TaSWEET-6A protein and its encoding gene in regulating plant kernel traits
By introducing the TaSWEET-6A protein or its encoding gene, wheat grain traits were regulated, solving the problems of insufficient grain width and weight, and thus increasing wheat yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIANGHU LABORATORY
- Filing Date
- 2025-09-26
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies are insufficient to effectively increase the width and weight of wheat grains, thus hindering yield improvement.
By introducing the TaSWEET-6A protein or its encoding gene, plant grain traits can be regulated, increasing grain width and weight.
It significantly increased the grain width and weight of wheat grains, thereby enhancing wheat yield.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of crop genetics and breeding, and relates to the application of TaSWEET-6A protein and its encoding gene in regulating plant grain traits. Background Technology
[0002] Wheat is one of the three major food crops, and the study of its yield traits has broad application prospects in the field of wheat breeding. It is of great significance for breeding high-yielding wheat varieties. Selecting high-yielding varieties and continuously improving yield per unit area are the main directions of wheat breeding in my country.
[0003] Wheat yield traits are complex traits composed of multiple yield factors, which can be broken down into three parts: thousand-grain weight, number of spikes per acre, and number of grains per spike. Among these, thousand-grain weight is most affected by genetic factors and makes a significant contribution to yield improvement in breeding practice. Zhuang Qiaosheng's research found that the thousand-grain weight increased from 31.4g in the 1950s to 40.5g in the 1970s, indicating that the improvement of thousand-grain weight plays an important role in yield growth. Summary of the Invention
[0004] The purpose of this invention is to provide the application of TaSWEET-6A protein and its encoding gene in regulating plant grain traits.
[0005] This invention provides the application of TaSWEET-6A protein in regulating plant grain traits, specifically grain width and / or grain weight. The regulation is positive; increased TaSWEET-6A protein abundance leads to increased grain width and / or grain weight.
[0006] The present invention also provides TaSWEET-6A Application of genes in regulating plant grain traits; the grain traits are grain width and / or grain weight. The regulation is positive regulation. TaSWEET-6A Increased gene abundance leads to increased grain width and / or grain weight in plant seeds.
[0007] The present invention also provides TaSWEET-6A Gene or TaSWEET-6A Application of gene-related biological materials in the preparation of transgenic plants with altered grain traits; the grain traits are grain width and / or grain weight. Altered grain traits refer to an increase in grain width and / or grain weight.
[0008] This invention also provides a method for cultivating transgenic plants with altered seed traits, comprising the following steps: introducing [the following steps] into a recipient plant. TaSWEET-6A Gene or TaSWEET-6A Gene-related biological materials were used to obtain transgenic plants with increased grain width and / or increased grain weight.
[0009] The present invention also provides a method for cultivating transgenic plants with increased yield, comprising the following steps: introducing into a recipient plant TaSWEET-6A Gene or TaSWEET-6A Gene-related biological materials are used to obtain transgenic plants with increased yields.
[0010] This invention also provides TaSWEET-6A protein or TaSWEET-6A Gene or TaSWEET-6A Application of gene-related biomaterials in plant breeding.
[0011] This invention also provides the application of any of the methods described above in plant breeding.
[0012] The goal of any of the above-mentioned plant breeding programs is to cultivate high-yielding plants.
[0013] The goal of any of the above-mentioned plant breeding programs is to develop plants with increased grain width and / or increased grain weight.
[0014] Specifically, any of the TaSWEET-6A proteins mentioned above are as follows (a1), (a2), or (a3):
[0015] (a1) The protein shown in SEQ ID NO: 1;
[0016] (a2) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of (a1) and which is related to the characteristics of plant grains;
[0017] (a3) is a protein derived from wheat that shares more than 90% identity with (a1) and is associated with plant grain traits.
[0018] The grain characteristics are grain width and / or grain weight.
[0019] Specifically, any of the above TaSWEET-6A The gene is the gene encoding the TaSWEET-6A protein.
[0020] Specifically, any of the above TaSWEET-6A The genes are as follows (b1) or (b2) or (b3):
[0021] (b1) A DNA molecule with a coding region as shown in positions 202-969 of SEQ ID NO: 2;
[0022] (b2) The DNA molecule shown in SEQ ID NO: 2;
[0023] (b3) A DNA molecule derived from wheat that has more than 90% identity with (b1) or (b2) and that encodes the protein thereon.
[0024] The above-mentioned 90% or more identity can be 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity, 99% or more identity, or 99.9% or more identity.
[0025] Any of the above TaSWEET-6A Gene-related biological materials are as follows: (c1) or (c2) or (c3) or (c4) or (c5) or (c6) or (c7):
[0026] (c1) has TaSWEET-6A Gene expression cassettes;
[0027] (c2) has TaSWEET-6A Recombinant plasmids of genes;
[0028] (c3) has TaSWEET-6A Recombinant cells of genes;
[0029] (c4) A recombinant plasmid having the expression cassette described in (c1);
[0030] (c5) Recombinant cells having the expression cassette described in (c1);
[0031] (c6) Recombinant cells having the recombinant plasmid described in (c2);
[0032] (c7) Recombinant cells having the recombinant plasmid described in (c4);
[0033] For example, in any of the expression boxes described above, the Ubi promoter initiates the process. TaSWEET-6A Gene expression.
[0034] For example, any of the above-described recombinant plasmids may be the following plasmids: the double-stranded DNA molecule shown in SEQ ID NO: 3 is homologously recombined with plasmid pLGY-OE3 to obtain a circular recombinant plasmid having the double-stranded DNA molecule shown in SEQ ID NO: 3.
[0035] For example, any of the above-mentioned recombinant cells are recombinant Agrobacterium obtained by introducing recombinant plasmids into Agrobacterium (e.g., Agrobacterium tumefaciens EHA105).
[0036] Specifically, the grain weight mentioned above refers to the weight of 1,000 grains.
[0037] Any of the plants mentioned above are either monocotyledons or dicotyledons.
[0038] Specifically, any of the plants mentioned above belong to the Poaceae family.
[0039] Specifically, any of the plants mentioned above belong to the genus Triticum.
[0040] Specifically, any of the plants mentioned above is wheat, such as wheat fielder.
[0041] This invention discovered that the TaSWEET-6A protein and its encoding gene are associated with wheat grain weight, and used transgenic methods to improve wheat grain weight. TaSWEET-6A The transcriptional level of genes can increase wheat grain weight. This invention provides application value for high-yield wheat breeding and genetic improvement of grain traits in new varieties. Attached Figure Description
[0042] Figure 1 This is an example photograph of a transgenic plant selected from seedling T0 generation plants.
[0043] Figure 2 For identification TaSWEET-6A Statistical results of gene expression levels.
[0044] Figure 3 An exemplary photograph of the seeds.
[0045] Figure 4 The results are statistical results for grain length and grain width.
[0046] Figure 5 This is the statistical result for the weight of a thousand grains. Detailed Implementation
[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0048] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged. In the embodiments, significant differences are indicated by asterisks: one asterisk (*) indicates a p-value less than 0.05, two asterisks (**) indicate a p-value less than 0.01, and three asterisks (***) indicate a p-value less than 0.001.
[0049] The plasmid pLGY-OE3 (overexpression vector) (i.e., "pTCK303 vector" in the literature) is described in the following literature: Arare gain of function mutation in a wheat tandem kinase confers resistance topowdery mildew; NATURE COMMUNICATIONS, (2020) 11:680.
[0050] Example 1: Preparation of transgenic plants
[0051] I. Preparation of recombinant plasmids
[0052] The double-stranded DNA molecule shown in SEQ ID NO: 3 was homologously recombined with plasmid pLGY-OE3 to obtain a circular recombinant plasmid containing the double-stranded DNA molecule shown in SEQ ID NO: 3, which was named plasmid pLGY-OE3-TaSWEET-6A.
[0053] The plasmid pLGY-OE3-TaSWEET-6A has been sequenced and validated. The plasmid pLGY-OE3-TaSWEET-6A is started by the Ubi promoter. TaSWEET-6A Gene expression.
[0054] II. Preparation of transgenic plants
[0055] 1. Plasmid pLGY-OE3-TaSWEET-6A was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.
[0056] 2. The recombinant Agrobacterium obtained in step 1 was used to perform Agrobacterium-mediated genetic transformation on the embryogenic callus of wheat Fielder (the steps were: infection, co-culture, recovery culture, herbicide resistance screening culture and rooting culture in sequence) to obtain rooted regenerated plants, which are the T0 generation plants.
[0057] 3. Select transgenic plants from seedling T0 generation plants.
[0058] Screening method: Take leaves from plants with 4-5 leaves and extract genomic DNA; use the genomic DNA as a template and perform PCR amplification using primer pair consisting of TaSWEET-6A-OE-F (SEQ ID NO: 4) and TaSWEET-6A-OE-R (SEQ ID NO: 5), and then perform agarose gel electrophoresis; if the electrophoresis shows the target band (about 440 bp), the plant is a transgenic plant.
[0059] TaSWEET-6A-OE-F (corresponding to...) TaSWEET-6AGene): 5'-ATCCTCTGCGTCCTCTTCG-3';
[0060] TaSWEET-6A-OE-R (corresponding vector skeleton): 5'-gtgtgcgcaatgaaactgat-3'.
[0061] See exemplary electrophoresis diagram. Figure 1 (Fielder was used as the wild-type control, plasmid pLGY-OE3-TaSWEET-6A was used as the positive control, and the other lanes corresponded to different plants).
[0062] 4. The T0 generation transgenic plants selected in step 3 are self-pollinated and the seeds are harvested. The seeds are then cultivated into plants, which are the T1 generation plants.
[0063] 5. Select transgenic plants from seedling T1 generation plants (the selection method is the same as the selection method in step 3).
[0064] 6. The T1 generation transgenic plants selected in step 5 are self-pollinated and their seeds are harvested. The seeds are then cultivated into plants, which are the T2 generation plants.
[0065] 7. Select transgenic plants from seedling T2 generation plants (the selection method is the same as the selection method in step 3).
[0066] For a given T1 generation transgenic plant, if all T2 generation plants obtained through self-pollination are transgenic plants, then the T1 generation plant is a homozygous transgenic plant, and the self-pollinated offspring of the T1 generation plant are homozygous transgenic lines.
[0067] Three transgenic lines were obtained and named TaSWEET-6A-OE1, TaSWEET-6A-OE2 and TaSWEET-6A-OE3 respectively.
[0068] III. Identification TaSWEET-6A Gene expression level
[0069] The tested plants were: T2 generation plants of the TaSWEET-6A-OE1 line, T2 generation plants of the TaSWEET-6A-OE2 line, T2 generation plants of the TaSWEET-6A-OE3 line, and wheat Fielder plants.
[0070] From test plants cultured under parallel conditions, seeds at the 8-day post-application (8 days after flowering) stage were collected, and total RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, ... TaActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. TaSWEET-6A Relative gene expression levels (2-ΔΔCT method).
[0071] Used for detectionTaSWEET-6A The primers for the gene are as follows:
[0072] TaSWEET-6A-qF (SEQ ID NO: 6): 5'-CGACGTTCATCAGTATCTGGAA-3';
[0073] TaSWEET-6A-qR (SEQ ID NO: 7): 5'-GATGTAGGTGAGCTCGATGAG-3'.
[0074] Used for detection TaActin The primers for the gene are as follows:
[0075] TaActin-qF (SEQ ID NO: 8): 5'-GTTGGTGATGAGGCCCAATC-3';
[0076] TaActin-qR (SEQ ID NO: 9): 5'-GTGCTACACGGAGCTCATTG-3'.
[0077] See results Figure 2 (Sample size N=3). Figure 2 In this context, TaSWEET-OE1 represents the TaSWEET-6A-OE1 strain, TaSWEET-OE2 represents the TaSWEET-6A-OE2 strain, and TaSWEET-OE3 represents the TaSWEET-6A-OE3 strain.
[0078] Example 2, trait detection
[0079] The tested seeds were: 125 seeds obtained by self-pollination of T2 generation plants of TaSWEET-6A-OE1 line, 143 seeds obtained by self-pollination of T2 generation plants of TaSWEET-6A-OE2 line, 134 seeds obtained by self-pollination of T2 generation plants of TaSWEET-6A-OE3 line, and 150 wheat Fielder seeds.
[0080] On October 11, 2023, the test seeds were sown in the transgenic experimental field of the Gaoyi Experimental Station in Shijiazhuang and cultivated and managed normally.
[0081] At full maturity, harvest the grains and measure their length and width. See exemplary photographs of the grains. Figure 3 (Scale bar is 1 cm). Statistical results for grain length and width are shown below. Figure 4 (Sample size N=12; GL represents grain length, GW represents grain width). Compared with Fielder's grains, the transgenic lines showed no significant difference in grain length, but a significant increase in grain width.
[0082] At full maturity, harvest the grains and measure the total weight of 1000 grains (TWG). The statistical results of the TWG are shown below. Figure 5 Compared to Fielder's grains, the transgenic lines showed a significant increase in thousand-grain weight.
[0083] Figure 3 , Figure 4 and Figure 5 In Chinese: TaSWEET-OE1 represents the TaSWEET-6A-OE1 strain, TaSWEET-OE2 represents the TaSWEET-6A-OE2 strain, and TaSWEET-OE3 represents the TaSWEET-6A-OE3 strain.
[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. Application of TaSWEET-6A protein in regulating plant grain traits; The grain characteristics are grain width and / or grain weight; The TaSWEET-6A protein is the protein shown in SEQ ID NO: 1; The regulation is positive, increasing the grain width and / or grain weight of plant seeds by increasing the abundance of TaSWEET-6A protein. The plant in question is wheat.
2. TaSWEET-6A Application of genes in regulating plant grain traits; The grain characteristics are grain width and / or grain weight; The TaSWEET-6A The gene is the gene encoding the TaSWEET-6A protein as described in claim 1; The regulation is positive regulation, achieved by increasing... TaSWEET-6A Gene abundance increases the width and / or weight of plant seeds; The plant in question is wheat.
3. The application as described in claim 2, characterized in that: The TaSWEET-6A The genes are either (b1) or (b2): (b1) A DNA molecule with a coding region as shown in positions 202-969 of SEQ ID NO: 2; (b2) The DNA molecule shown in SEQ ID NO:
2.
4. A method for cultivating transgenic plants with altered grain traits, comprising the following steps: introducing [the following text is incomplete and likely refers to a separate process] into a recipient plant. TaSWEET-6A Gene or TaSWEET-6A Gene-related biological materials were used to obtain transgenic plants with increased grain width and / or increased grain weight; TaSWEET-6A The gene is as described in claim 2 or 3; The TaSWEET-6A Gene-related biological materials are as follows: (c1) or (c2) or (c3) or (c4) or (c5) or (c6) or (c7): (c1) has TaSWEET-6A Gene expression cassettes; (c2) has TaSWEET-6A Recombinant plasmids of genes; (c3) has TaSWEET-6A Recombinant cells of genes; (c4) A recombinant plasmid having the expression cassette described in (c1); (c5) Recombinant cells having the expression cassette described in (c1); (c6) Recombinant cells having the recombinant plasmid described in (c2); (c7) Recombinant cells having the recombinant plasmid described in (c4); The plant in question is wheat.
5. A method for cultivating transgenic plants with increased yield, comprising the steps of: introducing into a recipient plant... TaSWEET-6A Gene or TaSWEET-6A Gene-related biological materials were used to obtain transgenic plants with increased yields; TaSWEET-6A The gene is as described in claim 2 or 3; TaSWEET-6A The gene-related biomaterial is as described in claim 4; the plant is wheat.
6. TaSWEET-6A protein or TaSWEET-6A Gene or TaSWEET-6A Application of gene-related biological materials or methods in plant breeding; The TaSWEET-6A protein is as described in claim 1; The TaSWEET-6A The gene is as described in claim 2 or 3; The TaSWEET-6A Gene-related biomaterials as described in claim 4; The method is as described in claim 4 or 5; The goal of the plant breeding program is to cultivate plants with increased grain width and / or increased grain weight. The plant in question is wheat.
7. The application as described in claim 6, characterized in that: The goal of this plant breeding program is to cultivate high-yielding plants.