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

Abstract

The present application relates to TaMADS7-D gene, coding protein and application thereof in improving wheat grain starch content and grain weight, and through gene engineering technology, the TaMADS7-D gene is overexpressed, so that the wheat variety with high grain starch content and grain weight is cultivated, and a new molecular breeding target gene and scheme are provided for high-yield breeding and starch quality improvement of wheat.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat breeding and relates to a TaMADS7-D gene, an encoded protein and applications thereof in improving the starch content and grain weight of wheat grains. Background Art

[0002] The primary storage substances in wheat grains are starch and protein. Starch, the primary storage carbohydrate in the endosperm, accounts for approximately 70% of the grain's dry weight and provides 80% of the energy consumed by humans. Starch is not only a key factor influencing grain yield but also an important raw material for food production and industrial processing. Therefore, overexpressing target genes through molecular breeding can rapidly increase wheat grain starch content and weight, thereby efficiently breeding high-yielding and high-quality wheat germplasm 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 use 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. Breeding application of overexpressing the aforementioned TaMADS7-D gene to increase 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 applications 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 wheat starch content and grain weight, comprising the following steps:

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

[0013] (2) Transform wheat using the overexpression vector via Agrobacterium-mediated method to obtain transgenic wheat plants.

[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 the Ubi promoter;

[0017] (1-3) Digestion with restriction enzymes: Digest the pWMB-110 plasmid with restriction enzymes to allow ligation 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 enzyme-digested pWMB-110 plasmid 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 its application in wheat breeding to increase starch content and grain weight. The present invention uses genetic engineering techniques to overexpress the TaMADS7-D gene, thereby breeding wheat varieties with increased starch content and grain weight. This provides a new molecular breeding gene and method for high-yield wheat breeding and quality improvement.

[0024] Experimental results showed that TaMADS7-D gene-overexpressing strains had increased 1000-grain weight and starch content compared to wild-type wheat. This suggests that the TaMADS7-D protein has the potential to regulate 1000-grain weight and starch content in wheat and can be applied to improve wheat quality. This invention aims to cultivate new high-yield and high-quality wheat varieties.

[0025] The present invention uses transgenic technology to overexpress the wheat TaMADS7-D gene to cultivate high-yield, high-quality wheat varieties. Specifically, a TaMADS7-D gene overexpression vector is constructed, comprising the CDS sequence of the TaMADS7-D gene and the Ubi promoter. Vector construction includes 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 via Agrobacterium-mediated transfection, thereby carrying the overexpression system. During subsequent generation separation, the transgenic wheat plants are identified and analyzed using various molecular biology techniques, including PCR and qRT-PCR analysis. These techniques determine whether plants overexpressing the TaMADS7-D gene have been obtained.

[0027] Transgenic wheat plants and wild-type plants were planted simultaneously in isolated fields, and a comparative analysis of thousand-grain weight and starch content was performed after the plants matured to determine that overexpression plants of the TaMADS7-D gene with increased thousand-grain weight and starch content were obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, 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 The data are for the 1000-grain weight and starch content of the TaMADS7-D gene overexpressing plants and wild-type Fielder, where A is the 1000-grain weight and B is the starch content.

[0032] In the accompanying 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 conserved primers (nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4) under the following reaction conditions: 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. The TaMADS7-D gene was linked to a cloning vector (purchased from Beijing Quanshijin Biotechnology, catalog number CT101-01) and transformed into E. coli. Single clones were selected, and the culture medium was tested by PCR and sequenced. Plasmids containing the correct sequence were extracted from the culture medium to obtain a plasmid containing the TaMADS7-D gene. Using this plasmid as a template, primers containing linker sequences (nucleotide sequences shown in SEQ ID NOs. 5 and 6) were used to further amplify the gene fragment containing the linker sequence of the overexpression vector.

[0036] With reference to “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 ligated into the pWMB110 plasmid fused with GFP to obtain an 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 performed using 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 hormone, such as 1 mg / L 2,4-D (Beijing Bao Biotechnology Co., Ltd.). Select appropriate plant material, such as leaves, stem segments, or embryos. Place the plant material in the culture medium, ensuring adequate coverage. Control temperature, humidity, and light conditions, such as incubating in the dark at 25°C, to facilitate the induction process.

[0040] Subculture: Removing plant tissue or cells from the original culture, isolating them, and transplanting them onto a new medium. Ensure fresh medium and aseptic technique are used to prevent contamination. Subculture regularly to prevent tissue aging and death.

[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] Ensure that 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 herbicide phosphinothricin resistance gene, bar, can be introduced through genetic transformation or other appropriate methods. 2 mg / L Basta (purchased from Beijing Bao Biotechnology Co., Ltd.) is added to the culture medium and incubated in the dark for 14 days to screen for calli that exhibit herbicide resistance.

[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 to simulate the developmental environment in plants, monitor the developmental status of cells and tissues, and ensure that the expected level of differentiation is achieved.

[0047] Rooting culture: Use a culture medium containing an appropriate amount of rooting hormone, such as 1 mg / L IAA (Beijing Bao Biotechnology Co., Ltd.), culture plant tissues 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 the development of the root system, monitor the rooting process, and ensure the healthy development of the root system.

[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 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, and place the plant seedlings in the cleaned culture medium. Ensure that sufficient water and nutrients are provided to support the growth of the plants, monitor the plant's adaptation process, and ensure that the transplant survives.

[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 wheat leaves at the seedling stage 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 using 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 it 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, mix thoroughly by inversion, 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 determined using a NanoDrop instrument and then stored in a -80°C freezer until 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 the order shown.

[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 incubated at 42°C for 2 minutes, cooled on ice, and then added as shown in Table 2.

[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 overexpressing wheat plants were positive, 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. 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 At maturity, it was observed that the grain width of the TaMADS7-D overexpressing lines (#1, #2, and #3) was significantly larger than that of the wild-type Fielder. Statistical analysis of the grain width of Fielder and TaMADS7-D overexpressing lines (#1, #2, and #3) showed that the grain width of the TaMADS7-D overexpressing lines (#1, #2, and #3) was significantly increased compared to 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 the maturity stage. 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 and are not limiting. 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. Overexpression TaMADS7-D The application of the gene in increasing the starch content of wheat is characterized in that: TaMADS7-D The CDS sequence of the gene is shown in SEQ ID NO.1.

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