TaMADS7-D gene, encoded protein and application of TaMADS7-D gene in increasing starch content and grain weight of wheat grains

Abstract

The invention relates to a TaMADS7-D gene, an encoded protein and an application of the TaMADS7-D gene in improving the starch content and the grain weight of wheat grains, the TaMADS7-D gene is over-expressed by utilizing a genetic engineering technology, so that a wheat variety with high starch content and grain weight is cultivated, and the yield of the wheat is improved. 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 invention belongs to the technical field of wheat breeding, and relates to a TaMADS7-D gene, a coded protein and an application thereof in improving the starch content and grain weight of wheat grains. Background Art

[0002] The main storage substances in grains are starch and protein. Starch is the main storage carbohydrate in the endosperm of cereals, accounting for about 70% of the dry weight of grains and providing 80% of the energy consumed by people. Starch is not only a key factor affecting grain yield, but also an important raw material for food production and industrial processing. Therefore, overexpressing target genes through molecular breeding can quickly increase the starch content and grain weight of wheat grains, thereby efficiently cultivating high-yield and high-quality wheat germplasm materials and varieties. Summary of the invention

[0003] In view of this, the object of the present invention is to provide the TaMADS7-D gene, the encoded protein and the application thereof in increasing the starch content and grain weight of wheat grains.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. TaMADS7-D gene, whose CDS sequence is shown in SEQ ID NO.1.

[0006] 2. The amino acid sequence of the protein encoded by the aforementioned TaMADS7-D gene is shown in SEQ ID NO.2.

[0007] 3. The breeding application of overexpressing the aforementioned TaMADS7-D gene in improving wheat starch content and grain weight.

[0008] As one of the preferred technical solutions, the starch content and grain weight of wheat grains are increased.

[0009] 4. Increasing the expression level of the aforementioned encoded protein or its homologous protein for breeding application in increasing the starch content and grain weight of wheat.

[0010] As one of the preferred technical solutions, the starch content and grain weight of wheat grains are increased.

[0011] 5. A breeding method for increasing starch content and grain weight of wheat, the specific steps are as follows:

[0012] (1) Construction of an overexpression vector of the TaMADS7-D gene;

[0013] (2) Using the overexpression vector to transform wheat via Agrobacterium-mediated method, transgenic wheat plants were obtained.

[0014] As one of the preferred technical solutions, the specific method of step (1) is:

[0015] (1-1) Specific amplification of the CDS sequence of the TaMADS7-D gene: used to overexpress the TaMADS7-D gene;

[0016] (1-2) Prepare pWMB-110 plasmid: a plasmid suitable for wheat overexpression system, which includes Ubi promoter;

[0017] (1-3) Digestion with restriction endonucleases: Digest the pWMB-110 plasmid with restriction endonucleases to connect it with the CDS sequence of the TaMADS7-D gene with the same sticky ends;

[0018] (1-4) Ligation: The CDS sequence of the TaMADS7 gene was ligated with the pWMB-110 plasmid that had been digested with enzymes to form the TaMADS7-D overexpression vector.

[0019] (1-5) Colony PCR detection: PCR detection was performed to ensure that the CDS sequence of the TaMADS7-D gene was successfully connected to the pWMB-110 plasmid;

[0020] (1-6) Sequencing analysis of the CDS sequence of the TaMADS7-D gene: The CDS sequence of the TaMADS7-D gene in the pWMB-110 vector was sequenced to confirm its correctness and pairing with the TaMADS7-D gene.

[0021] As one of the preferred technical solutions, step (2) specifically includes: induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, seedling hardening, and transplanting.

[0022] The beneficial effects of the present invention are:

[0023] The present invention relates to the TaMADS7-D gene (TraesCS7D02G261600), the encoded protein and the application thereof in wheat breeding for increasing starch content and grain weight. The present invention overexpresses the TaMADS7-D gene through genetic engineering technology, thereby cultivating wheat varieties with increased starch content and grain weight, and provides a new molecular breeding gene and method for high-yield breeding and quality improvement of wheat.

[0024] The experimental results showed that compared with the wild type, the TaMADS7-D gene overexpression strain had increased thousand-grain weight and starch content. This indicated that the TaMADS7-D protein had the potential to regulate the thousand-grain weight and starch content of wheat grains and could be applied to improve wheat quality. The present invention aims to cultivate new high-yield and high-quality wheat varieties.

[0025] The present invention overexpresses the TaMADS7-D gene of wheat through transgenic technology to cultivate high-yield and high-quality wheat varieties. Specifically, an overexpression vector of the TaMADS7-D gene is constructed, including the CDS sequence of the TaMADS7-D gene, the Ubi promoter, etc. The vector construction includes the steps of restriction endonuclease digestion, ligation, colony PCR detection, sequencing analysis and transformation.

[0026] The present invention introduces the constructed TaMADS7-D gene overexpression vector into the wheat material Fielder through Agrobacterium-mediated method, so that it carries the overexpression system. In the subsequent generation separation process, a variety of molecular biological techniques, including PCR and qRT-PCR analysis, are used to identify and analyze the transgenic wheat plants. Through these techniques, it is determined whether the TaMADS7-D gene overexpression plant is obtained.

[0027] The transgenic wheat plants were planted in an isolated field at the same time as the wild-type plants. After the plants matured, a comparative analysis of thousand-grain weight and starch content was performed to determine the overexpression plants of the TaMADS7-D gene with increased thousand-grain weight and starch content. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0029] Figure 1 To identify the genotype of TaMADS7-D gene overexpression materials, the relative expression levels of TaMADS7-D gene in overexpression plants and wild-type Fielder were displayed.

[0030] Figure 2 Statistics of grain appearance and grain width of TaMADS7-D gene overexpressing plants and wild-type Fielder, where A represents grain appearance and B represents grain width.

[0031] Figure 3 Statistics of thousand-grain weight and starch content of TaMADS7-D gene overexpressing plants and wild-type Fielder, where A is thousand-grain weight and B is starch content.

[0032] In the figures, **, P<0.01 indicates extremely significant differences. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0034] (1) Construction of overexpression vector and genetic transformation of wheat

[0035] TaMADS7-D gene was amplified using TaMADS7-D conservative primers (nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4), and the reaction conditions were: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min. PCR product was ligated to Cloning Vector (purchased from Beijing Quanshijin Biological, catalog number: CT101-01), transform Escherichia coli, pick single clones, perform PCR detection and sequencing of bacterial solution, extract plasmids from bacterial solution with correct sequencing, and obtain plasmids connected with TaMADS7-D gene. Using this plasmid as a template, using primer sequences containing adapters (nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6), amplify the gene fragment containing the adapter sequence of the overexpression vector again.

[0036] According to the literature “Optimization of Agrobacterium-mediated transformation inspring bread wheat using mature and immature embryos” (Kumar R., Mamrutha HM, Kaur A., ​​Venkatesh K., Sharma D., Singh GP (2019) Molecular Biology Reports 46: 1845-1853), the TaMADS7-D gene was connected to the pWMB110 plasmid fused with GFP to obtain the overexpression vector of TaMADS7-D.

[0037] Reference "Generation of marker-free transgenic hexaploid wheat via an

[0038] Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties" (Wang K., Liu H., Du L. and Ye X. (2017) Plant Biotechnol. J. 15: 614-623), the vector was transformed into the spring wheat variety Fielder using the Agrobacterium-mediated method, and genetic transformation was carried out by the transgenic platform of our research group. The formulas of various culture media refer to "Ishida, Y., Tsunashima, M., Hiei, Y. and Komari, Y. (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In Agrobacterium Protocols: Volume 1. Methods in Molecular Biology, vol. 1223 (Wang, K., ed), pp. 189–198. New York: Springer Science The transformation process includes induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, seedling hardening and transplanting. The specific operations are as follows:

[0039] Induction: Prepare a culture medium containing an appropriate amount of hormones, such as 1 mg / L 2,4-D (Beijing Bao Biotechnology Co., Ltd.), and select appropriate plant materials, such as leaves, stem segments, or embryos. Place the plant materials in the culture medium and ensure that they are fully covered. Control the temperature, humidity, and light conditions, such as dark culture at 25°C, to promote the induction process.

[0040] Subculture: Removing plant tissue or cells from the original culture, isolating them, and transplanting them onto new media. Be sure to use fresh media and sterile techniques to prevent contamination. Subculture regularly to prevent aging and death of the tissue.

[0041] Pre-cultivation: Place the plant material in a culture medium containing basic nutrients for a period of pre-cultivation, adjust the temperature and humidity, allow the plant material to gradually adapt to the culture conditions, and gradually reduce the plant material's dependence on external conditions before transitioning to the formal culture medium.

[0042] Co-cultivation: Select the plant materials that need to be co-cultivated and place them together on the culture medium.

[0043] Make sure the hormones and nutrients in the culture medium are suitable for the growth needs of both plants and monitor their interaction or cooperative growth.

[0044] Resistance screening: The resistance gene bar for the herbicide glufosinate can be introduced by gene transformation or other appropriate methods. 2 mg / L Basta (purchased from Beijing Bao Biotechnology Co., Ltd.) is added to the culture medium and cultured in the dark for 14 days to screen out callus tissues that show resistance to the herbicide.

[0045] Differentiation: The hormone type and concentration in the culture medium were adjusted, such as containing 1 mg / L trans-zeatin and 1 mg / L IAA (Beijing Bao Biotechnology Co., Ltd.) to promote callus differentiation and budding.

[0046] Provide appropriate light and temperature, simulate the developmental environment in plants, monitor the developmental status of cells and tissues, and ensure that the expected differentiation level is achieved.

[0047] Rooting culture: Use a culture medium containing an appropriate amount of rooting hormone, such as adding 1 mg / L IAA (Beijing Bao Biotechnology Co., Ltd.) to the culture medium, culture the plant tissue or seedlings on this culture medium, and provide appropriate temperature and light, such as 25°C and light 100 μmol / m 2 / s to promote root development, monitor the rooting process, and ensure healthy root development.

[0048] Hardening of seedlings: Increase external light and wind stimulation, such as 25℃ and light 300μmol / m 2 / s, strengthen the plant's structure, gradually reduce its dependence on the culture medium, and allow wheat plant seedlings to adapt to the soil or other growth media.

[0049] Transplantation: Carefully remove the transgenic plant seedlings, make grooves or holes in the target growth medium, place the plant seedlings in the cleaned medium, ensure that sufficient water and nutrients are provided to support the growth of the plants, monitor the plant's adaptation process, and ensure the survival of the transplant.

[0050] Wheat pWMB110 plasmid reference "Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outerglumes and grains" (Jing L., Zhaoyan C., Zhihui W., Zhaoheng Z., Xiaoming X., Zihao W., Lingling C., Long S., Xuejiao C., Man F., Xiaobo W., Yanhong L., Zhaorong H., Jiewen X., Zhenqi S., Huiru P., Mingming X., Yingyin Y., Weilong G., Qixin S., Jie L., and Zhong N. (2021) Molecular Plant 14:1-17) were obtained.

[0051] Three independent overexpression lines were obtained through expression level identification.

[0052] Total RNA from these overexpressing wheat plants was extracted using the Trizol method.

[0053] Using fresh leaves of wheat seedlings as materials, the main steps are as follows:

[0054] Take a small amount of fresh sample and freeze it in liquid nitrogen, then grind it thoroughly with a grinder, then quickly add 1 mL of Trizol reagent, gently shake to mix, and let it stand at room temperature for 5 minutes to lyse.

[0055] Add 200 μL of chloroform to extract proteins and other substances, immediately shake for 15 seconds, and let stand at room temperature for 3 minutes.

[0056] Use a centrifuge set at 4°C, 12,000 rpm, and centrifuge for 15 minutes.

[0057] Carefully pipette the supernatant (about 500 μL) into a new centrifuge tube, add an equal volume of isopropanol, invert to mix, and let stand at room temperature for 10 minutes.

[0058] Use a centrifuge set at 4°C, 12,000 rpm, and centrifuge for 15 minutes to precipitate RNA.

[0059] The following operations were performed on ice:

[0060] Pour off the supernatant, add 1 mL of 75% ethanol aqueous solution to wash the RNA precipitate, and mix gently.

[0061] Use a centrifuge set at 4°C, 7500 rpm, and centrifuge for 5 minutes.

[0062] The supernatant was discarded, the tube was centrifuged again, the residual ethanol was aspirated, and the centrifuge tube containing the RNA precipitate was dried on ice in a fume hood for 15 minutes.

[0063] Add an appropriate amount of DEPC-water to dissolve the RNA precipitate and place on ice for 30 minutes to fully dissolve the RNA.

[0064] The RNA concentration was measured using a NanoDrop instrument and then stored in a -80°C refrigerator for later use.

[0065] cDNA Synthesis:

[0066] RNA was reverse transcribed into cDNA using the Takara Biotech reverse transcription kit (Cat. No.: RR047A). The specific steps are as follows:

[0067] Add the reagents listed in Table 1 to a 200 μL centrifuge tube in order.

[0068] Table 1 Reaction system for removing genomic DNA

[0069]

[0070] All components were collected to the bottom of the tube by instant centrifugation, mixed and kept at 42°C for 2 min, cooled on ice, and then added as shown in Table 2 according to the table below.

[0071] Table 2 Reverse transcription system

[0072]

[0073]

[0074] All components were collected to the bottom of the tube by instant centrifugation, mixed and reacted under the following conditions: 37°C, 15 min; 85°C, 5 s; cooled on ice, and the obtained cDNA was stored at -20°C for later use.

[0075] In order to determine whether these overexpression wheat plants were positive, the transgenic plants were identified by amplifying specific primers of the pWMB110 vector backbone (nucleotide sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8).

[0076] The TaMADS7-D gene was quantitatively amplified using qPCR-TaMADS7-D primers (nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10) to determine the specific expression level. The results were ( Figure 1) showed that the expression levels of TaMADS7-D overexpression lines (#1, #2 and #3) were significantly increased.

[0077] (2) Phenotypic identification of overexpressing plants

[0078] refer to Figure 2 , it was observed that the grain width of TaMADS7-D overexpression lines (#1, #2 and #3) was significantly larger than that of wild-type Fielder at maturity. Statistical analysis of the grain width of Fielder and TaMADS7-D overexpression lines (#1, #2 and #3) showed that the grain width of TaMADS7-D overexpression lines (#1, #2 and #3) was significantly increased compared with Fielder.

[0079] refer to Figure 3 , the thousand-grain weight and starch content of wild-type Fielder and TaMADS7-D overexpression lines (#1, #2 and #3) were further counted at maturity. The results showed that compared with Fielder, the thousand-grain weight and starch content of TaMADS7-D overexpression lines (#1, #2 and #3) were significantly increased. This indicates that TaMADS7-D overexpression has a significant effect on increasing starch content. These results indicate that TaMADS7-D overexpression causes the characteristic of increased starch content in wheat, which has important potential for breeding high-yield wheat varieties.

[0080] 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 it. 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. TaMADS7-D gene, characterized in that Its CDS sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the TaMADS7-D gene according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. The breeding application of overexpressing the TaMADS7-D gene described in claim 1 to improve the starch content and grain weight of wheat.

4. The application of increasing the expression level of the encoded protein or its homologous protein according to claim 2 in breeding to increase the starch content and grain weight of wheat.

5. A breeding method for increasing wheat starch content and grain weight, characterized in that: The specific steps are as follows: (1) Construction of an overexpression vector of the TaMADS7-D gene; (2) Using the overexpression vector to transform wheat via Agrobacterium-mediated method, transgenic wheat plants were obtained.

6. The breeding method according to claim 5, characterized in that The specific method of step (1) is: (1-1) Specific amplification of the CDS sequence of the TaMADS7-D gene: used to overexpress the TaMADS7-D gene; (1-2) Prepare pWMB-110 plasmid: a plasmid suitable for wheat overexpression system, which includes Ubi promoter; (1-3) Digestion with restriction endonucleases: Digest the pWMB-110 plasmid with restriction endonucleases to connect it with the CDS sequence of the TaMADS7-D gene with the same sticky ends; (1-4) Ligation: The CDS sequence of the TaMADS7-D gene was ligated with the pWMB-110 plasmid that had been digested with enzymes to form a TaMADS7-D overexpression vector. (1-5) Colony PCR detection: PCR detection was performed to ensure that the CDS sequence of the TaMADS7-D gene was successfully connected to the pWMB-110 plasmid; (1-6) Sequencing analysis of the CDS sequence of the TaMADS7-D gene: The CDS sequence of the TaMADS7-D gene in the pWMB-110 vector was sequenced to confirm its correctness and pairing with the TaMADS7-D gene.

7. The breeding method according to claim 5, characterized in that Step (2) specifically includes: induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, seedling hardening, and transplanting.

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