Application of SnRK2.6 gene in regulating and controlling protein content of crops

Abstract

The invention discloses application of a SnRK2.6 gene in regulating and controlling the protein content of crops, and belongs to the technical field of gene engineering. According to the invention, through overexpression of the SnRK2.6 gene, the content of prolamin, non-prolamin and total protein in crops can be increased. A corn SnRK2.6 overexpression strain is created through a transgenic technology, and in the overexpression strain, the content of prolamin, non-prolamin and total protein is remarkably increased, but in a knockout mutant, the content of prolamin, non-prolamin and total protein is opposite.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an application of SnRK2.6 gene in regulating the protein content of crops. Background Art

[0002] SnRK (Sucrose Non-Fermenting 1-Related Protein Kinase) is a class of Ser / Thr protein kinases with high conservation. According to sequence similarity, structure and function, it can be divided into three subfamilies: SnRK1, SnRK2 and SnRK3. SnRK1 has high similarity with yeast sucrose non-fermenting 1 (SNF1) and mammalian AMPK (AMP-activated protein kinase) in structure and function, and plays an important role in metabolic regulation. SnRK2 participates in osmotic stress and abscisic acid (ABA) response and plays an important role in stress signal transduction. SnRK3, also known as CBL-interacting protein kinases (CIPK), participates in various biotic and abiotic stress responses and regulates plant growth and development by binding to calcineurin B-like proteins (CBL).

[0003] SnRK2 family proteins contain about 341 to 365 amino acids and are hydrophilic proteins. Their Ser / Thr protein kinase structure is conservative, with a molecular weight of about 40 kDa, which is relatively small in the protein kinase subfamily. The amino acid sequence of SnRK2 consists of two parts: the N-terminal kinase domain and the C-terminal regulatory domain. The N-terminal kinase domain is highly conserved and is related to SNF1 and AMPK; while the C-terminal regulatory domain has significant evolutionary differences and has a regulatory function. The C-terminal domain I is about 30 amino acids in size and exists in all SnRK2 members and is affected by adverse stress; the C-terminal domain II is about 40 amino acids after domain I and is of great significance in responding to ABA responses. It is unique to ABA-dependent SnRK2 members. SnRK2 encodes mainly hydrophilic proteins of α-helix, β-turn and random coil, and the gene structure generally contains 9 to 10 coding sequences. It contains multiple cis-acting elements related to plant stress resistance, which can affect the expression of downstream genes by phosphorylating substrates and regulate protein activity, thereby achieving the effect of improving plant stress resistance.

[0004] SnRK2 family members are plant-specific Ser / Thr kinases that participate in the development of ABA-dependent plants and play an important role in various life activities of plants. They can regulate the stomatal aperture of plant leaves, respond to plant drought stress and salt stress, and participate in seed germination and development. Under salt stress, SnRK2 can promote root development, regulate sulfur metabolism and sugar metabolism, and play an important regulatory role in the growth and development of ABA-dependent plants; SnRK2 can also respond to the induction of osmotic stress through protein phosphorylation / dephosphorylation; SnRK2 overexpression plants can improve the root system, enhance the tolerance of plants to drought, salt and freezing stress, increase the expression of abiotic stress response genes, improve physiological indicators, reduce water loss rate, and enhance cell membrane stability. Therefore, SnRK2 has great application prospects in transgenic breeding and new variety resistance breeding, and the tolerance of crops to abiotic stress can be improved by overexpressing the SnRK2 gene.

[0005] At present, many studies have shown that SnRK2 is evolutionarily conserved and is a key signal that depends on ABA to respond to water stress. However, so far, research on the mechanism of SnRK2 is still relatively limited, mainly focusing on the study of the ABA signaling pathway; there are few studies on non-ABA-dependent signaling pathways that respond to abiotic stress, and there is no research based on its effect on the content of proteins in organisms. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of the SnRK2.6 gene in regulating the protein content of crops. The present invention finds that SnRK2.6 can effectively increase the content of alcohol-soluble protein, non-alcohol-soluble protein and total protein in corn.

[0007] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] The purpose of the present invention is to provide a use of SnRK2.6 gene in regulating the protein content of crops.

[0009] Furthermore, the crop is a monocotyledonous crop of the Poaceae family.

[0010] Furthermore, the crop is corn.

[0011] Furthermore, overexpression of the SnRK2.6 gene increased the content of alcohol-soluble protein, non-alcohol-soluble protein and total protein in crops.

[0012] Another object of the present invention is to provide a use of a preparation for promoting the expression of SnRK2.6 gene in the preparation of a product for increasing the protein content in crops.

[0013] Another object of the present invention is to provide an agent for increasing the protein content in crops, which comprises a component that promotes the expression of the above SnRK2.6 gene as an active ingredient, or comprises a protein encoded by the above SnRK2.6 gene as an active ingredient.

[0014] Another object of the present invention is to provide the use of the SnRK2.6 gene in improving corn germplasm resources or cultivating transgenic corn plants with high protein content.

[0015] Beneficial effects of the present invention:

[0016] The present invention creates a corn SnRK2.6 overexpression strain by transgenic technology. In the overexpression strain, the contents of alcohol-soluble protein, non-alcohol-soluble protein and total protein are significantly increased, while the opposite is true in the knockout mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the tissue expression pattern of ZmSnRK2.6 gene in maize;

[0018] Figure 2 The results of subcellular localization of ZmSnRK2.6;

[0019] Figure 3 It is the map of ZmSnRK2.6 knockout mutants;

[0020] Figure 4 The map of ZmSnRK2.6 overexpression vector and the detection and analysis of mRNA and protein; wherein, a is the map of ZmSnRK2.6 overexpression vector; b is the analysis of the mRNA level of ZmSnRK2.6 in wild-type and ZmSnRK2.6 overexpression seeds; c is the immunoblot analysis of proteins in wild-type and ZmSnRK2.6 overexpression seeds;

[0021] Figure 5 Detection of protein content in mature seeds of ZmSnRK2.6 knockout mutants; a is the phenotype observation of wild-type and ZmSnRK2.6 knockout material seeds; b is SDS-PAGE analysis of endosperm alcohol-soluble protein content of wild-type and ZmSnRK2.6 knockout material seeds; c is the statistical analysis of alcohol-soluble protein content of wild-type and ZmSnRK2.6 knockout material seeds; d is SDS-PAGE analysis of non-alcohol-soluble protein content of wild-type and ZmSnRK2.6 knockout material seeds; e is the statistical analysis of non-alcohol-soluble protein content of wild-type and knockout material seeds;

[0022] Figure 6Detection of protein content in mature seeds overexpressing ZmSnRK2.6; wherein, a is the phenotypic observation of wild-type and ZmSnRK2.6 overexpressing grains; b is the SDS-PAGE analysis of the alcohol-soluble protein content in the wild-type and ZmSnRK2.6 overexpressing materials; c is the statistical analysis of the alcohol-soluble protein content in the wild-type and ZmSnRK2.6 overexpressing materials; d is the SDS-PAGE analysis of the non-alcohol-soluble protein content in the wild-type and ZmSnRK2.6 overexpressing materials; e is the statistical comparison of the non-alcohol-soluble protein content in the wild-type and ZmSnRK2.6 overexpressing materials; f is the statistical comparison of the total protein content in the wild-type and ZmSnRK2.6 overexpressing materials. DETAILED DESCRIPTION

[0023] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0024] Example 1

[0025] 1. Tissue expression pattern of ZmSnRK2.6 gene in maize

[0026] We used the maize GDP website to predict the expression of ZmSnRK2.6 in different developmental stages and tissues of maize. Figure 1 (The picture is from the maize GDP website, which shows the tissue expression pattern of ZmSnRK2.6 in different tissues and seeds of corn at different developmental stages.).

[0027] Figure 1 The results showed that the ZmSnRK2.6 gene was highly expressed in maize leaves, roots, seed coats and embryos, indicating that ZmSnRK2.6 plays an important role in the growth and development of maize.

[0028] 2. mRNA extraction and fluorescence quantitative PCR

[0029] Freeze corn kernels at a fixed number of days after pollination. When ready for extraction, take them out and place them in a 2ml EP tube containing RNAase free. Add 200μl of RNA extraction solution (50mM Tris-HCl (PH 8.0), 100mM LiCl, 0.5mM EDTA (PH 8.0) 100μl 0.1% SDS). After centrifugation, add 200μl of chloroform to extract RNA, and add 1ml TRIZOL to extract RNA. Then add chloroform for further extraction, wash with 70% ethanol, and dissolve in RNAase free water for reverse transcription. Use NanoDrop Spectrophotometer to measure the RNA concentration, and use Promega's ImProm-II TM Reverse Transcription System (cat#A3800) was used to transcribe and synthesize cDNA. The reverse transcribed cDNA was diluted to 30 ng / μl for later use. The PCR reaction used TAKARA's SYBR Premix Ex Taq system (cat#RR420A). The PCR used a two-step amplification standard procedure (Step 1: 95℃for 30s; Step 2: 95℃for 5s, 60℃for 30s, +plate, 40 cycles followed by 95℃for 5s, Melt Curve 65℃to 95℃(increment 0.5℃ / 5s), +plate, end).

[0030] 3. Subcellular localization

[0031] Subcellular localization vectors were constructed, and the CDS sequence of SnRK2.6 was linked to pCAMBIA1300-35S-nGFP and pCAMBIA1300-35S-cGFP vectors. The constructed vectors were respectively transferred into Agrobacterium for injection into tobacco leaves. After 48-72 hours of culture, the site of injection of Agrobacterium was cut out and placed on a slide, and the subcellular localization of the protein was observed using a fluorescence confocal microscope. The results showed that similar to the control group, ZmSnRK2.6 was expressed in tobacco leaves, and ZmSnRK2.6 was detected in both the nucleus and cytoplasm ( Figure 2 , (Bottom) Subcellular localization of Free-eGFP in tobacco leaves. (Top) Subcellular localization of ZmSnRK2.6 in tobacco leaves. Bar=100 μm), indicating that ZmSnRK2.6-GFP is a nuclear and cytoplasmic localized protein.

[0032] Example 2

[0033] 1. Creation of genetic material of ZmSnRK2.6 knockout mutant

[0034] To verify the effect of ZmSnRK2.6 on maize quality and yield traits, the CRISPR-Cas9 technology was used to create zmsnrk 2.6 knockout mutants, and two different types of mutants were obtained, named s2.6-c1 and s2.6-c2. The s2.6-c1 homozygous mutant lacked 197bp, and the s2.6-c4 homozygous mutant lacked 118bp and had an additional 4bp sequence. Both types caused the ZmSnRK2.6 translation process to terminate prematurely ( Figure 3 ).

[0035] 2. Creation of ZmSnRK2.6 overexpression materials

[0036] The CDS of ZmSnRK2.6 was constructed into the pCAMBIA3300-Ubi-Flag vector ( Figure 4 a), and transformed into the young embryos of the inbred line B104. After two rounds of self-pollination, the obtained overexpression materials were homozygous overexpression plants and corresponding wild-type materials. In this experiment, we selected two groups of overexpression plants for subsequent phenotypic investigation, named OE1 and OE2.

[0037] Example 3

[0038] 1. Western blot detection of ZmSnRK2.6 mRNA and protein levels in wild-type and ZmSnRK2.6 overexpressing plants

[0039] The sample to be extracted was lysed with nonzein buffer (12.5mM sodium borate, 5% SDS, 2% 2-mercaptoethanol) at room temperature for 1 hour, and then centrifuged for western blot analysis. After electrophoresis, the protein was transferred to the PVDF membrane. After the transfer, the PVDF membrane was removed and rinsed with TBST for 5 minutes; the membrane was placed in a clean box, and 5% skim milk or 3% BSA was poured to completely submerge the PVDF membrane, and shaken at room temperature for 1-2 hours, or shaken at 4°C overnight. Remove the membrane and wash it in TBST for 5 minutes; dilute the antibody in 10ml TBST solution according to the concentration recommended in the instructions, and incubate at room temperature for 1 hour or at 4°C overnight (depending on the antibody binding effect). Remove the membrane and wash it in TBST 4 times, 15 minutes each time; dilute the antibody in 10ml TBST solution according to the concentration recommended in the instructions, and incubate at room temperature for 1 hour. The membrane was then taken out and washed 4 times in TBST, each time for 15 min. The membrane was dried with filter paper and placed on plastic wrap. The prepared ECL reaction solution was evenly spread on the membrane and kept at room temperature for 5 min (ECL reaction solution was from Invitrogen, cat# WP20005). After that, the Tanon-5200 chemiluminescence analyzer was used for imaging detection. The results are shown in the figure. Figure 4 .

[0040] like Figure 4 As shown, compared with the wild type, the mRNA level of ZmSnRK2.6 in ZmSnRK2.6 overexpressing grains increased significantly ( Figure 4 b). Flag-tagged protein can be detected in overexpressed material, but not in wild type ( Figure 4 c).

[0041] 2. Analysis and determination of protein content in corn kernels

[0042] The ears of ZmSnRK2.6 knockout mutant and overexpression plants with normal fruiting rate were selected for testing and the protein content in the grains was detected. The specific process is as follows:

[0043] Take a few mature dry seeds, dry them and grind them into powder. Weigh 100 mg of dry powder and place it in a 2 ml centrifuge tube. Add 1 ml of alcohol-soluble protein extract (1 ml 70% ethanol, 2% 2-mercaptoethanol (vol / vol), 3.75 mM sodium borate (PH 10), 0.3% SDS) and place it at room temperature for more than 2 hours, shaking or shaking overnight. Centrifuge the centrifuge tube containing the sample at 15000 rcf for 10 minutes. Pipette 0.1 ml of the supernatant into a 1.5 ml centrifuge tube, add 10 μl of 10% SDS solution, and mix well. Use a centrifugal concentrator to vacuum the sample at 65°C for 75 minutes. Add 100 μl of ddH2O to the centrifuge tube and gently mix or vortex to dissolve the protein.

[0044] Collect the lower precipitate from which alcohol-soluble proteins have been extracted once, add 1 ml of alcohol-soluble protein extracting solution again, and repeat the extraction of alcohol-soluble proteins three times so that no alcohol-soluble proteins remain in the sample. Place the sample precipitate in a vacuum concentrator at 65°C for 75 minutes for vacuum extraction. Add 1 ml of non-alcohol-soluble protein extracting solution (12.5 mM sodium borate, 5% SDS, 2% 2-mercaptoethanol) to the drained lower precipitate, vortex and shake thoroughly to mix, and place the centrifuge tube at room temperature for more than 2 hours or overnight to fully extract non-alcohol-soluble proteins. Place the centrifuge tube in a centrifuge and centrifuge thoroughly to separate the supernatant liquid and the lower precipitate. The supernatant liquid is the extracted non-alcohol-soluble protein liquid.

[0045] Denature the extracted alcohol-soluble and non-alcohol-soluble proteins and perform electrophoresis and staining analysis or use the BCA protein quantification kit (P0006C) to quantify the protein. Figure 5 and Figure 6 .

[0046] like Figure 5 and Figure 6The results of the test showed that the contents of alcohol-soluble proteins and non-alcohol-soluble proteins in the ZmSnRK2.6 knockout mutant were both reduced ( Figure 5 ae). In contrast, the contents of alcohol-soluble proteins, non-alcohol-soluble proteins and total protein in the grains overexpressing ZmSnRK2.6 were significantly increased ( Figure 6 af);

[0047] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. The use of SnRK2.6 gene in regulating the protein content of crops.

2. The use according to claim 1, characterized in that The crops are monocotyledonous crops of the Poaceae family.

3. The use according to claim 2, characterized in that: The crop is corn.

4. The use according to any one of claims 1 to 3, characterized in that: Overexpression of the SnRK2.6 gene increases the content of alcohol-soluble protein, non-alcohol-soluble protein and total protein in crops.

5. Use of a preparation for promoting SnRK2.6 gene expression in the preparation of a product for increasing the protein content in crops.

6. An agent for increasing the protein content in crops, characterized in that: The active ingredient is a component that promotes the expression of the SnRK2.6 gene in claim 1, or the protein encoded by the SnRK2.6 gene in claim 1.

7. Use of the SnRK2.6 gene described in claim 1 in improving corn germplasm resources or cultivating transgenic corn plants with high protein content.

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