Application of taPHFT gene related biological material in regulating plant architecture and / or heading date

Abstract

The application discloses TaPHFT The application discloses application of a gene-related biological material in regulation of plant plant type and / or heading stage, and belongs to the technical field of plant genetic engineering and plant genetic breeding. TaPHFT The application finds that overexpression of the gene significantly reduces the plant height of wheat, and simultaneously can significantly advance the heading stage of the wheat. The above results have important significance for crop breeding, and provide a target site for precise molecular breeding of crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and plant genetic breeding technology, specifically involving TaPHFT Applications of gene-related biomaterials, especially TaPHFT Application of genes in improving wheat plant architecture and / or regulating heading time. Background Technology

[0002] wheat( Triticum aestivum Wheat (L.) is one of the world's major food crops, therefore, high and stable wheat yields are crucial for ensuring global food security. Heading date and plant height are two important agronomic traits affecting wheat yield and environmental adaptability. Heading date determines the efficiency of wheat varieties in utilizing light and temperature resources and their adaptability to different planting areas. Heading too early may result in late frost damage, leading to damage to young ears; heading too late makes them susceptible to high-temperature stress or hot, dry winds during the grain-filling stage, resulting in shriveled grains and a decrease in thousand-grain weight. Precisely controlling the heading date to ensure wheat completes heading and flowering within the optimal light and temperature window is key to mitigating climate risks and achieving stable yields. Plant height is another crucial trait affecting wheat yield potential. Appropriately reducing plant height and cultivating semi-dwarf varieties to improve lodging resistance are prerequisites for achieving high and stable yields. This plant type optimization also enhances wheat's adaptability to high-fertility and high-water cultivation conditions, enabling it to tolerate higher planting densities and nitrogen fertilizer application rates, thereby achieving greater yield per unit area. Synergistic optimization of wheat heading period and plant type is conducive to ensuring high and stable wheat yields.

[0003] In the prior art, for example, patent application with publication number CN118185954A constructs overexpression OsPRMT6a Gene vectors were introduced into rice using Agrobacterium-mediated transformation, and positive transgenic plants were obtained through screening with hygromycin markers. Further selection yielded positive plants with high expression levels. Under the Nipponbare background... OsPRMT6a Compared to wild-type, the overexpressed gene material has an earlier heading date and increased grain weight.

[0004] For example, patent application CN120796358A utilizes gene editing technology to knock out [a specific gene] in the wheat genome. TaWAK3-B Genes were used to breed dwarf wheat varieties, providing a new molecular breeding method for improving wheat yield and quality. Experimental results show that, compared to wild-type wheat, TaWAK3-B After knockout, the wheat plants in the knockout lines showed significantly reduced plant height and significantly delayed heading date.

[0005] Therefore, there is an urgent need for more methods to collaboratively optimize wheat heading period and plant type in order to ensure high and stable wheat yields. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide... TaPHFT Application of gene-related biomaterials.

[0007] TaPHFT The gene (Plant Height and Flowering Time) is located on chromosome 7B (Chr7B:18207990-18218252), with the following locus number: TraesCS7B03G0054200 Its full-length genome sequence is 11,510 bp, including 8 exons, 7 introns, a 5′ untranslated region (5′UTR), and a 3′ untranslated region (3′UTR). Its full-length cDNA is 684 bp (SEQ ID NO.1), encoding 227 amino acids (SEQ ID NO.3), and the nucleotide sequence of its promoter is shown in SEQ ID NO.2.

[0008] The objective of this invention is achieved through the following technical solution: TaPHFT The application of gene-related biomaterials in regulating plant architecture and / or heading time, including: The biomaterial is any one or more of the following substances: A, B, C, D, E, and F: A. TaPHFT Full-length genomic DNA of a gene or its homologous nucleic acid molecules; B. TaPHFT Gene cDNA or its homologous nucleic acid molecules; C. TaPHFT Proteins encoded by genes or their homologs; D. TaPHFT The promoter of gene expression or its homologous nucleic acid molecules; E. Substances that can alter the expression level and / or activity of substances A, B, or D; F. Substances that can alter the expression level and / or activity of substance C.

[0009] Furthermore, the nucleotide sequence of the full-length genomic DNA described in A, as shown by the gene locus number in the wheatomics database, is... TraesCS7B03G0054200 sequence is shown.

[0010] Furthermore, the homologous nucleic acid molecules mentioned in A refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with full-length genomic DNA.

[0011] Furthermore, the nucleotide sequence of the cDNA described in B is shown in SEQ ID NO.1.

[0012] Furthermore, the homologous nucleic acid molecules described in B refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.1.

[0013] Furthermore, the amino acid sequence of the protein described in C is shown in SEQ ID NO.3.

[0014] Furthermore, the homologous protein described in C refers to a protein that has at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.3.

[0015] Furthermore, the nucleotide sequence of the promoter described in D is shown in SEQ ID NO.2.

[0016] Furthermore, the homologous nucleic acid molecules described in D refer to nucleic acid molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the sequence shown in SEQ ID NO.2.

[0017] Furthermore, the substance E includes any one of the following substances I and II: I. Substances that can increase the expression level and / or activity of substances A, B, or D; II. Substances that can reduce the expression level and / or activity of substances A, B, or D.

[0018] Furthermore, the substance F includes any one of the following substances i and ii: i. Substances that can increase the expression level and / or activity of substance C; ii. Substances that can reduce the expression level and / or activity of substance C.

[0019] Furthermore, substance I or i is selected from: recombinant vectors containing corresponding nucleic acid molecules, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant bacteria.

[0020] Furthermore, substance II or ii is selected from: siRNA, sgRNA.

[0021] Furthermore, the plant is wheat, and the wheat variety includes Fielder.

[0022] Furthermore, by overexpressing the aforementioned TaPHFT Genes that reduce plant height; by inhibiting the aforementioned TaPHFT Gene expression increases plant height; And / or when applied, by overexpressing the TaPHFT Genes that advance the heading time of plants; by inhibiting the aforementioned TaPHFT Gene expression delays the heading time of plants.

[0023] This invention also provides a method for reducing wheat plant height and / or promoting earlier wheat heading time, comprising overexpressing in wheat TaPHFT The steps of gene-based reduction of wheat plant height and / or promotion of earlier wheat heading time; TaPHFT The nucleotide sequence of the gene cDNA is shown in SEQ ID NO.1.

[0024] The present invention also provides a transgenic plant in which the transgenic plant overexpresses TaPHFT Genes, the ones mentioned TaPHFT The nucleotide sequence of the gene cDNA is shown in SEQ ID NO.1.

[0025] The present invention has the following advantages and effects compared with the prior art: This invention discovered overexpression in wheat TaPHFT The gene significantly reduced wheat plant height and significantly advanced the heading date. These results are of great significance for crop breeding and provide target sites for precision molecular breeding of crops. Attached Figure Description

[0026] Figure 1 This is a PCR amplification band diagram. The left side of the image shows... TaPHFT The sample with gene overexpression is shown on the right, while the negative control is shown on the right.

[0027] Figure 2 For overexpression TaPHFT Figure showing the results of a study on the effects of genes on wheat heading date; in the left figure, the left side represents the Fielder wild type, and the right side represents... PHFT - OE The table shows three strains (scale bar = 1 cm). Different lowercase letters in the right figure indicate significant differences as determined by Duncan's test. p <0.05).

[0028] Figure 3 For overexpression TaPHFTFigure showing the results of a study on the effects of genes on wheat plant architecture; in the left figure, the left side represents the Fielder wild type, and the right side represents the [other type]. PHFT-OE The three strains in the table (scale bar = 1 cm). Different lowercase letters in the right figure indicate significant differences as determined by Duncan's test. p <0.05). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] All raw materials and equipment used in this invention are commercially available products that can be directly purchased from the market, and the primer sequences used are synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0032] TaPHFT The gene is located on chromosome 7B (Chr7B: 18207990-18218252), and its locus number is [missing information]. TraesCS7B03G0054200 Its full-length genome sequence is 11,510 bp, including 8 exons, 7 introns, a 5′ untranslated region (5′UTR), and a 3′ untranslated region (3′UTR). Its full-length cDNA is 684 bp (SEQ ID NO.1), encoding 227 amino acids (SEQ ID NO.3), and the nucleotide sequence of its promoter is shown in SEQ ID NO.2.

[0033] Example 1 TaPHFT Construction of gene overexpression vectors Using two-week-old young leaves of the new cultivar ZK331 (Zhongke 331), developed by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, total RNA was extracted and cDNA was obtained by reverse transcription. Specific primers were designed based on the TaPHFT coding region sequence, and upstream and downstream primers (SEQ ID NO.4 and SEQ ID NO.5) were introduced into the target area. BamHI and SacI Enzyme restriction sites and homologous sequences at the ends of the pLGYE-Ubi-003 vector were identified. PCR amplification was performed using cDNA as a template, and the product was purified by agarose gel electrophoresis. The pLGYE-Ubi-003 vector was then... BamHI and SacIThe linearized fragment was recovered after double enzyme digestion, and then ligated with the target gene fragment using the Uniclone One Step Seamless Cloning Kit at 50°C for 10 min. The resulting ligation product was transformed into DH5α competent cells, plated on LB agar containing kanamycin, and incubated overnight at 37°C. Single colonies were then picked for colony PCR detection. Colonies with the correct band size were selected for amplification and sequencing. After sequence verification, plasmids were extracted and transformed into Agrobacterium tumefaciens EHA105 for subsequent wheat genetic transformation.

[0034] Example 2 TaPHFT Creation of transgenic plants 1) Separation and pre-culture of immature embryos Select Fielder spikelets 12-14 days after pollination (gifted by Professor Li Genying of Shandong Academy of Agricultural Sciences). Under a sterile operating table, use micro-forceps to separate the embryos under a dissecting microscope. Arrange the embryos with the scutellum facing upwards on a pre-culture medium (containing auxins such as 2,4-D) and incubate in the dark at 22-25℃ for 3-5 days.

[0035] 2) Culture of Agrobacterium The recombinant vector pLGYE-Ubi-003 will be included: TaPHFT Agrobacterium EHA105 was streaked onto solid LB agar plates containing 50 mg / L kanamycin and incubated in the dark at 28°C for 2-3 days. The Agrobacterium cells were collected, suspended in LB liquid medium, and the cell concentration was adjusted to OD. 600 Add AS (acetylsuccinone) to a concentration of 0.3-0.5 to make the final AS concentration 100 µM, which is the Agrobacterium suspension for co-culturing and transforming wheat.

[0036] 3) Agrobacterium infection of immature embryos Collect the pre-cultured embryos into sterile Erlenmeyer flasks, pour in the Agrobacterium tumefaciens solution prepared in Example 1, and soak for 15-30 minutes. Gently agitate during this time to ensure full contact. Then filter out the bacterial solution, remove the infected embryos, and blot away excess solution with sterile filter paper. Place them on co-culture medium (containing AS and 2,4-D), with the scutellum facing upwards. Incubate in the dark at 22-24°C for 2-3 days.

[0037] 4) Screening and callus induction After co-culture, the embryos were thoroughly washed five times with sterile water in a 100 mL Erlenmeyer flask, blotted dry with sterile filter paper, and then transferred to a selection medium containing an antibacterial agent (2,4-D). Recovery culture was performed for 5-7 days. The recovered callus was then transferred to a selection medium (containing glyphosate) for selective culture. The selection medium was changed every two weeks, and cultured for 6-8 weeks until bright yellow, tightly structured callus tissue appeared.

[0038] 5) Differentiation The callus tissue was transferred to differentiation medium (+2 mg / L 6-benzylaminopurine + 250 mg / L carbenicillin + 10 mg / L glufosinate + 1 g / L hydrolyzed casein + 0.1 mg / L naphthaleneacetic acid) and cultured at 26-28℃ under light until regenerated plants differentiated.

[0039] 6) Identification of positive plants Total DNA was extracted from transgenic wheat. Using the DNA as a template, specific primers (SEQ ID NO. 6 and SEQ ID NO. 7) were designed based on the tag gene on the vector and our target gene for PCR amplification. The amplified bands were analyzed. Figure 1 (To determine the positive plants of the transgenic gene)

[0040] Example 3: Phenotypic Measurement and Statistical Analysis Fielder and TaPHFT-OE All plants were planted in the field, with a longitudinal spacing of 10-15 cm and a transverse spacing of 13-20 cm. Phenotypic photos and measurements were taken during the flowering and grain-filling stages, as detailed below: Analysis of flowering period: 20 plants were randomly collected for statistical analysis.

[0041] Plant height analysis: 20 plants were randomly collected for statistical analysis.

[0042] Figure 1 For overexpression TaPHFT Plant identification involves identifying plants that show DNA bands as overexpressing plants.

[0043] Figure 2 For overexpression TaPHFT A diagram showing the results of a study on the influence of genes on wheat heading time. (From...) Figure 2 It can be seen from this that overexpression TaPHFT The gene can significantly advance the heading stage of wheat, by about a week.

[0044] Figure 3 For overexpression TaPHFT A diagram showing the results of a study on the influence of genes on wheat plant architecture. (From...) Figure 3 It can be seen from this that overexpression TaPHFT Genes can significantly reduce wheat plant height.

[0045] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. TaPHFT Use of a gene in modulating plant architecture and heading date, characterized in that, When applied, by overexpressing the above TaPHFT Genes that reduce plant height and advance heading time; The plant is wheat; The TaPHFT The nucleotide sequence of the gene cDNA is shown as SEQ ID NO. 1; The TaPHFT The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.

3.

2. A method of reducing plant height and promoting early heading time in wheat, characterized by, overexpressing a gene in wheat TaPHFT steps to reduce wheat plant height and promote early heading time in wheat The TaPHFT The nucleotide sequence of the gene cDNA is shown as SEQ ID NO. 1.

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