Wheat grain starch content and grain weight related TabZIP9 gene, encoding protein and application of wheat grain starch content and grain weight related TabZIP9 gene

Abstract

The invention discloses a wheat grain starch content and grain weight related TabZIP9 gene, an encoded protein and an application thereof, the gene is knocked out or knocked down by utilizing a genetic engineering technology, so that a wheat variety with high grain starch content and grain weight is cultivated, and the yield of wheat is increased. A novel molecular breeding target gene and scheme are provided for high-yield breeding of wheat and starch quality improvement.

Description

Technical Field

[0001] The present invention belongs to the technical field of wheat breeding, and relates to the TabZIP9 gene related to wheat grain starch content and grain weight, the encoded protein and its application. Background Art

[0002] Starch is the most important storage substance in cereal crops, and the starch content in grains is of great significance for the formation of crop yield. In addition, the content and properties of starch are particularly important for the quality formation of cereal crop grains. The synthesis of starch in plants is a complex and delicate biological process involving a series of enzymatic reactions, which requires the participation of a large number of enzymes and related regulatory factors. At present, the direct synthesis pathway of starch has been studied relatively thoroughly, but the regulatory mechanism of this pathway is less understood. Therefore, cloning and identifying the key genes that regulate starch synthesis in plants has very important theoretical significance for enriching the regulatory network of starch synthesis. At the same time, it has potential application value for improving wheat yield and quality.

[0003] Studies have shown that several enzymes responsible for starch synthesis in several different tissues of synthetic plant starch are regulated at the transcriptional level. In recent years, while analyzing the functions of starch synthesis-related enzymes, a series of transcription factors involved in regulating starch synthesis genes have been successively identified in barley, rice, maize, and wheat. The bZIP transcription factor was the first transcription factor identified to regulate starch biosynthesis (Lohmer et al., 1991) and plays an important role in regulating starch synthesis. In rice, OsbZIP58 (also known as RISBZ1 or OsSMF1) can bind to the promoter regions of six starch synthesis genes (OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2) to regulate gene expression; the leucine zipper-structured transcription factor OsbZIP58 regulates the expression of multiple enzyme genes during rice endosperm starch synthesis and is a key regulatory factor in the rice endosperm starch synthesis pathway (Wang et al., 2013). In addition, OsbZIP58 can also activate the expression of the RPBF gene and interact with RPBF to jointly regulate starch synthesis (Yamamoto et al., 2006; Kawakatsu et al., 2009; Kim et al., 2017). In maize, ZmbZIP91 can bind to the promoters of key starch synthesis genes (such as AGPS1, SSI, SSIIIa, and SA1, etc.) to activate gene expression; the starch content of the mutant of the homologous gene vip1 of ZmbZIP91 in Arabidopsis thaliana decreases, while ZmbZIP91 can restore the phenotype of the vip1 mutant and enable normal starch synthesis (Chen et al., 2016). The ZmbZIP22 transcription factor can bind to the promoters of 8 key amylase genes. Overexpression of ZmbZIP22 increases the contents of soluble sugar and reducing sugar in rice and maize; the amylose content increases in the ZmbZIP22 mutant, indicating that ZmbZIP22 is a negative regulator of starch synthesis (Li et al., 2018; Dong et al., 2019). In wheat, 24 bZIP transcription factors that may be related to amylose synthesis were screened in the amylose mutant by combining transcriptome sequencing, qRT-PCR, and starch content (Kumar et al., 2018). TubZIP28 of Triticum urartu is preferentially expressed in the endosperm during the filling period. It can bind to the promoter of the cytosolic AGPase gene to activate its expression. Overexpression of TubZIP28 in common wheat increases the transcriptional level of cytosolic AGPase and the activity of AGPase enzyme, thereby increasing the starch content.The starch content of the TabZIP28 (the wheat homologous gene of TubZIP28) knockout line is reduced, and TubZIP28 and TabZIP28 play important roles in starch synthesis as activators of starch synthesis genes (Guo et al. 2020).

[0004] bZIP family transcription factors are generally considered to be related to plant response to stress, but their roles in starch synthesis are still unclear. In previous work, a new gene TabZIP9 was found by reverse genetics method. The wheat grain-specific transcription factor encoded by this gene may play a regulatory role in the synthesis of endosperm starch in grains. Studying the regulatory mechanism of TabZIP9 is of great significance for regulating the wheat starch synthesis pathway and then genetically improving wheat starch quality. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the TabZIP9 gene related to wheat grain starch content and grain weight, the encoded protein and their applications.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] 1. The TabZIP9 gene related to wheat grain starch content and grain weight, which comprises the following three homoeologous genes:

[0008] The TabZIP9-3A gene, whose coding region sequence is as shown in SEQ ID NO.1;

[0009] The TabZIP9-3B gene, whose coding region sequence is as shown in SEQ ID NO.2;

[0010] The TabZIP9-3C gene, whose coding region sequence is as shown in SEQ ID NO.3.

[0011] 2. The encoded protein TabZIP9 of the aforementioned TabZIP9 gene related to wheat grain starch content and grain weight, which comprises:

[0012] The encoded protein TabZIP9-3A of the TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.4;

[0013] The encoded protein TabZIP9-3B of the TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.5;

[0014] The encoded protein TabZIP9-3C of the TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.6.

[0015] 3. Application of knocking out the aforementioned TabZIP9 gene in increasing wheat grain starch content and grain weight.

[0016] 4. Application of inhibiting the aforementioned encoded protein TabZIP9 in increasing wheat grain starch content and grain weight.

[0017] 5. Application of the gRNA for knocking out the aforementioned TabZIP9 gene in increasing wheat grain starch content and grain weight.

[0018] 6. Application of the inhibitor of the aforementioned encoded protein TabZIP9 expression in increasing wheat grain starch content and grain weight.

[0019] 7. A breeding method for increasing wheat grain starch content and grain weight, the specific steps are as follows:

[0020] (1) Design sgRNA targeting the TabZIP9 gene, design inner primers according to pCBC-MT1T2 and sgRNA, and perform a first amplification using the MT1T2 plasmid as a template;

[0021] (2) Design outer primers MT1T2-F and MT1T2-R according to pBUE414 and sgRNA, and perform a second amplification using the first amplification product as a template to obtain a PCR product;

[0022] (3) Digest and ligate the PCR product to the pBUE414 vector, then transform Escherichia coli, perform colony PCR identification and sequence analysis of the sgRNA sequence in the CRISPR / Cas9 vector to obtain the knockout vector of the TabZIP9 gene;

[0023] (4) Use the Agrobacterium-mediated method to transform the knockout vector into wheat, identify the mutants with the TabZIP9 gene knocked out, and screen to obtain the transgenic positive plants with the TabZIP9 gene knocked out.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention relates to the TabZIP9 gene, the encoded protein and their applications related to wheat grain starch content and grain weight. By using genetic engineering techniques to knock out or knockdown this gene, wheat varieties with high grain starch content and grain weight are cultivated, providing a new molecular breeding target gene and scheme for high-yield breeding and starch quality improvement of wheat. Description of the Drawings

[0026] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0027] Figure 1 It is the knockout vector map of the TabZIP9 gene.

[0028] Figure 2 These are the target sites for TabZIP9 gene knockout and the editing status of mutant plants.

[0029] Figure 3 These are the phenotypic analyses of two TabZIP9 gene knockout lines and the wild-type Fielder; among them, A is the 1000-grain weight, and B is the grain starch content; * represents P < 0.05; ** represents P < 0.01; *** represents p < 0.001; **** represents p < 0.0001. Detailed implementation manners

[0030] Next, the preferred embodiments of the present invention will be described in detail in conjunction with the accompanying drawings.

[0031] The advantages and features of the present invention will become clearer as specific details are disclosed. However, the specific experimental methods involved in the following embodiments are all conventional methods or are implemented according to the conditions given in the manufacturer's user manual unless otherwise specified.

[0032] Unless otherwise specified, the technical means used in the embodiments are all conventional means mastered by those skilled in the art. The test methods in the following embodiments are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and consumables used in the experiments can be purchased on the market.

[0033] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any experimental methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation materials and methods mentioned in the text are only for demonstration purposes.

[0034] The pCBC-MT1T2, MT1T2 plasmid, and pBUE414 vector refer to the following literature: A CRISPR / Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14(1):327. 10.1186 / s12870-014-0327-y PubMed 25432517.

[0035] Examples

[0036] 1. Design sgRNAs targeting TabZIP9-A, TabZIP9-B, and TabZIP9-D simultaneously using WheatCrispr (https: / / crispr.bioinfo.nrc.ca / WheatCrispr / ), and align the found sgRNAs in Ensembl Plants (Ensembl plants) to check the sequence specificity. Select specific sgRNAs (Table 1). Design inner primers MT1T2-F0 and MT1T2-R0 (Table 2) based on the pCBC-MT1T2 and gRNA sequences, and perform a first amplification using the MT1T2 plasmid as a template. The reaction system is shown in Table 3.

[0037] Table 1

[0038]

[0039] Table 2

[0040]

[0041] Table 3

[0042]

[0043] The reaction program is as follows: First, pre-denature at 95°C for 1 min; denature at 98°C for 10 s, anneal at 58°C for 15 s, extend at 68°C for 40 s, for 35 cycles; finally, extend at 68°C for 5 min. After the amplification is completed, take 1 - 2 μL of the PCR product for electrophoresis detection. The band size is approximately 1 kb, and the remaining PCR product is used for the second amplification.

[0044] 2. Design outer primers MT1T2-F and MT1T2-R (Table 2) based on the pBUE414 and gRNA sequences, and perform a second amplification using the first amplification product as a template. The reaction system is shown in Table 4.

[0045] Table 4

[0046]

[0047]

[0048] The reaction program is the same as above. After the second amplification, the PCR product is electrophoresed in a 1% agarose gel and the target fragment (about 1 Kb) is cut out. Use a gel recovery kit to recover the target fragment. Store the recovered product at -20°C.

[0049] 3. Adopt a method of simultaneous digestion and ligation to ligate the PCR product to the pBUE414 vector (modified from pBUE411). The digestion and ligation system is shown in Table 5.

[0050] Table 5

[0051]

[0052] The reaction procedure was: 37 °C for 5 h, 50 °C for 5 min, 80 °C for 10 min.

[0053] Subsequently, Escherichia coli was transformed, colony PCR identification was carried out, and the sgRNA sequence in the CRISPR / Cas9 vector was sequenced and analyzed. After correct sequencing, plasmid extraction was performed according to the operation instructions of the plasmid extraction kit (OMEGA Bio-Tek D6943-01). The vector map is shown in Figure 1 .

[0054] 4. Agrobacterium-mediated genetic transformation

[0055] 0.25 μg of the plasmid obtained in the previous step was added to 50 μL of EHA105 Agrobacterium competent cells, and they were left standing on ice for 5 min, frozen in liquid nitrogen for 5 min, incubated in a water bath at 37 °C for 5 min, cooled on ice for 2 min, 700 μL of antibiotic-free LB (Coolaber PM0010-500g) was added, and they were shaken on a shaker at 28 °C for 2 - 3 h, centrifuged at 6000 r / min for 1 min, part of the supernatant was discarded, the remaining supernatant and the precipitate were pipetted and mixed evenly, spread on an LB solid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and after culturing at 28 °C for about 2 d, single colonies were picked for PCR verification. 100 μL of the bacterial liquid with a positive test result was added to a 50% (mass concentration) sterile glycerol aqueous solution and stored in a -80 °C refrigerator. This vector was transformed into the spring wheat variety Fielder using the Agrobacterium-mediated method.

[0056] 5. Identification of mutant plants and types of editing sites

[0057] Identification and screening of mutants:

[0058] When the T0 generation plants grew to the seedling stage with 4 - 5 leaves, a small amount of leaves were taken for genotype identification, and wheat genomic DNA was extracted by the improved CTAB method. The specific operations were as follows:

[0059] a. Fresh leaves were put into a 2.0 mL EP tube, and at the same time, steel beads with a diameter of 6 mm were put in. After cooling in liquid nitrogen, the tissue was ground into powder with a grinding machine;

[0060] b. 800 μL of preheated 2×CTAB (cetyltrimethylammonium bromide) solution (Coolaber SL2071) was added, and after shaking well, it was placed in a water bath at 65 °C for 30 min, and during this period, it was inverted up and down every 10 min to prevent insufficient water bath;

[0061] c. Add 600 μL of chloroform-isoamyl alcohol mixture (24:1, v / v), invert to mix well and let stand for stratification, then centrifuge at 10,000 r / min for 10 min at room temperature;

[0062] d. Carefully aspirate the supernatant, add an equal volume of pre-cooled isopropanol solution, invert to mix well and DNA precipitation can be seen, or cool at 4 °C for more than 30 min and centrifuge at 10,000 r / min for 10 min;

[0063] e. Discard the supernatant, add 1 mL of 75% ethanol aqueous solution by volume to wash and pipette the precipitate, centrifuge at 10,000 r / min for 5 min, and repeat once;

[0064] f. Discard the supernatant, air-dry at room temperature and dissolve in water. After complete dissolution, it can be used for experimental analysis or stored short-term at -20 °C.

[0065] Design primers according to the Cas9 sequence on the vector (Table 6) to detect whether the expression vector has been successfully inserted into the genome of the obtained transgenic plants. If the T0 generation plants are Cas9 positive, then specifically amplify the sequence near the target to detect gene editing.

[0066] Table 6

[0067]

[0068] The identification results are shown in Figure 2 .

[0069] 5. Agronomy, yield, grain and chemical composition of KO plants

[0070] At maturity, further count the 1000-grain weight and starch content of wild-type Fielder and this gene knockout line (tabzip9-1, tabzip9-2). The results show that ( Figure 3 ), compared with Fielder, the 1000-grain weight and starch content of the knockout line are significantly increased. This indicates that the knockout of this gene has an obvious effect of increasing the 1000-grain weight and starch content. These results show that the knockout of this gene has caused the characteristics of increased starch content and increased grain weight in wheat, which has important potential for breeding high-yield wheat varieties.

[0071] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. The TabZIP9 gene related to wheat grain starch content and grain weight, characterized in that, It comprises the following three parts of homologous genes: TabZIP9-3A gene, whose coding region sequence is as shown in SEQ ID NO.1; TabZIP9-3B gene, whose coding region sequence is as shown in SEQ ID NO.2; TabZIP9-3C gene, whose coding region sequence is as shown in SEQ ID NO.

3.

2. The encoded protein TabZIP9 of the TabZIP9 gene related to wheat grain starch content and grain weight according to claim 1, characterized in that, It comprises: The encoded protein TabZIP9-3A of TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.4; The encoded protein TabZIP9-3B of TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.5; The encoded protein TabZIP9-3C of TabZIP9-3A gene, whose amino acid sequence is as shown in SEQ ID NO.

6.

3. The application of knocking out the TabZIP9 gene described in claim 1 in increasing the starch content and grain weight of wheat grains.

4. The application of inhibiting the encoded protein TabZIP9 described in claim 2 in increasing the starch content and grain weight of wheat grains.

5. The application of the gRNA for knocking out the TabZIP9 gene described in claim 1 in increasing the starch content and grain weight of wheat grains.

6. The application of the inhibitor of the encoded protein TabZIP9 expression described in claim 2 in increasing the starch content and grain weight of wheat grains.

7. A breeding method for increasing the starch content and grain weight of wheat grains, characterized in that, The specific steps are as follows: (1) Design the sgRNA targeting the TabZIP9 gene described in claim 1, design the inner primers according to pCBC-MT1T2 and the sgRNA, and perform a first amplification using the MT1T2 plasmid as a template; (2) Design the outer primers MT1T2-F and MT1T2-R according to pBUE414 and the sgRNA, and perform a second amplification using the first amplification product as a template to obtain a PCR product; (3) Digest and ligate the PCR product to the pBUE414 vector, then transform Escherichia coli, perform colony PCR identification and sequence analysis on the sgRNA sequence in the CRISPR / Cas9 vector to obtain the knockout vector of the TabZIP9 gene; (4) Use the agrobacterium-mediated method to transform the knockout vector into wheat, identify the mutants with the TabZIP9 gene knocked out, and screen to obtain the transgenic positive plants with the TabZIP9 gene knocked out.

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