Wheat grain protein content-related protein, encoding gene thereof and application

Abstract

The present application relates to the technical field of plant genetic breeding, and particularly relates to a wheat grain protein content related protein, a coding gene thereof and application.The wheat grain protein content related protein is a TaNPF5.5-2A protein and / or a TaNPF5.5-2B protein.The research shows that the grain protein content and the total nitrogen content of the TaNPF5.5-2B overexpression transgenic wheat strain have a significant advantage.The present application screens and mines a new wheat grain protein content related gene, which not only provides a theoretical clue for the molecular genetic research of the wheat grain protein content, but also provides important gene resources for the molecular breeding of the wheat quality.

Description

Technical Field

[0001] This invention relates to the field of plant genetics and breeding technology, and in particular to proteins related to the protein content of wheat grains, their encoding genes, and their applications. Background Technology

[0002] Protein is a fundamental substance in human tissues, participating in various metabolic processes under the action of enzymes. Wheat provides 19% of the protein nutrition needed by humans, making it one of the main sources of protein. Wheat grain proteins are classified into four types based on their solubility in different solvents: glutenin, gliadin, globulin, and albumin. Glutenin and gliadin account for approximately 80% of the total grain protein, and their content and ratio are the main factors affecting the nutritional and processing quality of wheat. The protein content of wheat grains is closely related to their nutritional and processing quality. Therefore, wheat grain protein content can serve as an important indicator for evaluating the nutritional and processing quality of wheat. Improving wheat grain protein content (GPC) is an important goal of wheat quality genetic breeding. However, to date, there are few reports on the cloning of genes related to wheat grain protein content.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides proteins related to wheat grain protein content, their encoding genes, and their applications.

[0005] Specifically, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides the application of a protein or its encoding gene in increasing the protein content of wheat grains, wherein the protein is TaNPF5.5-2A protein and / or TaNPF5.5-2B protein; the amino acid sequence of the TaNPF5.5-2A protein is shown in SEQ ID NO.04; and the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.02.

[0007] Preferably, the present invention increases the protein content of wheat grains by increasing the expression level and / or activity of TaNPF5.5-2A and / or TaNPF5.5-2B proteins in wheat.

[0008] More preferably, the present invention increases the protein content of wheat grains by overexpressing the expression level and / or activity of TaNPF5.5-2B protein in spring wheat Fielder.

[0009] Preferably, the nucleotide sequence of the gene encoding the TaNPF5.5-2A protein of the present invention is shown in SEQ ID NO.03.

[0010] Preferably, the nucleotide sequence of the gene encoding the TaNPF5.5-2B protein of the present invention is shown in SEQ ID NO.01.

[0011] Secondly, the present invention provides a method for increasing the protein content of wheat grains by introducing the encoding genes of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein into recipient wheat, thereby increasing the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in recipient wheat.

[0012] Thirdly, the present invention provides the application of the method for increasing the protein content of wheat grains in wheat breeding.

[0013] Fourthly, the present invention provides an application of a protein or its encoding gene in the early detection of protein content in wheat grains, wherein the protein is TaNPF5.5-2A protein and / or TaNPF5.5-2B protein; the amino acid sequence of the TaNPF5.5-2A protein is shown in SEQ ID NO.04; and the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.02.

[0014] Preferably, the present invention determines the protein content of wheat grains by detecting the expression level and / or activity of TaNPF5.5-2A and / or TaNPF5.5-2B proteins in wheat; the determination method includes: if the expression level and / or activity of TaNPF5.5-2A and / or TaNPF5.5-2B proteins in wheat are high, then the protein content of wheat grains is high.

[0015] Preferably, the primer pair sequences for detecting the TaNPF5.5-2B overexpression transgenic line are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0016] Alternatively, the primer pair sequences for detecting TaNPF5.5-2B protein expression levels are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0017] Beneficial effects:

[0018] This invention provides proteins related to wheat grain protein content, their encoding genes, and their applications. The proteins are TaNPF5.5-2B and its homolog TaNPF5.5-2A. Research shows that transgenic wheat lines overexpressing TaNPF5.5-2B exhibit significant advantages in grain protein content and total nitrogen content. This invention screens and mines new genes related to wheat grain protein content, providing not only theoretical clues for molecular genetic research on wheat grain protein content but also important gene resources for molecular breeding of wheat quality. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0020] Figure 1 for TaSKP1 Gene PCR results; where M is the marker, and the band sizes from top to bottom are 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0021] Figure 2 This is a positive result for identifying the transcriptional level of transgenic wheat overexpression.

[0022] Figure 3 The results show the protein content of overexpressed transgenic wheat grains.

[0023] Figure 4 The results show the total nitrogen content of overexpressed transgenic wheat grains. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0025] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0027] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0028] The sequences involved in the following embodiments include:

[0029] Sequence 1: TaNPF5.5-2B CDS sequence 1623bp (SEQ ID NO.01).

[0030]

[0031] Sequence 2: Amino acid sequence of TaNPF5.5-2B, 540aa (SEQ ID NO.02).

[0032] MAAVDAEAPFLEGKEEPLAGVSDFRGRPVYRATSGGWRSAIFVAVVEGAGSFVYYGVSANLITYMTGPLGHSNAEAAAAVNVWTGTARLMPLLGAFVADSWLGRYWSIILACTLYVLGYGMITLASTLLTQRPSSTLDNDPSSNPLSPQVAFFYVSLYLIALAQGADKPCGLAFAADQFDAEHPRECAARSSFFNWWHFSISIGIAVAIIAVSYIQENLGWGICFGMLCTVMICAFIVFLLGIPTYRLHVSIIGSGSPFIHLGCNLITLARNSGFSSHAKTHMHEDEDATTNLEEARGVLRLLPIWVACLAYGVVFVQITTLFNKQGHTLDCHIFGSLVLPPAMLQTFWPASVLLFVPFYDRVLVPTLRCLTGIPTGLTQLQRVGTGMAMSLAAMCVAALVETQRLEMARVHNLVEDTDATVPMSWSWLVPQYVMVGVADVFVIVGMQEFFYDQMPSELRSLGIALYCSVIGIGGFISGTLISLIDRITREGGGDSWFSDNLNRAHLDYFYWLLAALSAVELALYIFFARAYTYKDKRDF*。

[0033] Sequence 3: TaNPF5.5-2A CDS sequence 1617 bp (SEQ ID NO.03).

[0034]

[0035] Sequence 4: TaNPF5.5-2A amino acid sequence 538 aa (SEQ ID NO.04).

[0036] *

[0037] In the above sequences, sequence 3 is a homolog of sequence 1; sequence 4 is a homologous amino acid of sequence 2. Given this homology, sequences 3 and 4 have functions similar to sequences 1 and 2, respectively.

[0038] Example 1

[0039] This embodiment describes the discovery process and cloning method of the gene encoding TaNPF5.5-2B, a protein related to wheat grain protein content.

[0040] Downstream genes regulated by TaNAM-6A were predicted and analyzed using the comprehensive database of wheat gene transcription regulatory networks from the bioinformatics platform of the Wheat Research Center of China Agricultural University (http: / / wheat.cau.edu.cn / wGRN / ). After screening, the potential downstream gene TaNPF5.5-2B regulated by TaNAM-6A was identified and validated using molecular biology methods. Subsequently, flag leaves 25 days after flowering of *Fielder* wheat were used as experimental samples, and total RNA was extracted from the leaves using the Trizol method. Wheat leaf cDNA was obtained by reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (RR047A; TaKaRa) from TaKaRa. The wheat leaf cDNA was used as a template, and primers synthesized by Beijing Qingke Biotechnology Co., Ltd. were used.

[0041] F: 5'-GAGCAAGAACGTACCGGCAT-3', SEQ ID NO.05;

[0042] R: 5'-TTCCTGGCATACATTCTCAAGT-3', SEQ ID NO. 06.

[0043] PCR amplification was performed using the following reaction program: 94°C pre-denaturation for 5 min; 98°C for 10 s, 60°C for 15 s, 68°C for 2 min, 35 cycles; 68°C extension for 5 min.

[0044] After identification by agarose gel electrophoresis, a PCR amplification product of approximately 1700 bp was obtained. Figure 1 The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the nucleotide sequence of the PCR product included the DNA sequence shown in Sequence 1, and it was named... TaNPF5.5-2B The gene was identified, and the amino acid sequence encoded by the gene was named TaNPF5.5-2B protein.

[0045] Example 2

[0046] This embodiment is... TaNPF5.5-2B The process of obtaining transgenic wheat through overexpression is explained.

[0047] (1) Use adapter primers:

[0048] F: 5'-AGGTCGACTCTAGAGGATCCATGGCAGCGGTGGATGCCGA-3', SEQ ID NO.07;

[0049] R: 5'-AGCTCGGTACCCGGGGATCCAAAGTCTCTCTTGTCCTTGT-3', SEQ ID NO. 08.

[0050] Amplification TaNPF5.5-2B The CDS sequence was used to purify the PCR product for enzyme digestion and ligation reactions.

[0051] (2) The expression vector pWMB110 was digested with BamHI I. The total reaction volume was 50 µL: 1 µg of pWMB110 vector plasmid, 5 µL of 10×NEBuffer, 1 µL of BamHI-HFv2, and 50 µL of Nuclease-free water. The reaction conditions were 37℃ for 3 h, and the mixture was cooled on ice after the reaction was completed.

[0052] (3) Homologous recombination of the products from steps (1) and (2) was performed using the homologous recombinase from Beijing Bomei Gene Technology Co., Ltd. The total reaction volume was 10 µL: 5 µL homologous recombinase, 3 µL vector fragment, and 2 µL PCR product. Reaction conditions: 50℃, 30 min. After the reaction, the product was placed on ice for later use.

[0053] (4) Following the E. coli transformation instructions from Beijing Bomei Gene Technology Co., Ltd., the product from step 3) was transformed into DH5α, and evenly spread on LB plates containing 50 mg / ml Kana. The plates were then incubated overnight at 37°C with the plates inverted. The detection primers were then used.

[0054] 110-seq-F: 5'-TAGCCCTGCCTCATACGCT-3', SEQ ID NO.09;

[0055] 110-seq-R: 5'-AAGACCGGCAACAGGATTCA-3', SEQ ID NO. 10.

[0056] Colony PCR was performed, and positive products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmid with the correct sequence was extracted and transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria.

[0057] (5) The recombinant bacteria were sent to the transgenic platform of the Wheat Research Center of China Agricultural University to transform the recipient wheat Fielder, and T0 generation plants were obtained. The DNA and RNA of the plants were extracted by the CTAB method and the Trizol method, respectively, and primers were used:

[0058] qPCR-F: 5'-CGCCTCCACGTATCCATCAT-3', SEQ ID NO.11;

[0059] qPCR-R: 5'-GCACGAATACCACACCATATGC-3', SEQ ID NO. 12.

[0060] Identification was performed at the transcriptome level, and after two generations of screening and identification, overexpression transgene-positive lines were obtained. OE- 1 and OE-2 ( Figure 2 ).

[0061] Example 3

[0062] This embodiment is... TaNPF5.5-2B The protein content and total nitrogen content of the grains of the overexpression transgenic wheat lines were determined.

[0063] (1) Harvest TaNPF5.5-2B Overexpression transgenic lines OE-1 , OE-2 And the mature seeds of wild-type Fielder.

[0064] (2) The protein content of wheat grains of different strains was determined using a DA7200 near-infrared grain analyzer. The results were repeated three times and the average value was used for data analysis.

[0065] (3) The total nitrogen content in wheat grains of different strains was determined by the Kjeldahl method. The results were repeated three times and the average value was used for data analysis.

[0066] Through analysis of wild-type Fielder and overexpression transgenic lines OE-1 and OE-2 A comparison of seed protein content revealed that, compared to wild-type Fielder, OE-1 and OE-2 The protein content of wheat grains in both lines was significantly increased. Figure 3 Further analysis of the total nitrogen content in the grains revealed that... OE-1 and OE-2 The total nitrogen content in the grains of both wheat lines was also significantly higher than that of the wild-type Fielder. Figure 4 The above results indicate that TaNPF5.5-2B Genes can influence the protein content of wheat grains by affecting nitrogen transport within plants.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a protein or its encoding gene in increasing the protein content of wheat grains, characterized in that, The protein is TaNPF5.5-2B protein; the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.

02.

2. The application according to claim 1, characterized in that, Increase the protein content of wheat grains by increasing the expression level of TaNPF5.5-2B protein in wheat.

3. The application according to claim 1 or 2, characterized in that, The nucleotide sequence of the gene encoding the TaNPF5.5-2B protein is shown in SEQ ID NO.

01.

4. A method for increasing the protein content of wheat grains, characterized in that, The gene encoding the TaNPF5.5-2B protein was introduced into recipient wheat to increase the expression level of the TaNPF5.5-2B protein in recipient wheat.

5. The application of the method for increasing the protein content of wheat grains as described in claim 4 in wheat breeding.

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