Use of taBLGU1-5a gene or biological material containing the gene in regulating protein content of plant seeds

Abstract

The application relates to the technical field of plant genetic engineering, and discloses application of a TaBLGU1-5A gene or biological material containing the gene in regulating the protein content of plant seeds. The gene TaBLGU1-5A is cloned from a Chinese spring wheat genome, and is overexpressed in Fielder wheat to obtain TaBLGU1-5A transgenic overexpression plants; and the detection result shows that, compared with a wild type control variety, the TaBLGU1-5A gene overexpression material can significantly increase the protein content in the wheat seeds. Therefore, the TaBLGU1-5A gene has the function of regulating the protein content of plant seeds, and can be applied to plant breeding or quality improvement.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of the TaBLGU1-5A gene or biological materials containing the gene in regulating the protein content of plant seeds. Background Technology

[0002] wheat( Triticum aestivum Wheat (L.) is one of the most widely cultivated food crops globally, a staple food for human survival, providing approximately 20% of the calories and protein needed for human life activities. As people's living standards continue to improve, the requirements for wheat quality are also gradually increasing. While focusing on wheat yield, it is also important to improve wheat grain quality. Wheat grains contain approximately 10% to 15% protein by weight, including a variety of essential amino acids. Grain protein content is one of the important quality indicators of wheat, directly affecting its nutritional and processing quality. Therefore, effectively improving the protein content in wheat grains will help improve the nutritional and processing quality of wheat.

[0003] Extensive research has been conducted on the genetic mechanisms of wheat grain protein content. However, to date, few genes regulating wheat grain protein content with significant practical value have been reported, and the mechanism of wheat grain protein formation remains unclear, greatly limiting the progress of wheat quality improvement breeding. Therefore, it is urgent to further discover and effectively utilize genes regulating wheat grain protein content. Discovering key genes regulating wheat grain protein content through genetic methods and exploring their functions can deepen our understanding of the genetic mechanisms of wheat grain protein content and provide an important theoretical foundation and genetic resources for wheat quality improvement breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the TaBLGU1-5A gene or biological materials containing the gene in regulating the protein content of plant seeds.

[0005] In order to achieve the purpose of this invention, in a first aspect, this invention provides the application of the TaBLGU1-5A gene or biological materials containing said gene in regulating the protein content of plant seeds.

[0006] The TaBLGU1-5A gene of this invention is a gene encoding either (a) or (b) the following protein:

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0008] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0009] The CDS sequence of the TaBLGU1-5A gene is as follows:

[0010] i) the nucleotide sequence shown in SEQ ID NO: 1 ;

[0011] ii) a nucleotide sequence of which the nucleotide sequence shown in SEQ ID NO: 1 is substituted, deleted and / or increased by one or more nucleotides and expresses the same functional protein;

[0012] iii) a nucleotide sequence which hybridizes to the sequence shown in SEQ ID NO: 1 under stringent conditions, which are hybridization in 0.1 x SSPE or 0.1 x SSC containing 0.1% SDS at 65°C, and washing the membrane with the solution; and expresses the same functional protein; or

[0013] iv) a nucleotide sequence which has more than 90% homology to the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

[0014] Further, the regulation is positive regulation.

[0015] Further, the biological material includes, but is not limited to, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0016] Further, the plant includes, but is not limited to, wheat.

[0017] In a second aspect, the present application provides a method for increasing the protein content of wheat grains, which comprises: enhancing the TaBLGU1-5A gene in wheat by genetic engineering.

[0018] Further, the enhancement approach can be selected from the following 1) ~ 6), or optional combinations:

[0019] 1) enhanced by introducing a plasmid having the gene;

[0020] 2) enhanced by increasing the copy number of the gene on the chromosome;

[0021] 3) enhanced by changing the promoter sequence of the gene on the chromosome;

[0022] 4) enhanced by operably linking a strong promoter to the gene;

[0023] 5) enhanced by introducing an enhancer;

[0024] 6) enhanced by using a gene or allele having a gene encoding a highly active corresponding enzyme or protein.

[0025] The expression vector carrying the gene of interest can be introduced into plant cells by using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation and other conventional biotechnological methods (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2nd Edition). In a third aspect, the present application provides the use of the transgenic wheat obtained according to the method in plant breeding.

[0026] The breeding method includes, but is not limited to, transgenesis, crossing, backcrossing, selfing or asexual reproduction.

[0027] In a fourth aspect, the present application provides the use of TaBLGU1-5A gene or biological material containing the gene in plant breeding or quality improvement.

[0028] Further, the purpose of breeding or quality improvement is to increase the protein content in plant seeds.

[0029] Further, the plant includes, but is not limited to, wheat.

[0030] By means of the above technical solution, the present application has at least the following advantages and beneficial effects:

[0031] The present application discloses the use of TaBLGU1-5A gene and related biological material in regulating the protein content in plant seeds. The gene TaBLGU1-5A is cloned from the genome of Chinese spring wheat, and is overexpressed in Fielder wheat to obtain TaBLGU1-5A transgenic overexpression plants. The detection results show that, compared with the wild type control variety, the overexpression of TaBLGU1-5A gene can significantly increase the protein content in wheat seeds. Therefore, the TaBLGU1-5A gene has the function of regulating the protein content in plant seeds, and can be applied to plant breeding or quality improvement. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The figure is the vector map of TaOE (i.e. TaOE-PPT) in the preferred embodiment of the present application.

[0033] Figure 2 The figure is the vector map of TaOE-TaBLGU1-5A in the preferred embodiment of the present application.

[0034] Figure 3RT-PCR detection results of relative expression of TaBLGU1-5A gene in wheat overexpression lines and wild type plants in the preferred embodiments of the present application.

[0035] Figure 4 Results of detection of total protein content in mature seeds of wheat overexpression lines and wild type plants using near infrared spectrometer in the preferred embodiments of the present application. DETAILED DESCRIPTION

[0036] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means well known to those skilled in the art, and the raw materials used are commercially available.

[0037] Example 1 Cloning and functional verification of gene TaBLGU1-5A

[0038] 1. Experimental materials and reagents:

[0039] Wheat material “Fielder”: provided by Fu Xiangdong group of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0040] Reagents:

[0041] Escherichia coli Trans1-T1 Phage Resistant competent cells: fullzyme, item number: CD501-03;

[0042] RNA reverse transcription kit EasyScript® One-Step RT-PCR SuperMix: fullzyme, item number: AE411-02;

[0043] Seamless cloning kit pEASY®-Uni Seamless Cloning and Assembly Kit: fullzyme, item number: CU101-01;

[0044] Wheat overexpression vector TaOE, provided by Professor Liu Zhiyong of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Li, M., Zhang, H., Xiao, H. et al. (2024). A membrane associated tandem kinase from wild emmer wheat confers broad-spectrum resistance to powdery mildew. Nat. Commun. 15, 3124.

[0045] Agrobacterium EHA109 competent cells: Huaiyueyang, item number: GT707.

[0046] 2. Experimental method:

[0047] In the early stage, through the investigation of 350 germplasm resource materials and the whole genome association analysis (GWAS), our research group obtained the candidate gene TaBLGU1-5A related to the regulation of wheat grain protein content. Through literature review, it was found that TaBLGU1 family members are a kind of β-glucosidase, which is a kind of hydrolase widely existing in microorganisms, plants and animals, belongs to glycoside hydrolase family (GH1, GH3, etc.), can specifically hydrolyze β-glycosidic bond, release glucose or other active molecules, regulate glycoside hydrolysis balance, affect sugar signal pathway, and participate in plant organ development and stress response. In addition, β-glucosidase can hydrolyze glycoside precursors in plants, such as regulating the deglycosylation process of flavonoids, anthocyanins and other compounds, thereby affecting their stability and biological activity, and participating in the regulation of plant secondary metabolism. In addition, β-glucosidase is involved in the regulation of gibberellin, abscisic acid and other hormone signal pathways, and directly or indirectly participates in key processes such as growth and development, stress response, defense mechanism and metabolic regulation. Therefore, we cloned the TaBLGU1-5A gene in wheat and constructed related biological materials and phenotype analysis.

[0048] (1) The total RNA of wheat material Chinese spring leaf was extracted, and after reverse transcription into cDNA, it was used as a template to clone and sequence the wheat TaBLGU1-5A gene using forward primer (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGGGAGTTCGCGCA-3', SEQ ID NO:3) and reverse amplification primer (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCATTTCATCGCCTCGGTGG-3', SEQ ID NO:4). The CDS sequence of TaBLGU1-5A gene is shown in SEQ ID NO:1. The protein sequence encoded by TaBLGU1-5A gene is shown in SEQ ID NO:2.

[0049] The PCR reaction program is as follows: 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 20 seconds, 58℃ recombination for 30 seconds, 68℃ extension for 2 minutes, 40 cycles; 68℃ extension for 5 minutes.

[0050] (2) Construction of wheat TaBLGU1-5A overexpression vector Figure 1The TaOE vector sequence is shown in CN119776368A). The specific method is as follows: first, the TaOE vector is digested with restriction endonuclease BamH I and Sac I at 37°C for 3 hours, and then recovered and purified. Then, the PCR product (3 uL), the linearized TaOE fragment (2 uL) and 2 x Assembly Mix (5 uL) are mixed, and the ligation reaction is carried out at 50°C for 30 minutes. Finally, the ligation product is transformed into E. coli Trans1-T1 competent cells for transformation, and cultured at 37°C for 12 hours to obtain recombinant E. coli. Positive monoclonal colonies are screened and sequenced.

[0051] The correct monoclonal colony is amplified in the corresponding resistant LB liquid medium (containing kanamycin 50 mg / L), and the recombinant plasmid TaOE-TaBLGU1-5A is extracted Figure 2 ). Second, the TaOE-TaBLGU1-5A plasmid (10 uL) is transformed into Agrobacterium EHA105 competent cells, and cultured on LB solid medium (containing kanamycin 50 mg / L and rifampicin 25 mg / L) at 28°C for 2 days. Positive clones are selected and identified by PCR using forward primer F: 5'-GATGATGGCATATGCAGCAGCT-3' and reverse primer R: 5'-TGTCGAAACCGATGATACGAACGA-3' (SEQ ID NO: 5-6). Finally, the correct positive bacteria liquid obtained after PCR identification is mixed with 50% glycerol at an equal ratio and stored in a -80°C refrigerator for standby use.

[0052] (3) Obtaining of transgenic plants: Using the wheat genetic transformation system established by Yuji Ishida et al. (Ishida Y, Tsunashima M, Hiei Y, Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol Biol. 2015; 1223: 189-98.), the wheat variety Fielder was used as the recipient material, and TaOE-TaBLGU1-5A overexpression transgenic plants were obtained by Agrobacterium-mediated method. The identification of transgenic plants was performed by PCR detection using Bar gene-specific primers (forward primer: 5'-ATGAGCCCAGAACGACGCCCG-3' and reverse primer: 5'-AACTCGAGTCAAATCTCGGT-3', SEQ ID NO: 7-8), and the length of the amplified product was 560 bp. A total of 31 independent T0 generation transgenic lines were obtained. The T0 positive plants were selfed to obtain T1 generation segregation populations, and the positive single plants were screened by PCR detection. The harvested T1 generation seeds were further germinated and planted, and T2 generation homozygous transgenic lines were obtained by PCR detection.

[0053] Detection of the expression amount of TaBLGU1-5A gene in TaOE-TaBLGU1-5A transgenic T2 overexpression wheat lines. We used real-time fluorescent quantitative PCR (Real-time RT-PCR) technology to detect the relative expression amount of TaBLGU1-5A gene in different background materials. Three independent biological replicates were set to enhance the reliability of the data, and the specific operation of each replicate was as follows: total RNA was extracted from the young leaves of three independent TaOE-TaBLGU1-5A transgenic overexpression lines and wild type (WT) wheat Fielder, respectively, and cDNA was synthesized after reverse transcription. Using wheat-actin as the internal reference, the relative expression level of TaBLGU1-5A gene was determined by Real-time RT-PCR.

[0054] The primer sequences of the internal reference gene wheat-actin were: forward primer 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer 5'-CCAGACAACTCGCAACTTAGA-3' (SEQ ID NO: 9-10); the primer sequences of the target gene TaBLGU1-5A were: forward primer 5'-GGTTTTGTGATTACAGACTA-3' and reverse primer 5'-TGGCTGCCAATTTCACCGGCGA-3' (SEQ ID NO: 11-12).

[0055] The results are shown in Figure 3 As shown in

[0056] The protein content in the grains of the above-mentioned three TaOE-TaBLGU1-5A transgenic overexpression lines and wild-type plant (WT) wheat Fielder was determined using a near-infrared spectrometer (WaveScan DA7200).

[0057] The results are shown in Figure 4 As shown in

[0058] It can be seen that overexpression of wheat TaBLGU1-5A can significantly increase the protein content in wheat seeds, and the encoded protein TaBLGU1-5A can regulate the protein content in wheat seeds, which can be applied to wheat breeding and variety improvement.

[0059] The above has been described in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the experiments involved in the present application can be carried out in a wider range under the same parameters, concentrations and conditions.

[0060] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. Use of TaBLGU1-5A gene or biological material containing the gene in regulating protein content in plant seeds; the TaBLGU1-5A gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2; the plant is wheat.

2. Use according to claim 1, characterized in that, The regulation is positive regulation.

3. Use according to claim 1, characterized in that, The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

4. A method of increasing the protein content of wheat grain, characterized in that, The method comprises: using genetic engineering means to enhance the TaBLGU1-5A gene in wheat; the TaBLGU1-5A gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO:

2.

5. The method of claim 4, wherein, The enhancement approach is selected from the following 1) ~ 6), or optional combinations: 1) enhancement by introducing a plasmid with the gene; 2) enhancement by increasing the copy number of the gene on the chromosome; 3) enhancement by changing the promoter sequence of the gene on the chromosome; 4) enhancement by operably linking a strong promoter to the gene; 5) enhancement by introducing an enhancer.

6. Use of transgenic wheat obtained by the method of claim 4 or 5 in plant breeding.

7. Use according to claim 6, characterized in that, The breeding method comprises transgenesis, crossing, backcrossing, selfing or vegetative reproduction.

8. Use of TaBLGU1-5A gene or biological material containing the gene in plant breeding or quality improvement; The purpose of breeding or quality improvement is to increase the protein content in plant seeds; the TaBLGU1-5A gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2; the plant is wheat.

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