Gene capable of regulating starch content and storage protein accumulation in corn kernels and application thereof

Abstract

The invention discloses a gene capable of regulating starch content and storage protein accumulation in corn kernels and application of the gene, and belongs to the technical field of crop molecular biology. The invention discloses corn protein kinase capable of regulating and controlling starch content and storage protein accumulation in corn kernels. The corn protein kinase comprises corn protein kinase CKA1 and CKA3; the nucleotide sequence of the gene for coding the corn protein kinase CKA1 is as shown in SEQ ID NO. 1; the nucleotide sequence of the gene for coding the corn protein kinase CKA3 is as shown in SEQ ID NO. 2. Results of the invention show that corn protein kinases CKA1 and CKA3 can phosphorylate an important transcription factor Opaque2 of corn so as to achieve the purposes of regulating and controlling the starch content in corn kernels and storage protein accumulation, so that the protein content of the corn kernels is increased, and a reference is provided for obtaining a corn variety with high protein content.

Description

Technical Field

[0001] The invention belongs to the technical field of crop molecular biology, and in particular relates to a gene capable of regulating starch content and storage protein accumulation in corn kernels and an application thereof. Background Art

[0002] Corn is one of the most important food crops in the world. At present, corn is the largest crop in my country in terms of both planting area and total output. In addition to being one of the important food sources for Chinese people, most of my country's corn is used as feed in animal husbandry and poultry industry, providing an important source of energy and protein for farmed animals. It is also an important initial nitrogen source for Chinese people to obtain animal protein such as meat, eggs and milk. Therefore, increasing the protein content of corn has important economic benefits.

[0003] Corn endosperm accounts for 90% of the weight of the kernel and is the main storage place for nutrients such as starch and protein. Storage proteins in corn kernel endosperm account for 8% to 10% of the endosperm mass, including albumin, glutelin, globulin and prolamin, of which prolamin accounts for about 50% to 70% of the total mass of storage proteins.

[0004] Opaque2 (O2) transcription factor is a bZIP type transcription factor. It not only directly activates almost all prolamin coding genes except 16kD γ prolamin, but also cooperates with other transcription factors to activate the expression of various prolamin proteins. Prolamin-box Binding Factor 1 (PBF1) is a DOF type transcription factor that can bind to the P-box in the promoter of most prolamin genes. RNAi silencing of PBF1 leads to a severe decrease in the abundance of 22-kD prolamin, indicating that PBF1 is involved in the regulation of 22kD prolamin. OHP1 and OHP2 are two bZIP type transcription factors that can form heterodimers with O2. They can bind to the O2-like box in the gene promoter of 27kD prolamin. RNAi silencing of OHPs leads to a significant decrease in the abundance of 27kD prolamin. ZmbZIP22 is also a bZIP type transcription factor that transcriptionally activates 27kD prolamin. ZmMADS47 is a transcription factor that binds to the MADS box and can interact with O2 and activate the expression of α-alcohol-soluble protein and 50kD alcohol-soluble protein genes through interaction. NAC128 and NAC130 can specifically activate the transcription of 27kD alcohol-soluble protein by directly directing its promoter. Given the core position of O2 transcription factors in regulating zein, finding its upstream regulatory factors will help increase the expression and accumulation of alcohol-soluble proteins, thereby increasing the protein content in the grain. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a gene that can regulate the starch content and storage protein accumulation in corn kernels and its application. The research results of the present invention show that the proteins encoded by the maize protein kinase CKA1 and CKA3 gene sequences can phosphorylate the important transcription factor Opaque2 of corn, thereby achieving the purpose of regulating the starch content and storage protein accumulation in corn kernels.

[0006] The present invention provides the following technical solutions:

[0007] Technical solution 1: A corn protein kinase that can regulate the starch content and storage protein accumulation in corn kernels, the corn protein kinase includes corn protein kinase CKA1 and CKA3; the nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.2.

[0008] Technical solution 2: A gene encoding corn protein kinase CKA1 and CKA3, the nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown as SEQ ID NO.2.

[0009] Technical solution three: an expression vector containing the gene.

[0010] Technical solution 4: Application of the corn protein kinase, the gene or the expression vector in regulating the starch content in corn kernels.

[0011] Technical Solution 5: Application of the corn protein kinase, the gene or the expression vector in regulating the accumulation of storage proteins.

[0012] Technical Solution 6: Application of the corn protein kinase, the gene or the expression vector in agricultural high-protein corn breeding.

[0013] Technical Solution 7: A method for increasing storage protein in corn kernels, comprising knocking out corn protein kinases CKA1 and CKA3 to increase storage protein in corn kernels, the nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.2.

[0014] Technical Solution 8: A method for reducing the starch content in corn kernels, comprising knocking out corn protein kinases CKA1 and CKA3 to reduce the starch content in corn kernels, the nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.2.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] The present invention provides a gene capable of regulating the accumulation of storage proteins in corn kernels and its application, and relates to the application of two genes encoded by corn protein kinases CKA1 and CKA3 in regulating corn kernel starch and alcohol-soluble protein. The gene sequence encoded by CKA1 is shown in SEQ ID NO: 1, and the gene sequence encoded by CKA3 is shown in SEQ ID NO: 2. The results of the present invention show that the proteins CKA1 and CKA3 encoded by the gene sequence can phosphorylate Opaque2, an important transcription factor of corn. In addition, the present invention also utilizes CRISPR-Cas9 technology and adopts the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 to edit CKA1 and CKA3 respectively, and obtains CKA1 and CKA3 double gene deletion mutant plants after hybridization. The results show that compared with wild-type kernels, the starch content of mature kernels of transgenic mutants is significantly reduced, and the alcohol-soluble protein content is significantly increased, thereby increasing the protein content of corn kernels, and providing a reference for obtaining corn varieties with high protein content. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is an experiment to verify the interaction between protein kinases CKA1, CKA 3 and O2; A and B respectively represent the LCI protein interaction verification between protein kinases CKA1, CKA3 and O2; C and D respectively represent the GST pulldown protein interaction verification between protein kinases CKA1, CKA3 and O2;

[0019] Figure 2 In vitro phosphorylation O2 experiments of protein kinases CKA1 and CKA 3; A and B represent in vitro phosphorylation O2 experiments of protein kinases CKA1 and CKA3, respectively;

[0020] Figure 3 This is a test for CKA1 phosphorylation O2 transcriptional activation; **: P < 0.05; ns: no significant difference, where A and B represent CKA1 K63M and CKA3 K63M that have lost kinase activity, respectively;

[0021] Figure 4 Phenotypic observation of mature grains of wild type and cka1 cka3 double mutant; A is the comparison of mature grain size between wild type and cka1cka3 double mutant; B is the detection of 100-grain weight of mature grains of wild type and cka1 cka3 double mutant; C is the detection of starch content in mature grains of wild type and cka1 cka3 double mutant; D is the detection of total protein, alcohol-soluble protein and non-alcohol-soluble protein content in mature grains of wild type and cka1 cka3 double mutant; ***: P<0.001; **: P<0.0; *: P<0.05. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The technical means in the following examples, unless otherwise specified, were carried out according to the methods shown in Cloning: a Laboratory Manual, 2001, ISBN: 0879695773, or were operated according to the conditions recommended in the manufacturer's instructions.

[0028] Example 1

[0029] Maize protein kinases CKA1 and CKA3 interact with O2 protein

[0030] The present invention uses O2 rabbit polyclonal antibody to perform immunoprecipitation combined with mass spectrometry analysis on 18DAP immature endosperm proteins of the wild type and o2 mutant of the W22 inbred line background. The mass spectrometry identification results contain protein kinases CKA1 and CKA3 (the CKA1 gene sequence encoding protein kinase CKA1 is shown in SEQ ID NO: 1; the CKA3 gene sequence encoding protein kinase CKA3 is shown in SEQ ID NO: 2).

[0031] CKA1 gene sequence:

[0032]

[0033]

[0034] In order to test whether corn protein kinases CKA1 and CKA3 can directly interact with O2 in plants, the present invention uses luciferase complementation imaging (LCI) to detect the interaction between corn protein kinases CKA1 and CKA3 in tobacco, respectively constructs the semi-luciferase fusion expression vectors corresponding to corn protein kinases CKA1, CKA3 and O2 and transforms Agrobacterium, then infects tobacco leaves with the corresponding combination of Agrobacterium, and develops after culturing for 48 to 72 hours after infection. The results show that corn protein kinases CKA1, CKA3 and O2 interact with each other (see Figure 1 The present invention also uses GST-pulldown technology to verify that CKA1, CKA3 and O2 interact with each other (see Figure 1 C and D).

[0035] Example 2

[0036] Maize protein kinases CKA1 and CKA3 phosphorylate transcription factor O2 protein

[0037] In order to determine whether corn protein kinases CKA1 and CKA3 phosphorylate O2, the present invention conducted an in vitro phosphorylation test. The results of autoradiography showed that compared with the blank control, radioactive signals were detected in the corresponding band (about 90kD) of HIS-O2 protein treated with kinases, indicating that these two kinases can phosphorylate [γ]- 32 The addition of Pi to O2 proteins showed that they could phosphorylate O2 in vitro (see Figure 2 A and B).

[0038] Example 3

[0039] Effects of maize protein kinases CKA1 and CKA3 on the activity of O2 transcription factor

[0040] In order to detect the influence of corn protein kinase CKA1 and CKA3 on the activity of O2 transcription factor, the present invention carried out a dual luciferase transient transcription activity test. O2 is driven by CaMV 35S promoter as a transcription factor driving the reporter gene. CKA1 and CKA3 are also driven by CaMV 35S promoter and co-transformed with O2 into protoplasts. The active sites of lysine (Lysine, K) at position 63 of CKA1 and CKA3 kinases are mutated to methionine (Methionine, M) (CKA1K63M, CKA3 K63M) to block their kinase activity and serve as controls for the lack of CKA1 and CKA3 activity. Protoplasts are also co-transformed with O2, and the expression of kinases CKA1 K63M and CKA3 K63M and the normalized internal reference Renilla luciferase REN is driven by CaMV 35S promoter. The reporter gene uses firefly luciferase LUC, which is driven by the 22kDzein gene promoter sequence. The results of transient transcriptional activity assays showed that CKA1 and CKA3 inhibited the transcriptional activation activity of O2, while CKA1K63M and CKA3 K63M, which lost their kinase activity, could not inhibit the transcriptional activation activity of O2 (see Figure 3 A and B in FIG. 1 ). The results indicate that CKA1 and CKA3 can negatively regulate the transcriptional activation activity of O2. The CKA1 kinase protein sequence is shown in SEQ ID NO: 3; the CKA3 kinase protein sequence is shown in SEQ ID NO: 4.

[0041] CKA1 kinase protein sequence (the bold part is the mutation site)

[0042] MSKARVYADVNVLRPKEYWDYEALTVQWGEQDDYEVVRKVGRGKYSEVFEGINVNNNEKCIIKILKPVKKKKIKREIKILQNLCGGPNIVKLLDIVRDQHSKTPSLIFEYVNNTDFKVLYPTLTDYDIRYYIYELLKALDYCHSQGIMHRDVKPHNVMIDHELRKLRL IDWGLAEFYHPGKEYNVRVASRYFKGPELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNHDQLVKIAKVLGTDGLNVYLNKYRIELDPQLEALVGRHSRKPWLKFMNADNQHLVSPEAIDFLDKLLRYDHQERLTALEAMTHPYFQQVRAAENSRTRA(SEQ ID NO: 3).

[0043] CKA3 kinase protein sequence (the bold part is the mutation site)

[0044] MSKARVYTDVNVLRPKEYWDYEALTVQWGEQDNYEVVRKVGRGKYSEVFEGINVNNNEKCIIKILKPVKKKKIKREIKILQNLYGGPNIVKLLDIVRDQHSKTPSLIFEYVNNTDFKVLYPTLTDYDIRYYIYELLKALDYCHSQGIMHRDVKPHNVMIDHELRKLRL IDWGLAEFYHPGKEYNVRVASRYFKGPELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNHDQLVKIAKVLGTDGLNVYLNKYRIELDPQLEALVGRHSRKPWTKFINADNQHLVSHEAIDFLDKLLRYDHQDRLTAREAMTHPYFQQVRAAENSRTRA(SEQ ID NO: 4).

[0045] Example 4

[0046] O2 upstream kinases CKA1 and CKA3 can regulate the accumulation of grain proteins

[0047] The above shows that CKA1 and CKA3 can phosphorylate O2 protein in the grain and negatively regulate the activity of O2. It is speculated that CKA1 and CKA3 double mutant corn may have changes related to grain protein. Therefore, taking the corn B104 inbred line as an example, the present invention compares the phenotypes related to wild-type and cka1 cka3 double mutant corn grains. Using CRISPR-Cas9 technology, CKA1 and CKA3 were gene-edited using the sequences shown in SEQID NO: 5 and SEQ ID NO: 6, respectively, to obtain gene-edited corn materials with CKA1 and CKA3 functional deficiency, respectively. The offspring generation (F1) obtained after hybridization of the edited corns with CKA1 and CKA3 functional deficiency, the F1 material was self-pollinated to obtain F2, and F2 contained CKA1 and CKA3 double gene deletion mutants.

[0048] GTTGTCAGGAAGGTCGGGAG (SEQ ID NO: 5);

[0049] GAGGCGCTCACCGTTCAATG (SEQ ID NO: 6).

[0050] There is no significant difference in the survival ability of CKA1 and CKA3 double gene deletion mutant (cka1 cka3 double mutant) plants and wild-type plants. Therefore, the phenotype of corn kernels of CKA1 and CKA3 double gene deletion mutant (cka1 cka3 double mutant) and wild-type were compared, and it was found that the length and width of the kernels of cka1 cka3 double mutant were reduced compared with those of wild-type kernels (see Figure 4 Since the grains became smaller, the present invention further measured the 100-grain weight of the cka1 cka3 double mutant grains, and the results showed that the 100-grain weight of the cka1 cka3 double mutant grains decreased by 16% ( Figure 4 (middle B).

[0051] In addition, the present invention also compared the starch and protein content in the grains of the two plants. The results showed that the starch content of the cka1cka3 double mutant was lower than that of the wild type ( Figure 4 C), the total protein and alcohol-soluble protein contents increased compared with the wild type, while the non-alcohol-soluble protein decreased ( Figure 4 (D) The results showed that the upstream kinases CKA1 and CKA3 of O2 can regulate the accumulation of alcohol-soluble proteins. The double mutant had significantly increased alcohol-soluble proteins and total protein compared with the wild type. Therefore, CKA1 and CKA3 can increase the storage protein content of maize grains.

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A corn protein kinase capable of regulating starch content and storage protein accumulation in corn kernels, characterized in that: The corn protein kinase includes corn protein kinase CKA1 and CKA3; the nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.

2.

2. A gene encoding corn protein kinases CKA1 and CKA3, characterized in that: The nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.

2.

3. An expression vector containing the gene according to claim 2.

4. Use of the corn protein kinase according to claim 1, the gene according to claim 2 or the expression vector according to claim 3 in regulating the starch content in corn kernels.

5. Use of the corn protein kinase according to claim 1, the gene according to claim 2 or the expression vector according to claim 3 in regulating the accumulation of storage proteins.

6. Use of the corn protein kinase according to claim 1, the gene according to claim 2 or the expression vector according to claim 3 in agricultural high-protein corn breeding.

7. A method for increasing storage protein in corn kernels, characterized in that: The method includes knocking out corn protein kinases CKA1 and CKA3 to increase storage protein in corn kernels. The nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.

2.

8. A method for reducing starch content in corn kernels, characterized in that: The starch content of corn kernels is reduced by knocking out corn protein kinases CKA1 and CKA3. The nucleotide sequence of the gene encoding the corn protein kinase CKA1 is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the corn protein kinase CKA3 is shown in SEQ ID NO.2.

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