

FIG. 11 shows the observation results of fluorescent signals co-expressed by NZP3 with Zera-22-mCherry and Zera-mCherry, respectively, in example 5 of the present invention. Wherein, the A picture of FIG. 11 is the observation result of the fluorescence signal of tobacco co-transformed by Zera-22-mCherry and NZP 3-GFP; FIG. 11B is a photograph showing the observation of fluorescence signals obtained by co-transforming tobacco with Zera-mCherry and NZP 3-GFP.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

1. Plant lines and strains

In the following examples, the opaque1 mutant (hereinafter abbreviated as o1), the opaque2 mutant (hereinafter abbreviated as o2), the opaque7 mutant (hereinafter abbreviated as o7), and the flory 1 mutant (hereinafter abbreviated as fl1) are described in non-patent documents "Yao D, Qi W, Li X, et al. maize opaque10 encodes a spatial-specific protein assay for the protein distribution of peptides in vivo proteins nucleotides [ J ]. Plos proteins nucleotides, 2016,12(8): 1006270". The fls 1, o1, o2 and o7 are all generated by single gene mutation (the fls 1 mutant gene is GRMZM2G094532, the o1 mutant gene is GRMZM2G449909, the o2 mutant gene is GRMZM2G016150, and the o7 mutant gene is GRMZM2G074759), and the seed of the mutant is normally developed. After the grains are mature, the mutant grains are opaque, but can normally germinate. Wild type materials corresponding to the o1 mutant, the o2 mutant and the o7 mutant are all maize inbred line W22, and wild type materials corresponding to the fl1 mutant are maize B73 inbred lines. The mutant material described above is publicly available from the university of agriculture in China to repeat the experiments of the present application and is not available for other uses.

The GV3101 Agrobacterium tumefaciens in the examples described below is a Shanghai unique habitat biological product, cat # AC 1001S.

2. Carrier

pGBKT7 and pGADT7 in the examples below are from the Matchmarker Gold Yeast-Two hybrid system Yeast Two-hybrid kit, product of Clontech, Cat. 630442.

The P19 vector in the following examples is described in the non-patent literature Golden Braid 2.0, a comprehensive DNA assembly frame 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.2013Jul; 162(3) 1618-31.10.1104/pp.113.217661, named pDG 3alpha2_35S, P19, Tnos. The public is available from the university of agriculture in China to repeat the experiments of the present application and not for other uses.

CLUC and NLUC vectors in the following examples are described in non-patent literature "Zhang Z, Yang J, Wu Y. transformation Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque2, protein-Box Binding Factor, and O2 heterologous Proteins [ J ] Plant Cell,2015,27(4): 1162", publicly available from the national university of agriculture to repeat the experiments of the present application, and are not useful for other applications.

In The following examples pHB is described in The non-patent literature "Mao J, Zhang YC, Sang Y, Li QH, Yang HQ. from The Cover: A role for Arabidopsis cryptochromes and COP1 in The regulation of stock exposure. Proc Natl Acad Sci U S A.2005; 102(34):12270-12275.doi:10.1073/pnas.0501011102 "support methods and materials, publicly available from the university of agriculture to repeat the experiments of this application, not for other uses.

3. Antibodies and enzymes

The 27-kD γ -zein antibody, 22-kD α -zein antibody, 16-kD γ -zein antibody and 19-kD α -zein antibody of the examples described below are described in the non-patent literature "Yao D, Qi W, Li X, et al, maize opaque10 Encodes a center-Specific Protein at which Is Essential for the protocol Distribution of Zeins in Endosperm Protein diodes [ J ]. Plos Genetics,2016,12(8): e1006270", available from university of agriculture for the repetition of the experiments of the present application, and are not available for other uses.

The colloidal gold labeled goat anti-rabbit IgG (D17537) is a product of Beijing Wacky Biotech Co., Ltd, and the NZP1 antibody is made by ABClonal, and has the project number WG-01036D.

The polymerase used in the following examples is Phanta high fidelity DNA polymerase, a product of Biotech, Nanjing Novowed, having a product number P511-01.

4. Reagent

The radix asparagi polyphenol polysaccharide total RNA extraction kit in the following examples is a product of radix asparagi biotechnology company, and the product number is DP 360.

The seamless cloning kit in the following examples is available from ABClonal, Inc., under the accession number RK 21020.

The yeast-Leu/-Trp solid medium (cat. No. 630317), yeast-Leu/-Trp liquid medium (cat. No. 630316), and yeast-Ade/-His/-Leu/-Trp solid medium (cat. No. 630323) in the following examples are all products of Clontech.

The LUC fluorescein substrate in the following examples is available from Promega under the reference P1041.

The ABClonal multi-fragment recombination kit of the examples described below was purchased from Botetaike Biotech, Wuhan, Inc. under the accession number RK 21020.

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 performed in triplicate and the results averaged to give a significance analysis result of P < 0.05.

Example 1, NZP1, NZP2 and NZP3 interaction with 22-kD α -zein in Yeast systems

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

Extracting total RNA of the maize B73 inbred line immature grain by using a radix angelicae sinensis polyphenol polysaccharide total RNA extraction kit, and performing reverse transcription to obtain cDNA of the maize 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 the sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table). Designing a 5 'end primer NZP1-F and a 3' end primer NZP1-R according to the coding sequence of the NZP1 gene:

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

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

a coding DNA fragment of the NZP1 gene is amplified from cDNA of an immature seed grain of a maize B73 inbred line by using a primer pair consisting of NZP1-F and NZP 1-R.

The fragment obtained by amplification replaces the fragment between restriction endonuclease BamHI and EcoRI cutting sites (small fragment including BamHI recognition site and EcoRI enzyme recognition site) of pGBKT7 vector by using a seamless cloning kit, and other sequences of pGBKT7 vector are kept unchanged to obtain a recombinant expression vector of NZP1 protein, which is named as pGBKT7-NZP 1.

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 coding DNA fragment of the NZP2 gene is amplified from cDNA of an immature seed grain of a maize B73 inbred line by using a primer pair consisting of NZP2-F and NZP 2-R.

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 primer pair consisting of NZP3-F and NZP3-R is used for amplifying a DNA fragment of the NZP3 gene from cDNA of an immature seed grain of a maize B73 inbred line.

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

The coding sequence of the 22-kD alpha-zein gene is shown as a sequence 7 in a sequence table, and the coded protein is 22-kD alpha-zein (a sequence 8 in the sequence table). Designing a 5 'end primer 22-kD alpha-zein-F and a 3' end primer 22-kD alpha-zein-R 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 a coding DNA fragment of the 22-kD alpha-zein gene from cDNA of immature grains of an inbred line of the corn B73.

The fragment obtained by the above amplification was substituted for the fragment between the restriction endonuclease BamHI and EcoRI cleavage sites of pGADT7 vector (small fragments including the recognition site for BamHI and the recognition site for EcoRI enzyme) using a seamless cloning kit, and the other sequences of pGADT7 vector were kept unchanged to obtain a recombinant expression vector of 22-kD α -zein protein, which was named pGADT7-22-kD α -zein.

2. The interaction between NZP1, NZP2 and NZP3 and 22-kD alpha-zein in a yeast system is verified

2.1, NZP1 and 22-kD alpha-zein interacted and verified in a yeast system

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 yeast competent AH109 strain on a yeast-Leu/-Trp solid culture medium, culturing for 3-5 days at 30 ℃, selecting a single clone to culture in a yeast-Leu/-Trp liquid culture medium under the conditions of 30 ℃ and 220rpm until OD is reached₆₀₀When the concentration is 1.0, the culture broth is subjected to gradient dilution, and the diluted culture broth is applied to a yeast-Leu/-Trp solid medium and a yeast-Ade/-His/-Leu/-Trp solid medium, respectively, to detect the interaction. The results are shown in panel A of FIG. 1, indicating that NZP1 interacts with 22-kD α -zein in the yeast system.

2.2, NZP2 and 22-kD alpha-zein in the yeast system interaction verification

The interaction was detected by substituting pGBKT7-NZP2 constructed in step 1.2 for pGBKT7-NZP1 constructed in step 2.1 and pGADT7-22-kD α -zein constructed in step 1.4 to co-transform the yeast competent AH109 strain, maintaining the other conditions in step 2.1, and the results are shown in panel A of FIG. 2, indicating that NZP2 interacts with 22-kD α -zein in the yeast system.

2.3, NZP3 and 22-kD alpha-zein interacted and verified in a yeast system

The interaction was detected by substituting pGBKT7-NZP3 constructed in step 1.3 for pGBKT7-NZP1 in step 2.1 and pGADT7-22-kD α -zein constructed in step 1.4 to co-transform the yeast competent AH109 strain, while keeping the other conditions in step 2.1 unchanged, and the results are shown in FIG. 2A, which indicates that NZP3 interacts with 22-kD α -zein in the yeast system.

Example 2, NZP1, NZP2 and NZP3 interaction with 22-kD α -zein in LCI systems

1. Construction of 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 the sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table). Designing a 5 'end primer NZP 1-F' and a3 'end primer NZP 1-R' according to the coding sequence of the NZP1 gene:

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

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

a DNA fragment for coding the NZP1 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP1-F 'and NZP 1-R'.

The fragment obtained by the above amplification was substituted for a fragment (a small fragment including a recognition site for BamHI and a recognition site for SalI) between restriction endonuclease BamHI and SalI sites of the CLUC vector using a seamless cloning kit, and the other sequences of the CLUC vector were kept unchanged to obtain a recombinant expression vector of NZP1 protein, which was named CLUC-NZP 1.

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 a 5 'end primer NZP 2-F' and a3 'end primer NZP 2-R' according to the coding sequence of the NZP2 gene:

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

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

a DNA fragment for coding the NZP2 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP2-F 'and NZP 2-R'.

1.3 CLUC-NZP3 vector construction

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 NZP 3-F' and a3 'end primer NZP 3-R' according to the coding sequence of the NZP3 gene:

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

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

a DNA fragment for coding the NZP3 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP3-F 'and NZP 3-R'.

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

The coding sequence of the 22-kD alpha-zein gene is shown as a sequence 7 in a sequence table, and the coded protein is 22-kD alpha-zein (a sequence 8 in the sequence table). Designing a 5 'end primer 22-kD alpha-zein-F' and a3 'end primer 22-kD alpha-zein-R' 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 (3) replacing a fragment (a small fragment including a recognition site of KpnI and a recognition site of SalI enzyme) between restriction endonuclease KpnI and SalI enzyme cutting 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 the 22-kD alpha-zein protein, which is named as 22-kD alpha-zein-NLUC.

2. The interaction between NZP1, NZP2 and NZP3 and 22-kD alpha-zein in LCI system is verified

Tobacco seeds (Nicotiana benthamiana) were sterilized with 70% ethanol, plated on a petri dish containing MS +8g/L agar medium, and cultured in an incubator. Temperature 25 + -1 deg.C, humidity 70%, 16 hr light/8 hr dark cycle light. After the seeds germinate, transplanting the germinated plants into nutrient soil, keeping the culture temperature, humidity and illumination conditions unchanged, and after 3-4 weeks of culture, when 3-4 true leaves grow, the leaves can be used for instantaneous transformation.

2.1, NZP1 and 22-kD alpha-zein in the interaction verification of LCI system

Transforming Agrobacterium GV3101 with p19 vector by heat shock method to obtain GV3101 containing p19, named p19-GV3101, and culturing to obtain OD₆₀₀P19-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, which is named as CLUC-NZP1-GV3101, and culturing to obtain OD₆₀₀A CLUC-NZP1-GV3101 suspension of 0.5. Transforming Agrobacterium tumefaciens GV3101 with the 22-kD alpha-zein-NLUC constructed in step 1.4 by 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 OD₆₀₀22-kD α -zein-NLUC-GV3101 suspension 0.5.

Mixing p19-GV3101 suspension, CLUC-NZP1-GV3101 suspension, and 22-kD alpha-zein-NLUC-GV 3101 suspension at a volume ratio of 0.5:1:1, injecting into leaf blade from tobacco back side by disposable injector for infection, culturing at 25 + -1 deg.C with 70% humidity in dark for 8 hr, and culturing under 16 hr light/8 hr dark cyclic light for 24-48 hr. LUC fluorescein substrate (Promega, 10mM) was injected into the affected area and observed using Tanon-5200image system. The results are shown in panel B of FIG. 1, indicating that NZP1 interacts with 22-kD α -zein in the LCI system.

2.2, NZP2 and 22-kD alpha-zein in the interaction verification of LCI system

The interaction was detected by replacing CLUC-NZP2 constructed in step 1.2 with CLUC-NZP1 in step 2.1, keeping the other conditions unchanged in step 2.1, and the results are shown in panel B of FIG. 2, indicating that NZP2 interacts with 22-kD α -zein in the LCI system.

2.3, NZP3 and 22-kD alpha-zein in the interaction verification of LCI system

The interaction was detected by replacing CLUC-NZP3 constructed in step 1.3 with CLUC-NZP1 in step 2.1, keeping the other conditions unchanged in step 2.1, and the results are shown in panel B of FIG. 2, indicating that NZP3 interacts with 22-kD α -zein in the 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.

Respectively extracting endosperm total protein of wild type grains, fl1 mutant, o2 mutant and o7 mutant grains, and carrying out immunoblotting detection by using 22-kD alpha-zein antibody, 16-kD gamma-zein antibody, 19-kD alpha-zein antibody and NZP1 antibody.

As shown in FIG. 3, in the o2 mutant, 19-kD α -zein and 22-kD α -zein were significantly reduced, and the content of NZP1 was also significantly reduced. In the o7 mutant, the levels of 19-kD α -zein and 16-kD α -zein were significantly reduced, and the level of NZP1 was not significantly changed. It is shown that the content of NZP1 correlates with the content of 22-kD α -zein.

Example 4 confirmation of the cellular localization of NZP1

1. Density gradient centrifugation and immunoblot detection confirmation

Taking endosperm parts of grains 20 days after selfing pollination of the maize inbred line B73, and extracting by using an extracting solution.

Extracting solution: 10mM Tris-cl, 10mM MgCl₂5mM KCl, 10mM PMSF, 1mM DTT, 7.2% sucrose.

The centrifugal tube is characterized in that the discontinuous density gradient of the sucrose laid from bottom to top is as follows: centrifugation at 0.6M, 0.9M, 1.2M, 1.45M and 1.8M sucrose at 36900rpm for 1h 30min at 4 ℃ separated the different cell fractions (FIG. 4, Panel A) to give L1(0M/0.6M centrate), L2(0.6M/0.9M centrate), L3(0.9M/1.2M centrate), L4(1.2M/1.45M centrate), L5(1.45M/1.8M centrate) for a total of 5 cell fractions.

The content of proteosome in each component is detected by using 22-kD alpha-zein antibody to warn immunoblotting.

The content of NZP1 in each fraction was detected by immunoblotting using NZP1 antibody.

Results as shown in B of fig. 4, significant accumulation of NZP1 was observed in the components of the proteosome (the proteosome was mainly located between the 1.45M/1.8M gradient, i.e., the L5 component).

2. Confirmation by immunoElectron microscopy

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

As shown in fig. 5, it was confirmed by immunoelectron microscopy that NZP1 was distributed in the proteosome.

Example 5 Agrobacterium-mediated transient transformation of tobacco and fluorescence Observation

Firstly, the coding sequences of NZP1 genes obtained on the MaizeGDB website are shown in sequence 1 in the sequence table, and the coded protein is NZP1 (sequence 2 in the sequence table). 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 a sequence 7 in a sequence table, and the coded protein is 22-kD alpha-zein (a sequence 8 in the sequence table). The coding sequence of the Zera gene is sequence 9 in the sequence table, and the protein sequence coded by the Zera gene is Zera (sequence 10 in the sequence table). Similarly, the coding sequence of the mCherry gene obtained from the GenBank of the NCBI website is shown as a sequence 11 in the sequence table, and the amino acid sequence of the encoded protein is a sequence 12 in the sequence table. 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 a maize B73 self-line, extracting total RNA by using a total RNA extraction kit of a radix puerariae polyphenol polysaccharide sample, carrying out agarose gel electrophoresis, and detecting the extraction quality of the total RNA. And (3) carrying out reverse transcription on the total RNA by using a full-type gold One-step cDNA reverse transcription kit to obtain the cDNA of the immature grains of the maize B73 inbred line.

1. Construction of recombinant expression vectors

1.1, construction of pHB-mCherry vector

The primers were designed based on the coding sequence of the mCherry gene (SEQ ID NO: 11 in the sequence listing) as follows:

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

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

and amplifying a coding DNA fragment of the mCherry gene from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of mCherry-F and mCherry-R.

And replacing the fragment (including the SacI enzyme recognition site) of the restriction endonuclease SacI enzyme cutting site of the pHB vector (the map of the pHB vector is shown in a picture A of figure 6) with the amplified fragment, and keeping other sequences of the pHB vector unchanged to obtain the recombinant expression vector of the mCherry, wherein the recombinant expression vector is named as pHB-mCherry.

1.2 construction of pHB-GFP vector

Primers were designed based on the gene coding sequence of GFP (SEQ ID NO: 13 of the sequence listing), as follows:

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

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

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

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

1.3, constructing pHB-Zera-mCherry and pHB-Zera-22-mCherry vectors

The primers were designed based on the Zera gene coding sequence (SEQ ID NO: 9 of the sequence listing) as follows:

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

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

and amplifying a coding DNA fragment of the Zera gene from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of Zera-F and Zera-R, and using the amplified coding DNA fragment to construct a pHB-Zera-mCherry vector.

The primers were designed based on the Zera gene coding sequence (SEQ ID NO: 9 of the sequence listing) as follows:

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

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

and amplifying a DNA fragment encoding the Zera gene from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of Zera (22) -F and Zera (22) -R, and using the DNA fragment to construct a pHB-Zera-22-mCherry vector.

The primers were designed based on the coding sequence of the 22-kD α -zein gene (SEQ ID NO: 7 of the sequence listing) as follows:

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

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

a primer pair consisting of 22-kD alpha-zein-F and 22-kD alpha-zein-R is used for amplifying a coding DNA fragment of a 22-kD alpha-zein gene from cDNA of immature grains of an inbred line of corn B73 and is used for constructing a pHB-Zera-22-mCherry vector. .

1.3.1 construction of pHB-Zera-mCherry vector

The 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 of figure 6: replacing the fragment (small fragment including BamHI enzyme recognition site and SacI enzyme recognition site) between the multiple cloning site BamHI enzyme cutting site and SacI enzyme cutting site of pHB-mCherry vector with the amplified coding DNA fragment of the Zera gene by using an ABClonal multi-fragment recombination kit and taking pHB-mCherry as a framework, keeping other sequences of the pHB-mCherry vector unchanged, obtaining the recombination expression vector of the fusion protein of Zera-mCherry, and naming the recombination expression vector as pHB-Zera-mCherry.

1.3.2 construction of pHB-Zera-22-mCherry vector

The DNA fragment encoding the Zera gene amplified above and the DNA fragment encoding the 22-kD α -zein gene amplified above were ligated in vitro using fusion PCR in the order of the second fusion gene from top to bottom as shown in panel B in fig. 6, to obtain DNA fragments encoding the Zera and 22-kD α -zein fusion protein genes, and cloned into the pHB-mCHerry vector constructed in step 1.1: replacing a fragment (a small fragment comprising a recognition site of BamHI enzyme and a recognition site of SacI enzyme) between a multiple cloning site BamHI enzyme cutting site and a SacI enzyme cutting site of a pHB-mCherry vector with a coding DNA fragment of the fusion protein gene obtained by amplification by using an ABClonal multi-fragment recombination kit and taking pHB-mCherry as a framework, keeping other sequences of the pHB-mCherry vector unchanged, obtaining a recombination expression vector of the fusion protein of Zera-22-mChery, and naming the recombination expression vector as pHB-Zera-22-mChery.

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’。

a DNA fragment for coding the NZP1 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP1-F 'and NZP 1-R'.

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 a fragment between the restriction endonuclease BamHI cleavage site and the SacI cleavage site of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using an ABClonal multi-fragment recombination kit with the pHB-GFP vector as a backbone, and was named pHB-NZP 1-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’。

a DNA fragment for coding the NZP2 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP2-F 'and NZP 2-R'.

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 of FIG. 6: the amplified fragment was substituted for a fragment between the restriction endonuclease BamHI cleavage site and the SacI cleavage site of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using an ABClonal multi-fragment recombination kit with the pHB-GFP vector as a backbone, and was named pHB-NZP 2-GFP.

1.4.3 construction of pHB-NZP3-GFP

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

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

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

a DNA fragment for coding the NZP3 gene is amplified from the cDNA of the immature grain of the maize B73 inbred line by using a primer pair consisting of NZP3-F 'and NZP 3-R'.

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 of FIG. 6: the amplified fragment was substituted for a fragment between the restriction endonuclease BamHI cleavage site and the SacI cleavage site of the pHB-GFP vector (a small fragment including the recognition site of BamHI enzyme and the recognition site of SacI enzyme) using an ABClonal multi-fragment recombination kit with the pHB-GFP vector as a backbone, and was named pHB-NZP 3-GFP.

2. Gene expression profile in transiently transformed tobacco

2.1, NZP1 with Zera-22-mCherry and Zera-mCherry transient transformation tobacco gene

2.1.1 Agrobacterium-mediated transient transformation of tobacco

Transforming Agrobacterium GV3101 with p19 vector by heat shock method to obtain GV3101 containing p19, named p19-GV3101, and culturing to obtain OD₆₀₀P19-GV3101 suspension 0.5.

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-mChery, which is named as pHB-Zera-mChery-GV 3101, and culturing to obtain OD₆₀₀0.5 pHB-Zera-mCherry-GV3101 suspension.

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-mChery, which is named as pHB-Zera-22-mChery-GV 3101, and culturing to obtain OD₆₀₀0.5 pHB-Zera-22-mCherry-GV3101 suspension.

Transforming the pHB-NZP1-GFP vector constructed in step 1 into Agrobacterium GV3101 by heat shock to obtain GV3101 containing pHB-NZP1-GFPNamed pHB-NZP1-GFP-GV3101, cultured to obtain OD₆₀₀0.5 pHB-NZP1-GFP-GV3101 suspension.

A suspension of p19-GV3101 (designated p19), a suspension of pHB-Zera-mCherry-GV3101 (designated Zera-mCherry), a suspension of pHB-Zera-22-mCherry-GV3101 (designated Zera-22-mChery) and a suspension of pHB-NZP1-GFP-GV3101 (designated NZP1-GFP) were mixed in the following combinations, all in volume:

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

and (3) combination 2: Zera-22-mCherry + NZP1-GFP + p19, and the mixing ratio of the suspension is 1:1: 0.5;

and (3) combination: Zera-mCherry + NZP1-GFP + p19, the suspension mixing ratio is 1:1: 0.5.

Injecting the mixed solution of each combination into the leaf blade from the back of the tobacco by using a disposable syringe for infection, culturing at the temperature of 25 +/-1 ℃ and the humidity of 70% for 8 hours in dark, and then culturing under the circulating illumination of 16 hours illumination/8 hours in dark for 24-48 hours to obtain fresh transgenic tobacco sheets. Wherein, the combination 1 is infected to obtain a transgenic tobacco sheet independently expressed by Zera-22-mCherry; the combination 2 is infected to obtain a transgenic tobacco sheet co-expressed by Zera-mCherry and NZP 1; infection with combination 3 yielded a tobacco sheet co-expressing the transgene by Zera-22-mCherry and NZP 1.

2.1.2 fluorescence Observation

Fluorescence observation was performed on the transgenic tobacco sheets 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 could be formed. As shown in fig. 8, NZP1 could not accumulate in the proteosome formed by Zera when Zera-mCherry and NZP1 were co-expressed (combination 3, see panel B of fig. 8). When Zera-22-mchery and NZP1 were co-expressed (combination 2, see panel a of fig. 8), NZP1 was able to accumulate in the Zera-forming proteosome and the fluorescent signals of NZP1-GFP and Zera-22-mchery were consistently located, indicating that NZP1 was able to relocate to the Zera-22-forming proteosome.

2.1.3 Western immunoblot detection

And (3) extracting total protein from the transgenic tobacco sheet 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, when NZP1-GFP was co-expressed with Zera-22-mCherry, there was significant accumulation of NZP 1-GFP.

As shown in panel B of FIG. 9, when NZP1-GFP was co-expressed with Zera-mCherry, there was no significant accumulation of NZP 1-GFP.

2.2, NZP2 with Zera-22-mCherry and Zera-mCherry transient transformation tobacco gene

Replacing the pHB-NZP1-GFP vector in the step 2.1.1 with the pHB-NZP2-GFP vector constructed in the step 1, and keeping the other methods in the step 2.1.1 unchanged to obtain a transgenic tobacco sheet co-expressed by Zera-mCherry and NZP2 and a transgenic tobacco sheet co-expressed by Zera-22-mCherry and NZP 2. The obtained transgenic tobacco pieces were subjected to fluorescence observation using GFP expression.

As a result, as shown in FIG. 10, when Zera-mCherry and NZP2 were co-expressed (see FIG. 10, panel B), NZP2 could not be accumulated in the proteosome formed by Zera. When Zera-22-mCherry and NZP2 were co-expressed (see Panel A of FIG. 10), the fluorescent signals of NZP2-GFP and Zera-22-mCherry were consistently located, indicating that NZP2 was able to relocate to the proteosome formed by Zera-22.

2.3, NZP3 with Zera-22-mCherry and Zera-mCherry transient transformation tobacco gene

Replacing the pHB-NZP1-GFP vector in the step 2.1.1 with the pHB-NZP3-GFP vector constructed in the step 1, and keeping the other methods in the step 2.1.1 unchanged to obtain a transgenic tobacco sheet co-expressed by Zera-mCherry and NZP3 and a transgenic tobacco sheet co-expressed by Zera-22-mCherry and NZP 3. The obtained transgenic tobacco pieces were subjected to fluorescence observation using GFP expression.

As a result, as shown in FIG. 11, when Zera-mCherry and NZP3 were co-expressed (see FIG. 11B), NZP3 could not be accumulated in the proteosome formed by Zera. When Zera-22-mCherry and NZP3 were co-expressed (see Panel A of FIG. 11), the fluorescent signals of NZP3-GFP and Zera-22-mCherry were consistently located, indicating that NZP3 was able to relocate to the proteosome formed by Zera-22.

In conclusion, the invention verifies that the target proteins NZP1, NZP2 and NZP3 respectively interact with prolamin 22-kD alpha-zein by a yeast two-hybrid system or an LCI system. The detection of an immunoelectron microscope of a corn kernel protein body proves that NZP1 is accumulated in the protein body, and the accumulation amount of NZP1 in a 22kD gliadin-deficient mutant material is obviously reduced. Further, it was confirmed that the proteins NZP1, NZP2 and NZP3 of interest can be accumulated in a protein body formed by Zera-22 by the interaction with 22-kD α -zein, as a result of expressing Zera-22 (fusion protein of Zera and 22-kD α -zein) and the protein NZP1, NZP2 or NZP3 of interest in tobacco and experiments expressing Zera and the protein NZP1, NZP2 or NZP3 of interest in tobacco, showing that Zera-22 induces protein formation in tobacco and the protein of interest is localized in the protein body formed by Zera-22.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<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

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

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