TaOSBP1-B gene for improving thousand grain weight of wheat grains as well as CAPS marker and application of TaOSBP1-B gene
Through the application of TaOSBP1-B gene and its CAPS marker, the problem of improving wheat grains with a thousand grain weight is solved, efficient molecular marker assisted breeding is achieved, and the wheat grains with a thousand grain weight and breeding efficiency is improved.
Patent Information
- Application Number
- CN202410111399.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-26
- Publication Date
- 2025-07-29
AI Technical Summary
The functions of the 100-grain weight-related genes in the prior art are limited, and the molecular marker assisted selection effect is poor, making it difficult to meet the needs of high yield breeding.
The TaOSBP1-B gene and its CAPS marker were discovered and provided, and improved wheat grain weights were achieved through PCR amplification and restriction enzyme detection.
It improves the weight of wheat grains, enhances the accuracy and efficiency of molecular marker-assisted breeding, and promotes the selection and breeding process of high-yield wheat varieties.
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Figure CN120384081A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a TaOSBP1-B gene for improving the 1000-grain weight of wheat grains and a CAPS marker and application thereof, belonging to the technical field of plant genetic engineering and breeding. Background Art
[0003] Grain weight QTLs have been identified on all 21 wheat chromosomes, but most have low phenotypic contributions, poor reproducibility, poor comparability between results, and high false-positive rates, making them insufficient for marker-assisted selection. Because wheat is an allohexaploid, relatively few genes related to grain weight have been identified. Identified genes associated with 1000-grain weight include TaSus (Jiang et al., 2011; Hou et al., 2014), TaGW2 (Su et al., 2011, Yang et al., 2012; Zhai et al., 2018), TaGS3 (Zhang et al., 2014), TaCKX (Zhang et al., 2012; Lu et al., 2015), TaGASR7 (Ling et al., 2013; Dong et al., 2014), TaSnRK2 (Zhang et al., 2017), TaGS1 (Guo et al., 2013), and TaFL02 (Sajjad et al., 2017). The discovery of these superior alleles and the development of molecular markers have provided important guidance for high-yield wheat breeding.
[0004] Cleaved amplified polymorphism sequence (CAPS) markers are a method for detecting SNPs. The principle is that some SNP sites together with several nearby nucleotides constitute specific restriction enzyme cleavage sites, so that amplified products carrying different SNP types can produce DNA band length differences after restriction enzyme cleavage.
[0005] However, the functions of these genes are limited, and there are few wheat grain weight and molecular markers closely linked to it for actual molecular breeding. Therefore, it is very necessary to develop a new gene and molecular marker to improve wheat thousand-grain weight. Summary of the Invention
[0006] In response to the shortcomings of the existing technology, the present invention has discovered the TaOSBP1-B gene that can increase wheat grain weight after long-term research and exploration, and provides a TaOSBP1-B gene for improving the thousand-grain weight of wheat grains and its CAPS marker and application.
[0007] The technical solution of the present invention is as follows:
[0008] In the first aspect of the present invention, there is provided a TaOSBP1-B gene for improving the thousand-grain weight of wheat grains, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.
[0009] In the second aspect of the present invention, there is provided a biological material containing the above TaOSBP1-B gene, and the biological material is an expression cassette containing the TaOSBP1-B gene, a recombinant expression vector containing the TaOSBP1-B gene expression cassette, a recombinant microorganism containing the TaOSBP1-B gene recombinant expression vector, and a transgenic cell line containing the TaOSBP1-B gene.
[0010] In the third aspect of the present invention, there is provided the application of the above TaOSBP1-B gene or the protein encoded by the above TaOSBP1-B gene in increasing wheat grains, improving the thousand-grain weight of wheat grains or cultivating wheat varieties with high thousand-grain weight.
[0011] In the fourth aspect of the present invention, there is provided a molecular marker CAPS-TaOSBP1-B of the above TaOSBP1-B gene, and the nucleotide sequence of the molecular marker CAPS-TaOSBP1-B is shown in SEQ ID NO.3.
[0012] In the fifth aspect of the present invention, there is provided the application of the above molecular marker CAPS-TaOSBP1-B in molecular-assisted breeding.
[0013] Preferably according to the present invention, the application refers to using the molecular marker CAPS-TaOSBP1-B to identify whether wheat is a wheat variety or strain in which the TaOSBP1-B gene increases wheat grains and improves the thousand-grain weight of wheat, including the following steps:
[0014] (1) Extract the genomic DNA of the wheat sample to be identified;
[0015] (2) Perform PCR amplification on the wheat genomic DNA with the forward primer and reverse primer of the molecular marker CAPS-TaOSBP1-B, electrophoretically detect the amplification product, and obtain a target band with a molecular weight of 501bp; digest the amplification product with BsrDI enzyme, and electrophoretically separate the enzyme digestion product to obtain two target bands with molecular weights of 339bp and 162bp respectively;
[0016] (3) In the amplification results, if the sample to be tested is detected using the molecular marker CAPS-TaOSBP1-B and a target band with a molecular weight of 501 bp is obtained, and after detection by BsrDI enzyme digestion, two target bands with molecular weights of 339 bp and 162 bp are obtained, then the sample is a wheat variety or strain in which the TaOSBP1-B gene increases wheat grain size and thousand-grain weight.
[0017] Further preferably, in step (2), the sequences of the forward primer and the reverse primer are as follows:
[0018] Forward primer: 5′-TGCTCCGATCGATTCGCTCC-3′,
[0019] Reverse primer: 5′-CCATGCACAATCAAGCTCAG-3′.
[0020] In a sixth aspect, the present invention provides a kit for detecting the TaOSBP1-B gene for improving the 1000-grain weight of wheat grains, comprising the above-mentioned molecular marker CAPS-TaOSBP1-B.
[0021] The use of the above kit in any of the following a) to e):
[0022] a) Used to detect TaOSBP1-B gene in wheat or hybrid wheat progeny;
[0023] b) Used to identify or assist in identifying the thousand-grain weight trait of wheat;
[0024] c) for cultivating, screening or assisting in screening wheat lines, varieties or varieties with different thousand-grain weights;
[0025] d) preparing a product for detecting the TaOSBP1-B gene in wheat or hybrid offspring;
[0026] e) Products used for breeding, screening or assisting in screening wheat lines, varieties or varieties with different thousand-grain weights.
[0027] Beneficial effects:
[0028] 1. This invention, for the first time, discovered and disclosed a novel wheat gene, TaOSBP1-B, which can effectively enlarge wheat grains and increase their 1000-grain weight. Research on this gene revealed that transgenic wheat lines overexpressing the TaOSBP1-B gene exhibited an increased 1000-grain weight. Therefore, this gene could be used to improve important agronomic traits such as seed size and 1000-grain weight, thereby increasing crop yields, with significant economic and social benefits.
[0029] 2. The present invention provides a molecular marker CAPS-TaOSBP1-B for detecting the TaOSBP1-B gene, which is located in the wheat TaOSBP1 gene. Through the application of this specific CAPS-TaOSBP1-B, it is possible to detect whether a wheat variety or strain has the TaOSBP1-B gene that increases the 1,000-grain weight of wheat grains. This sub-marker CAPS-TaOSBP1-B can be used for screening high-yield wheat germplasm materials, providing an effective detection method for cultivating high-yield wheat varieties, improving the application efficiency of grain weight genes, and having important scientific significance and practical application value in accelerating my country's wheat breeding process and increasing wheat yield. At the same time, this molecular marker not only screens quickly and accurately, is not affected by the environment, and has a clear selection target, but also saves production costs and greatly improves the selection efficiency and quality of high-yield wheat varieties or strains. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 This is a comparison of the amino acid sequence encoded by the TaOSBP1-B gene with the amino acid sequences encoded by homologous genes in rice, corn, and Arabidopsis.
[0031] Figure 2 Phylogenetic tree analysis of proteins encoded by homologous genes of TaOSBP1-B gene.
[0032] Figure 3 This is the agarose gel electrophoresis detection diagram of TaOSBP1-B gene.
[0033] Figure 4 Schematic diagram of the structure of the wheat overexpression vector pUbi::TaOSBP1-B.
[0034] Figure 5 This is the detection and screening results of transgenic wheat overexpressing the TaOSBP1-B gene.
[0035] Figure 6 Comparative analysis of transgenic wheat overexpressing TaOSBP1-B gene and wild-type wheat;
[0036] In the figure, A is plant height (cm), B is ear length (cm), C is grain length (cm), D is grain width (cm), and E is 1000-grain weight (g).
[0037] Figure 7 This is the result of enzyme-digested agarose gel detection using the molecular marker CAPS-TaOSBP1-B to identify whether wheat has the TaOSBP1-B genotype.
[0038] Figure 8 The relationship between TaOSBP1-B genotyping and 1000-grain weight in RIL population. DETAILED DESCRIPTION
[0039] Unless otherwise specified, all the materials and reagents used in the following examples are available from commercial sources unless otherwise specified.
[0040] Example 1: Cloning and vector construction of wheat TaOSBP1-B gene
[0041] 1. Using the wheat genome database website Gramene, the inventors discovered the TraesCS4B02G042300 gene on wheat chromosome 4B. The inventors discovered that TraesCS4B02G042300 is highly expressed during wheat grain development. Its full nucleotide sequence, 11,077 bp long, has been publicly available on the Gramene database website, with the ID TraesCS4B02G042300. Because it encodes the oxysterol-binding protein (OSBP), the inventors named this gene, TaOSBP1-B. Its sequence is shown in SEQ ID NO. 1, is 1,941 bp long, and encodes 646 amino acids. The encoded amino acid sequence is shown in SEQ ID NO. 2.
[0042] The functions of OSBP proteins in animals have been partially studied. They are proteins that bind to oxysterols and regulate their metabolism. OSBP is involved in membrane biosynthesis and participates in cell membrane biosynthesis. OSBP participates in cell signaling pathways, regulating cell growth, differentiation, and apoptosis. OSBP plays a crucial role in sterol transport between the endoplasmic reticulum and the Golgi apparatus, helping to regulate the lipid composition of cell membranes. However, less research has been conducted on OSBP proteins in plants. Some studies have hypothesized that OSBP plays an important role in crop growth and development by affecting lipid metabolism and transport in crops, but the biological function of the TaOSBP1-B gene in wheat has not been reported. Further research has revealed that OSBP in wheat may improve thousand-grain weight.
[0043] Therefore, the inventors of the present application compared the amino acids encoded by the TaOSBP1-B gene with the amino acid sequences encoded by homologous gene sequences of rice, corn, and Arabidopsis. Figure 1 The rice homologous amino acid sequence encodes the gene Os12t0285600, the maize homologous amino acid sequence encodes the gene Zm00001eb054410, and the Arabidopsis homologous amino acid sequence encodes the gene AT4G08180. The Zm00001eb054410, AT4G08180, and Qs12t0285600 genes are publicly available in the Grameen database.
[0044] Depend on Figure 1 It can be seen that the amino acids encoded by the wheat TaOSBP1-B gene have low sequence similarity with the amino acid encoded by the homologous genes in rice, maize, and Arabidopsis, indicating that the function of the amino acids encoded by the TaOSBP1-B gene in wheat is different from that of its homologous genes in rice, maize, and Arabidopsis.
[0045] Then, the proteins encoded by the homologous genes of TaOSBP1-B gene were analyzed by phylogenetic tree. The results are as follows Figure 2 shown.
[0046] Depend on Figure 2 It can be seen that the amino acids encoded by the TaOSBP1-B gene are distantly related to those of other crops, and the function of the TaOSBP1-B gene in wheat cannot be determined based on the function of the protein encoded by its homologous genes.
[0047] 2. Cloning of the wheat TaOSBP1-B gene
[0048] (1) The genomic RNA of wheat Shannong 01-35 and Gaocheng 9411 was extracted using the UltraPure RNA Extraction Kit provided by Beijing Kangwei Reagent Biotechnology Co., Ltd., and the purity and concentration of the purified RNA samples were detected by spectrophotometer and agarose gel electrophoresis;
[0049] Among them, the purity and concentration standards are: RNA purity is OD260 / 280 and OD260 / 230 both in the range of 1.8 to 2.0, and RNA concentration is in the range of 1.0 to 2.0 μg / μL;
[0050] (2) Using the FastQuantRT Kit (with gDNase) from Tiangen Biotechnology Co., Ltd., the genomic RNA obtained in step (1) was reverse transcribed according to the kit instructions to obtain reverse transcribed cDNA:
[0051] (3) Cloning of wheat TaOSBP1-B gene:
[0052] The reverse transcribed cDNA obtained in step (2) was used as a template and PCR amplification was performed using Ubi-TaOSBP1-BF / R primers and 2×PhantaMax MasterMix reagent to obtain the TaOSBP1-B gene sequence.
[0053] Amplification primers were designed based on the TaOSBP1-B gene sequence. The sequences of the primers are as follows:
[0054] Ubi-TaOSBP1-BF: 5′-ATGCACCCATTCTGCTGCG-3′,
[0055] Ubi-TaOSBP1-BR: 5′-GTACAACTGATGAGGATTGCGAT-3′.
[0056] The reaction system is: Mix 25 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA template 1 μL, and water to make up to 50 μL.
[0057] Amplification conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 2 minutes, repeated 35 cycles; post-extension at 72°C for 5 minutes; and incubation at 4°C.
[0058] After the reaction was completed, the amplified products were subjected to agarose gel electrophoresis. Figure 3 As shown, after the target band is detected, the gel is cut and the gel is recovered.
[0059] Depend on Figure 3 It can be seen that a 1990 bp amplification product was amplified from the genomic RNA of Shannong 01-35 and Gaocheng 9411. After sequencing analysis and verification, it was shown that the amplified product was the TaOSBP1-B gene, indicating that the TaOSBP1-B gene was successfully amplified in this example.
[0060] 3. Construction of expression vector
[0061] The empty expression vector pwmb110 was double-digested with restriction endonucleases SmaI / Spe, and the amplified TaOSBP1-B gene was ligated to the restriction endonuclease vector. The ligation product was transformed into Escherichia coli DH5α competent cells and cultured overnight on LB solid medium containing kanamycin (50 mg / L). White colonies were picked and cultured overnight in LB liquid medium containing spectinomycin (50 mg / L). Plasmid DNA was extracted by alkaline method to obtain the wheat overexpression vector pUBI::TaOSBP1-B, and the wheat overexpression vector pUBI::TaOSBP1-B was subjected to enzyme digestion identification and sequencing analysis. The structure of the wheat overexpression vector pUBI::TaOSBP1-B is shown in FIG. Figure 4 shown.
[0062] Example 2: Obtaining transgenic wheat overexpressing the TaOSBP1-B gene
[0063] 1. Transform wheat overexpression vector pUBI::TaOSBP1-B into Agrobacterium EHA105
[0064] The correctly identified wheat overexpression vector pUBI::TaOSBP1-B constructed in Example 1 was transformed into Agrobacterium tumefaciens EHA105 to obtain the Agrobacterium strain carrying the TaOSBP1-B gene for transformation.
[0065] The specific method for transferring the wheat overexpression vector pUBI::TaOSBP1-B into Agrobacterium is as follows:
[0066] (1) Take out 50 μL of Agrobacterium tumefaciens EHA105 competent cells stored in an -80 °C refrigerator, place them on the pre-prepared crushed ice, and wait for 30 minutes to melt. After melting, add 5 μL of plasmid, then gently mix, freeze in liquid nitrogen for 3 min, then place in a 37 °C water bath for 5 min, add 1 mL of MGL liquid, set the shaker to a temperature of 28 °C and a rotation speed of 200 rpm, and culture for 3 h to obtain Agrobacterium liquid.
[0067] (2) Centrifuge the Agrobacterium liquid at 5000 rpm for 1 min, aspirate 100 μL of the supernatant, pour out the remaining liquid, and pipette and mix the cell pellet. Spread the Agrobacterium evenly on MGL solid medium (containing 50 μg / mL kanamycin + 50 μg / mL rifampicin), and culture at 28 °C in the dark until single colonies grow on the MGL solid medium.
[0068] (3) Pick a single colony for colony PCR verification, and at the same time pick the target single colony and place it in 50 mL of MGL liquid medium (containing 50 μg / mL kanamycin + 50 μg / mL rifampicin), culture at 28 °C and 200 rpm in the dark for 2 days to obtain the Agrobacterium strain carrying the TaOSBP1-B gene.
[0069] 2. Construction of transgenic wheat overexpressing the TaOSBP1-B gene
[0070] For the detailed steps and methods of the Agrobacterium-mediated method for wheat immature embryos, refer to the published literature Wheat (Triticum aestivum L.) Transformation Using Immature Embryos, Ishida et al., 2015. The specific method is as follows:
[0071] Select wheat young embryos at 14 days after anthesis, and then use Agrobacterium tumefaciens carrying the TaOSBP1-B gene to infect the young embryos of wild-type wheat. After induction, recovery, and two rounds of screening with gradient concentrations, the callus is transferred to a regeneration medium to germinate seedlings, and resistant seedlings overexpressing the TaOSBP1-B gene are obtained. The obtained resistant seedlings overexpressing the TaOSBP1-B gene are then rooted, acclimatized to the environment, and cultivated in a greenhouse, and transgenic wheat seeds overexpressing the TaOSBP1-B gene are harvested. At the same time, the transgenic wheat overexpressing the TaOSBP1-B gene cultivated in this example is used as the Fielder T0 generation and divided into lines 1, 2, 3, and 4.
[0072] At the same time, wild-type wheat plants are cultivated and planted according to the same cultivation method, and wild-type wheat seeds are harvested.
[0073] Example 3: Detection and screening of transgenic wheat overexpressing the TaOSBP1-B gene, and comparison of the grain sizes of transgenic wheat carrying the TaOSBP1-B gene and wild-type grains
[0074] 1. Cut the leaves of the transgenic wheat plants overexpressing the TaOSBP1-B gene cultivated in Example 2, and use the Mag-Bind Plant DNA Kit M2327 for magnetic bead method to extract DNA according to the method described in the kit instructions. Design primers Ubi-TaOSBP1-B-pF / R, and screen positive plants by PCR amplification. During the PCR amplification process, the wheat overexpression vector pUBI::TaOSBP1-B is used as a positive control, and ddH2O is used as a negative control. Among them, the plants that can show the correct TaOSBP1-B gene band are positive plants. The detection and screening results of the transgenic wheat overexpressing the TaOSBP1-B gene cultivated in Example 2 are as Figure 5 shown.
[0075] The sequences of the primers Ubi-TaOSBP1-B-pF / R are as follows:
[0076] Ubi-TaOSBP1-B-pF: 5′-TCGAAAGAGTATGGATCTGC-3′,
[0077] Ubi-TaOSBP1-B-pR: 5′-ATGCTTCCGGCTCGTATGTT-3′.
[0078] By Figure 5It can be seen that the transgenic wheat overexpressing the TaOSBP1-B gene cultivated in Example 2 produced a band of the same size as the positive control, while no band was amplified in the negative control ddH2O, indicating that the positive transgenic wheat overexpressing the TaOSBP1-B gene was successfully screened in this example. 1, 2, 3, and 4 in the figure represent 4 lines, and CK is the wild-type wheat plant.
[0079] 2. Observe the plants and seeds of the transgenic wheat overexpressing the TaOSBP1-B gene and the wild-type wheat obtained by screening, and count the thousand-grain weight. The results are as Figure 6 shown.
[0080] As Figure 6 can be seen, there is no obvious difference in plant height between the transgenic wheat plants overexpressing the TaOSBP1-B gene and the wild type; however, the seeds of the transgenic wheat plants overexpressing the TaOSBP1-B gene are larger, and the thousand-grain weight is significantly increased. This indicates that the TaOSBP1-B gene can effectively increase the size of wheat grains and the thousand-grain weight of wheat grains. Therefore, this gene can be applied to improve important agronomic traits such as the size and thousand-grain weight of seeds, thereby increasing the yield of crops.
[0081] Example 4: Identification of wheat varieties or lines with increased wheat grain size and thousand-grain weight by the TaOSBP1-B gene using the molecular marker CAPS-TaOSBP1-B
[0082] The present invention provides a molecular marker CAPS-TaOSBP1-B for detecting the TaOSBP1-B gene, and its nucleotide sequence is as shown in SEQ ID NO.3. This marker is located within the TraesCS4B02G042300 gene on chromosome 4B of wheat. By applying this specific CAPS-TaOSBP1-B, it can be detected whether the TaOSBP1-B gene that increases the thousand-grain weight of wheat grains exists in wheat varieties or lines. The specific verification is as follows.
[0083] 1. Cross and self-cross the wheat varieties Shanong 01-35 and Gaocheng 9411, and use the single-seed descent method to obtain a RIL population containing 173 lines. The specific method can refer to the publicly available literature Liu K, Sun XX, Ning TY, ET AL., 2018 Genetic dissection of wheat panicle traits using linkage analysis and a genome-wide association study. Theoretical and Applied Genetics, 131: 1073-1090.
[0084] The genomic DNA of the RIL population was extracted by the CTAB method. After detecting the concentration and quality of the DNA, using the genomic DNA of the wheat to be tested as a template, PCR amplification was carried out with the CAPS-TaOSBP1-B marker primer pair to obtain an amplification product. The sequences of the primers are as follows:
[0085] Forward primer: 5′-TGCTCCGATCGATTCGCTCC-3′,
[0086] Reverse primer: 5′-CCATGCACAATCAAGCTCAG-3′.
[0087] The amplification system was: 1 μL of upstream primer, 1 μL of downstream primer, 10 μL of 1 Taq Mix, 1 μL of DNA template, and ddH2O was added to make the total volume up to 20 μL;
[0088] The amplification conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, for 34 cycles; extension at 72°C for 5 min.
[0089] 2. The above amplification product was subjected to agarose gel electrophoresis. The molecular weight of the amplification product was 501 bp. BsrDI specific endonuclease was added to the PCR amplification product, and it was incubated in a water bath at 37°C for 90 min. Then the enzyme digestion system was inactivated at 80°C for 20 min, and agarose gel electrophoresis was carried out. The results are as Figure 7 shown.
[0090] It can be Figure 7 seen that among the amplification results, 5 large-grain offspring have or are candidate to have the thousand-grain weight increasing gene TaOSBP1-B, meeting the identification criteria of first obtaining a single target band with a molecular weight of 501 bp (as shown in SEQ ID NO.3), and after detecting by digestion with BsrDI, obtaining two target bands with molecular weights of 339 bp and 162 bp respectively (as shown in SEQ ID NO.4 and SEQ ID NO.5). While 5 small-grain offspring do not have or are candidate not to have the thousand-grain weight increasing TaOSBP1-B gene, that is, they cannot obtain a single target band with a molecular weight of 501 bp, or can only obtain a single target band with a molecular weight of 501 bp, and cannot obtain two target bands with molecular weights of 339 bp and 162 bp after digestion with BsrDI.
[0091] The enzyme digestion system of the marker primer was: 17.6 μL of amplification product, 0.4 μL of BsrDI endonuclease (NEB), 2 μL of NEBuffer TM r2.1, with a total volume of 20 μL.
[0092] Example 5: Identification of the thousand-grain weight phenotype of the RIL population
[0093] The wheat variety Shanong 01-35 was crossed with Gaocheng 9411 and self-crossed, and a RIL population consisting of 173 lines was obtained by the single-seed descent method. The specific method can refer to the publicly available literature: Liu K, Sun XX, Ning TY, et al., 2018 Genetic dissection of wheat panicle traits using linkage analysis and a genome-wide association study. Theoretical and Applied Genetics, 131: 1073-1090.
[0094] Then, a field planting experiment was conducted on this RIL population of 173 lines. The field planting experiment was carried out at the experimental base of Shandong Agricultural University in Tai'an City, Shandong Province. The field planting adopted a randomized block design, and the specific process was carried out according to the general wheat planting method, with three replicates. The grains were harvested at maturity, and the thousand-grain weight was measured using a seed grader for three consecutive years (2019, 2020, and 2021). The results are as Figure 8 shown. At the same time, the 173-line RIL population was identified using the molecular marker CAPS-TaOSBP1-B, and it was found that the expression level of the TaOSBP1-B gene in the TaOSBP1-B genotype of Shanong 01-35 was higher than that in the TaOSBP1-B genotype of Gaocheng 01-35.
[0095] It can be Figure 8 seen that the thousand-grain weight of the offspring of the same type of Shanong 01-35 was significantly higher than that of the offspring of the same type of Gaocheng 9411. This indicates that overexpressing the TaOSBP1-B gene can effectively increase the size of wheat grains and improve the thousand-grain weight of wheat grains. The wheat lines overexpressing the TaOSBP1-B gene have the phenotype of increased thousand-grain weight of seeds. Therefore, this gene can be applied to improve important agronomic traits such as the size and thousand-grain weight of seeds, thereby increasing the yield of crops, and has important economic value and social benefits.
[0096] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.
Claims
1. A TaOSBP1-B gene for improving the thousand-grain weight of wheat grains, characterized in that, The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.
2.
2. A biological material containing the TaOSBP1-B gene according to claim 1, characterized in that: The biological material is an expression cassette containing the TaOSBP1-B gene, a recombinant expression vector containing the TaOSBP1-B gene expression cassette, a recombinant microorganism containing the TaOSBP1-B gene recombinant expression vector, or a transgenic cell line containing the TaOSBP1-B gene.
3. Use of the TaOSBP1-B gene according to claim 1 or the protein encoded by the TaOSBP1-B gene according to claim 1 in enlarging wheat grains, increasing the 1000-grain weight of wheat grains, or breeding wheat varieties with high 1000-grain weight.
4. The molecular marker CAPS-TaOSBP1-B of the TaOSBP1-B gene according to claim 1, characterized in that: The nucleotide sequence of the molecular marker CAPS-TaOSBP1-B is shown in SEQ ID NO.
3.
5. Use of the molecular marker CAPS-TaOSBP1-B according to claim 4 in molecular assisted breeding.
6. The use according to claim 5, characterized in that The application is to use the molecular marker CAPS-TaOSBP1-B to identify whether wheat is a wheat variety or strain with the TaOSBP1-B gene that increases wheat grain size and improves wheat thousand-grain weight, including the following steps: (1) Extracting genomic DNA from the wheat sample to be identified; (2) Wheat genomic DNA was amplified by PCR using the forward and reverse primers of the molecular marker CAPS-TaOSBP1-B. The amplified product was detected by electrophoresis to obtain a target band with a molecular weight of 501 bp. The amplified product was digested with BsrDI and the enzyme digestion products were separated by electrophoresis to obtain two target bands with molecular weights of 339 bp and 162 bp, respectively. (3) In the amplification results, if the sample to be tested is detected using the molecular marker CAPS-TaOSBP1-B and a target band with a molecular weight of 501 bp is obtained, and after detection by BsrDI enzyme digestion, two target bands with molecular weights of 339 bp and 162 bp are obtained, then the sample is a wheat variety or strain in which the TaOSBP1-B gene increases wheat grain size and thousand-grain weight.
7. The application according to claim 6, characterized in that In step (2), the sequences of the forward primer and the reverse primer are as follows: Forward primer: 5′-TGCTCCGATCGATTCGCTCC-3′, Reverse primer: 5′-CCATGCACAATCAAGCTCAG-3′.
8. A kit for detecting the TaOSBP1-B gene that improves wheat grain 1000-grain weight, the use according to claim 5, characterized in that: The molecular marker CAPS-TaOSBP1-B according to claim 5.
9. Use of the kit according to claim 9, characterized in that, Application in any of the following a) to e): a) Used to detect TaOSBP1-B gene in wheat or hybrid wheat progeny; b) Used to identify or assist in identifying the thousand-grain weight trait of wheat; c) for cultivating, screening or assisting in screening wheat lines, varieties or varieties with different thousand-grain weights; d) preparing a product for detecting the TaOSBP1-B gene in wheat or hybrid offspring; e) Products used for breeding, screening or assisting in screening wheat lines, varieties or varieties with different thousand-grain weights.