Wheat grain protein content related protein as well as coding gene and application thereof

Abstract

The invention relates to the technical field of plant genetic breeding, in particular to a wheat grain protein content related protein and a coding gene and application thereof. The protein content related to the protein content of the wheat grains disclosed by the invention is TaNPF5.5-2A protein and / or TaNPF5.5-2B protein. The research shows that the grain protein content and the grain total nitrogen content of the TaNPF5.5-2B overexpression transgenic wheat strain have remarkable advantages. The new wheat grain protein content related gene is screened and excavated, a theoretical clue is provided for molecular genetic research of the wheat grain protein content, and an important gene resource is provided for wheat quality molecular breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic breeding, and in particular to proteins related to protein content in wheat grains, coding genes and applications thereof. Background Art

[0002] Protein is the basic substance of human tissue and participates in various metabolic processes in the human body under the action of various enzymes. Wheat provides 19% of protein nutrition for humans and is one of the main sources of protein required by humans. The proteins in wheat grains are divided into four types: glutenin, gliadin, globulin and albumin according to their solubility in different solvents. Glutenin and gliadin account for about 80% of the total grain protein. The content and ratio of the two are the main factors affecting the nutritional quality and processing quality of wheat. The protein content of wheat grains is closely related to the nutritional quality and processing quality of wheat. Therefore, the protein content of wheat grains can be used as an important indicator to measure the nutritional quality and processing quality of wheat. Improving the protein content of wheat grains (Grain protein content, GPC) is an important goal of wheat quality genetic breeding. However, so far, there have been few reports on the cloning of genes related to wheat grain protein content.

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

[0004] In order to solve the above technical problems, the present invention provides proteins related to the protein content of wheat grains, their encoding genes and applications.

[0005] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides an 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; the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.02.

[0006] Preferably, the present invention increases the protein content of wheat grains by increasing the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat.

[0007] 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.

[0008] Preferably, the nucleotide sequence of the gene encoding the TaNPF5.5-2A protein of the present invention is as shown in SEQ ID NO.03.

[0009] Preferably, the nucleotide sequence of the gene encoding the TaNPF5.5-2B protein of the present invention is as shown in SEQ ID NO.01.

[0010] In a second aspect, the present invention provides a method for increasing the protein content of wheat grains by introducing the coding 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 the recipient wheat.

[0011] In a third aspect, the present invention provides application of the method for increasing the protein content of wheat grains in wheat breeding.

[0012] In the fourth aspect, the present invention provides an application of a protein or its encoding gene in early detection of wheat grain protein content, 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; the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.02.

[0013] Preferably, the present invention determines the protein content of wheat grains by detecting the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat; the judgment method includes: if the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat is high, the protein content of wheat grains is high.

[0014] Preferably, the primer pair sequences for detecting the TaNPF5.5-2B overexpressing transgenic strain are shown in SEQ ID NO.7 and SEQ ID NO.8; Alternatively, the primer pair sequences for detecting the expression level of TaNPF5.5-2B protein are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0015] Beneficial effects: The present invention provides a protein related to the protein content of wheat grains, a coding gene and an application thereof, wherein the protein related to the protein content of wheat grains is TaNPF5.5-2B protein and its homologous protein TaNPF5.5-2A protein. The present invention's research shows that the grain protein content and grain total nitrogen content of the transgenic wheat strain with TaNPF5.5-2B overexpression have significant advantages. The present invention screens and mines new genes related to the protein content of wheat grains, which not only provides theoretical clues for the molecular genetic research of the protein content of wheat grains, but also provides important gene resources for molecular breeding of wheat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0017] Figure 1 for TaSKP1 Gene PCR results; where: M is 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, respectively.

[0018] Figure 2 This is a positive identification result of the transcription level of overexpressed transgenic wheat.

[0019] Figure 3 The results are for determining the protein content in overexpressed transgenic wheat grains.

[0020] Figure 4 The results are for determining the total nitrogen content in grains of overexpressed transgenic wheat. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0024] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0025] The sequences involved in the following embodiments include: Sequence 1: TaNPF5.5-2B CDS sequence 1623 bp (SEQ ID NO.01).

[0026]

[0027] Sequence 2: TaNPF5.5-2B amino acid sequence, 540 aa (SEQ ID NO.02).

[0028] MAAVDAEAPFLEGKEEPLAGVSDFRGRPVYRATSGGWRSAIFVAVVEGAGSFVYYGVSANLITYMTGPLGHSNAEAAAAVNVWTGTARLMPLLGAFVADSWLGRYWSIILACTLYVLGYGMITLASTLLTQRPSSTLDNDPSSNPLSPQVAFFYVSLYLIALAQGADKPCGLAFAADQFDAEHPRECAARSSFFNWWHFSISIGIAVAIIAVSYIQENLGWGICFGMLCTVMICAFIVFLLGIPTYRLHVSIIGSGSPFIHLGCNLITLARNSGFSSHAKTHMHEDEDATTNLEEARGVLRLLPIWVACLAYGVVFVQITTLFNKQGHTLDCHIFGSLVLPPAMLQTFWPASVLLFVPFYDRVLVPTLRCLTGIPTGLTQLQRVGTGMAMSLAAMCVAALVETQRLEMARVHNLVEDTDATVPMSWSWLVPQYVMVGVADVFVIVGMQEFFYDQMPSELRSLGIALYCSVIGIGGFISGTLISLIDRITREGGGDSWFSDNLNRAHLDYFYWLLAALSAVELALYIFFARAYTYKDKRDF*。

[0029] Sequence 3: TaNPF5.5-2A CDS sequence, 1617 bp (SEQ ID NO.03).

[0030]

[0031] Sequence 4: TaNPF5.5-2A amino acid sequence 538 aa (SEQ ID NO.04).

[0032] *.

[0033] In the above sequences, sequence 3 is a homologous gene of sequence 1, and sequence 4 is a homologous amino acid of sequence 2. In view of the above homology, sequence 3 and sequence 4 have functions similar to sequence 1 and sequence 2, respectively.

[0034] Example 1 This example illustrates the discovery process and cloning method of the gene encoding the wheat grain protein content-related protein TaNPF5.5-2B.

[0035] The wheat gene transcriptional regulatory network comprehensive database (http: / / wheat.cau.edu.cn / wGRN / ) of the bioinformatics platform of the Wheat Research Center of China Agricultural University was used to predict and analyze the downstream genes regulated by TaNAM-6A. After screening, the downstream gene TaNPF5.5-2B that TaNAM-6A may regulate was found, and relevant verification was carried out by molecular biological methods. Subsequently, the flag leaves of Fielder 25 days after flowering were taken as experimental samples, and the total RNA of the leaves was extracted using the Trizol method. The PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit (RR047A; TaKaRa) of TaKaRa was used for reverse transcription to obtain wheat leaf cDNA. The cDNA of wheat leaves was used as a template, and the primers synthesized by Beijing Qingke Biotechnology Co., Ltd. were used: F: 5'-GAGCAAGAACGTACCGGCAT-3', SEQ ID NO.05; R: 5'-TTCCTGGCATACATTCTCAAGT-3', SEQ ID NO. 06.

[0036] PCR amplification was performed with the following reaction procedure: pre-denaturation at 94°C for 5 min; 35 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 2 min; and extension at 68°C for 5 min.

[0037] After agarose gel electrophoresis, a PCR amplification product of about 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, and the amino acid sequence encoded by the gene was named TaNPF5.5-2B protein.

[0038] Example 2 This embodiment TaNPF5.5-2B The process of obtaining over-expression transgenic wheat is described.

[0039] (1) Using adapter primers: F: 5'-AGGTCGACTCTAGAGGATCCATGGCAGCGGTGGATGCCGA-3', SEQ ID NO.07; R: 5'-AGCTCGGTACCCGGGGATCCAAAGTCTCTCTTGTCCTTGT-3', SEQ ID NO. 08.

[0040] Amplification TaNPF5.5-2B CDS sequence, the PCR product was purified and used for restriction digestion and ligation reaction.

[0041] (2) The expression vector pWMB110, with the restriction site BamHI. The total reaction system is 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. Reaction conditions: 37℃ for 3 h, and place on ice to cool after the reaction.

[0042] (3) The products of steps (1) and (2) were homologously recombined using homologous recombination enzymes from Beijing Biomed Gene Technology Co., Ltd. The total reaction system was 10 µL: 5 µL homologous recombination enzyme, 3 µL vector fragment, and 2 µL PCR product. Reaction conditions: 50°C, 30 min. After the reaction, place on ice for later use.

[0043] (4) According to the E. coli transformation instructions of Beijing Bomed Gene Technology Co., Ltd., the product of step 3) was transformed into DH5α, evenly spread on LB plates containing 50 mg / ml Kana, and incubated inverted at 37°C overnight; using detection primers: 110-seq-F: 5'-TAGCCCTGCCTCATACGCT-3', SEQ ID NO.09; 110-seq-R: 5'-AAGACCGGCAACAGGATTCA-3', SEQ ID NO. 10.

[0044] Perform colony PCR detection, and send the positive products to Beijing Qingke Biotechnology Co., Ltd. for sequencing; extract the cloned plasmids with correct sequencing and transform them into EHA105 Agrobacterium to obtain recombinant bacteria.

[0045] (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 to obtain T0 generation plants. The DNA and RNA of the plants were extracted by CTAB method and Trizol method respectively and the primers were used: qPCR-F: 5'-CGCCTCCACGTATCCATCAT-3', SEQ ID NO.11; qPCR-R: 5'-GCACGAATACCACACCATATGC-3', SEQ ID NO. 12.

[0046] Identification was performed at the transcriptome level, and after two generations of screening and identification, an overexpression transgenic positive strain was obtained. OE-1 and OE-2 ( Figure 2 ).

[0047] Example 3 This embodiment TaNPF5.5-2B The grain protein content and grain total nitrogen content of the overexpressing transgenic wheat lines were determined.

[0048] (1) Harvest TaNPF5.5-2B Overexpression transgenic lines OE-1 , OE-2 And the mature kernels of wild-type Fielder.

[0049] (2) The protein content of wheat grains of different strains was determined using a Botong DA7200 near-infrared grain analyzer. The results were repeated three times and the mean value was used for data analysis.

[0050] (3) The total nitrogen content in wheat grains of different strains was determined using the Kjeldahl method. Three biological replicates were performed and the mean value was taken for data analysis.

[0051] By expressing wild-type Fielder, overexpressing transgenic lines OE-1 and OE-2 The comparison of grain protein content showed that compared with wild-type Fielder, OE-1 and OE-2 The protein content of wheat grains in the two lines increased significantly ( 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 wheat grains of the two lines was also significantly higher than that of the wild type Fielder ( Figure 4 ), the above results show that TaNPF5.5-2B Genes can affect the protein content of wheat grains by affecting nitrogen transport within the plant.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a protein or its encoding gene in increasing the protein content of wheat grains, characterized in that: 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; 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: The protein content of wheat grains is increased by increasing the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat.

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

03.

4. The use 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.

5. A method for increasing the protein content of wheat grains, characterized in that: The coding genes of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein are introduced into the recipient wheat to increase the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in the recipient wheat.

6. Use of the method for increasing the protein content of wheat grains according to claim 5 in wheat breeding.

7. Use of a protein or its encoding gene in early detection of protein content in wheat grains, characterized in that: 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; the amino acid sequence of the TaNPF5.5-2B protein is shown in SEQ ID NO.

02.

8. The use according to claim 7, characterized in that: The protein content of wheat grains is determined by detecting the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat; the determination method comprises: if the expression level and / or activity of TaNPF5.5-2A protein and / or TaNPF5.5-2B protein in wheat is high, then the protein content of wheat grains is high.

9. The use according to claim 8, characterized in that: The primer pair sequences for detecting the TaNPF5.5-2B overexpression transgenic strain are shown in SEQ ID NO.7 and SEQ ID NO.8; Alternatively, the primer pair sequences for detecting the expression level of TaNPF5.5-2B protein are shown in SEQ ID NO.11 and SEQ ID NO.12.

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