Wheat grain carotenoid content related gene TaSUS21, recombinant plant overexpression vector, protein and application thereof

Abstract

The invention belongs to the technical field of wheat genetic engineering, and discloses a gene TaSUS21 related to the carotenoid content of wheat grains, a recombinant overexpression vector, a protein and application of the gene TaSUS21. The gene TaSUS21 related to the carotenoid content of the wheat is transferred into common wheat for overexpression by utilizing a transgenic technology for the first time, and the effect of the gene TaSUS21 in the breeding of varieties with high carotenoid content of the wheat is verified, so that the genetic regulation mechanism of the carotenoid content of the wheat is disclosed; in addition, improvement of wheat varieties by using a genetic engineering technology is promoted, and meanwhile, an important theoretical foundation is laid for breeding of wheat varieties with high carotenoid content.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat genetic engineering, and relates to a wheat grain carotenoid content-related gene TaSUS21, a recombinant plant overexpression vector, a protein and applications thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the most widely distributed, largest planted area, and highest yielding food crop in the world. It is the staple food of more than 35% of the world's population. The carotenoid content in wheat grains is a key indicator of wheat's appearance and nutritional quality, but the carotenoid content in wheat grains is relatively low. In humans and animals, carotenoids, as antioxidants and vitamin A precursors, are essential nutrients for maintaining normal life activities. A lack of carotenoids in the human body can lead to eye diseases such as night blindness and cataracts. Currently, carotenoid health products on the market are relatively expensive and difficult to extract. Therefore, if the carotenoid content in wheat can be directly increased and ingested by the human body, it can not only improve the nutritional quality of wheat, but also help improve human health.

[0003] However, the carotenoid content in the current main wheat varieties is low, and the cloned carotenoid-related genes are relatively scarce. Therefore, discovering more new genes that regulate carotenoid content and analyzing their genetic regulation mechanisms are of great application value for breeding new wheat varieties with high carotenoid content. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a wheat grain carotenoid content-related gene TaSUS21, which is applied to the breeding of new wheat varieties with high carotenoid content, providing more genetic resources and technical support for the breeding of new wheat varieties with high carotenoid content.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a gene TaSUS21 related to carotenoid content in wheat grains, the genomic nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] Furthermore, the full-length cDNA sequence of the above-mentioned wheat grain carotenoid content-related gene TaSUS21 is:

[0008] 1) the nucleotide sequence shown in SEQ ID NO.2; or,

[0009] 2) A nucleotide sequence with equivalent functions derived from the nucleotide sequence shown in SEQ ID NO.2, such a nucleotide sequence with equivalent functions includes a DNA molecule that hybridizes with the DNA sequence of SEQ ID NO.2 and encodes a protein that controls the carotenoid content trait of wheat, or a DNA molecule that has more than 90% homology with the DNA sequence of SEQ ID NO.2 and encodes a protein that controls the carotenoid content-related trait of wheat.

[0010] Furthermore, the CDS sequence of the above-mentioned wheat grain carotenoid content-related gene TaSUS21 is:

[0011] 1) the nucleic acid sequence shown in SEQ ID NO.3; or,

[0012] 2) A nucleic acid sequence having equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.3.

[0013] In a second aspect, the present invention provides a recombinant plant overexpression vector constructed by the wheat grain carotenoid content-related gene TaSUS21, specifically, the CDS sequence shown in SEQ ID NO.3 is inserted between the restriction sites BamHI and SacI of the plasmid pLGY-OE to obtain the pLGY-OE-TaSUS recombinant overexpression vector.

[0014] In a third aspect, the present invention provides a recombinant overexpression strain constructed by the above-mentioned recombinant plant overexpression vector.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned wheat grain carotenoid content-related gene TaSUS21 in the preparation of transgenic plant cells. Specifically, the recombinant plant overexpression vector carrying the above-mentioned gene is transformed into plant tissue by using an Agrobacterium-mediated genetic transformation method. The transformed plant tissue is then regenerated into a plant by tissue culture, which can be used for subsequent research. The target plant can be either a monocotyledonous plant or a dicotyledonous plant, such as common wheat Fielder.

[0016] In a fifth aspect, the present invention provides the use of the above-mentioned wheat grain carotenoid content-related gene TaSUS21 in wheat breeding.

[0017] In a sixth aspect, the present invention provides a protein encoded by the above-mentioned wheat grain carotenoid content-related gene TaSUS21, whose amino acid sequence is:

[0018] 1) the amino acid sequence shown in SEQ ID NO.4; or,

[0019] 2) Add, delete or replace one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO.4 to obtain the sequence of active fragments or conservative variants.

[0020] In a seventh aspect, the present invention provides the use of the protein encoded by the above-mentioned wheat grain carotenoid content-related gene TaSUS21 in wheat breeding.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention has identified a gene related to carotenoid content in wheat, named TaSUS21, which is located on wheat chromosome 7B and the protein expressed by it can regulate the carotenoid content in wheat grains.

[0023] 2. The present invention clarifies the DNA sequence, CDS sequence and protein coding sequence of the wheat carotenoid content-related gene TaSUS21, providing important gene resources for the breeding of new wheat varieties with high carotenoid content.

[0024] 3. The present invention uses transgenic technology to transfer the wheat carotenoid content-related gene TaSUS21 into common wheat for overexpression for the first time, verifying the role of the TaSUS21 gene in the breeding of wheat varieties with high carotenoid content. This not only helps to reveal the genetic regulation mechanism of wheat carotenoid content, but also promotes the use of genetic engineering technology to improve wheat varieties. At the same time, it also lays an important theoretical foundation for the breeding of wheat varieties with high carotenoid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 To use genome-wide association analysis (GWAS) to discover the carotenoid content-related gene TaSUS21.

[0026] Figure 2 is the location of the TaSUS21 gene on the chromosome.

[0027] Figure 3 This is the QTL positioning analysis of the TaSUS21 gene.

[0028] Figure 4 This is a map of the plant overexpression vector pLGY-OE.

[0029] Figure 5 is the relative expression level of TaSUS21 gene in T2 generation overexpression plants.

[0030] Figure 6 The carotenoid content in the grains of the TaSUS21 wheat overexpression line. DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment involved in the following examples are conventional instruments and equipment unless otherwise specified.

[0032] The phenotypic identification experiments in the following examples were all set up with three biological replicates, and the results were averaged. Primer synthesis and gene sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0033] The wheat materials involved in the following examples were all purchased from the market:

[0034] A natural population consisting of 168 main varieties promoted in recent years in the Huanghuai wheat region was genotyped using an 800KSNP chip covering the entire genome (Chengdu Tiancheng Future Technology Co., Ltd.). This population was used for genome-wide association studies (GWAS);

[0035] The F4 generation RIL population (XX population) was generated by hybridizing Xianyang Dasui and Xinmai 208, which have different gene nucleotide sequences, and this population was used for QTL positioning analysis;

[0036] Fielder: Low carotenoid content wheat varieties are used as overexpression transgenic receptor materials to verify the function of the TaSUS21 gene in regulating the carotenoid content of wheat grains.

[0037] Example 1 Location and acquisition of wheat carotenoid content-related gene TaSUS21

[0038] 168 wheat varieties from the Huanghuai wheat region were selected (as shown in Table 1), and the carotenoid content of the natural population composed of 168 wheat varieties from the Huanghuai wheat region was measured, and then a genome-wide association study (GWAS) was performed using the 800KSNP chip. A total of 359 significant SNP sites were identified, mainly distributed in the 751-754Mb interval of chromosome 7B (226, 63%). This interval contains a total of 12 HC genes, and the TaSUS21 gene (gene ID is TraesCS7B02G482200.1) is enriched with the most significant SNP sites in this section (61, 27.0%), indicating that the TaSUS21 gene is most significantly associated with carotenoid content ( Figure 1). The TaSUS21 genome nucleotide sequence is shown in SEQ ID NO.1, with a length of 9456 bp; the full-length cDNA sequence of TaSUS21 is shown in SEQ ID NO.2, with a length of 2966 bp; the full-length CDS sequence of TaSUS21 is shown in SEQ ID NO.3, with a length of 2586 bp, encoding 861 proteins, and the sequence is shown in SEQ ID NO.4.

[0039] Table 1 168 wheat varieties from the Huanghuai wheat region

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Parents with extreme differences in TaSUS21 gene nucleotide sequence and carotenoid content (Xianyang Dasui and Xinmai 208) were selected for hybridization to create a recombinant inbred line population (RIL, named XX population) containing 143 materials. The genetic linkage map constructed by the RIL population (Zhao, L., Wang, C., Wang, T., Liu, J., Qiao, Q., Yang, Y., Hu, P., Zhang, L., Zhao, S., Chen, D., et al. (2023). Identification of the candidate gene controlling tiller angle in common wheat through genome-wide association study and linkage analysis. Crop J. 11: 870-877.) was then combined with the measured carotenoid content phenotypic data for QTL positioning. The results showed that the QTL controlling carotenoid content was stably detected between markers AX-110580651 and MS-SUS7B in three replicate environments ( Figure 2 and Figure 3 ), indicating that the QTL positioning result of TaSUS21 gene was significant. In summary, TaSUS21 was significantly correlated with the carotenoid content in wheat.

[0046] Example 2 Acquisition and identification of wheat plants overexpressing TaSUS21

[0047] (1) Construction of TaSUS21 plant overexpression vector

[0048] According to the full-length CDS sequence of the wheat variety TaSUS21 in Chinese Spring, primers were designed using SnapGene software to amplify the CDS sequence of the TaSUS21 gene. The forward primer of the primer pair is the upstream primer TaSUS-F1 with a nucleotide sequence as shown in SEQ ID NO.5, and the reverse primer of the primer pair is the downstream primer TaSUS-R1 with a nucleotide sequence as shown in SEQ ID NO.6. TaSUS-F1: 5'-CGATGACAAG GGATCC ATGGCCTCCAAGCTCAGC-3' (SEQ ID NO.5);

[0049] TaSUS-R1: 5′-CGAAAGCTCT GAGCTC TCATTTGTTAGCGCGCCG-3' (SEQ ID NO. 6).

[0050] Total RNA from wheat seedlings of Chinese Spring was extracted using TRIzol reagent, and RNA was used as a template to reverse transcribe cDNA using SuperScript III reverse transcriptase (Invitrogen, Cat no. 18080-044). Then, cDNA was used as a template to PCR amplify the CDS sequence of the wheat TaSUS21 gene using primers TaSUS-F1 and TaSUS-R1. The PCR amplification system and amplification procedure are shown in Tables 2 and 3, respectively, and the amplified product was recovered and purified for later use.

[0051] Table 2 PCR amplification system of TaSUS21 gene CDS sequence

[0052]

[0053] Table 3 PCR amplification program of TaSUS21 gene CDS sequence

[0054]

[0055]

[0056] The plant overexpression vector pLGY-OE (provided by Shandong Academy of Agricultural Sciences) was selected, and its map is shown in Figure 4As shown. When constructing the plant overexpression vector, a strong promoter of the ubiquitin gene in corn is added before the transcription start nucleotide. In addition, a transcription enhancer or a translation enhancer is used in the region near the start codon to increase the transcription or translation strength of the gene. The expression vector also contains the 3' non-translated region of the foreign gene, that is, a polyadenylic acid signal and other DNA sequences involved in mRNA processing or gene expression. In order to facilitate the screening and identification of transgenic plants, a kanamycin resistance (kan) marker gene and a herbicide resistance (bar) marker gene that can be expressed in plants are also added.

[0057] The plant overexpression vector pLGY-OE was double-digested with BamHI and SacI, recovered and purified, and subjected to homologous recombination reaction with the recovered wheat TaSUS21 gene amplification product to complete the construction of the pLGY-OE-TaSUS recombinant overexpression vector.

[0058] (2) Obtaining TaSUS21 overexpression positive plants

[0059] pLGY-OE-TaSUS was genetically transformed into callus tissue of common wheat Fielder by Agrobacterium infection, and regenerated plants were obtained after screening, pre-differentiation and differentiation. The genomic DNA (gDNA) of the regenerated plants was extracted and PCR amplified using primers TaSUS-F2 and TaSUS-R2. The PCR amplification system and amplification program are shown in Tables 4 and 5, respectively. The amplified products were sequenced to identify T0 generation overexpression positive plants.

[0060] The forward primer of the primer pair is the upstream primer TaSUS-F2 whose nucleotide sequence is shown as SEQ ID NO.7, and the reverse primer of the primer pair is the downstream primer TaSUS-R2 whose nucleotide sequence is shown as SEQ ID NO.8.

[0061] TaSUS-F2: 5'-ATGGCCTCCAAGCTCAGC-3' (SEQ ID NO. 7);

[0062] TaSUS-R2: 5'-TCATTTGTTAGCGCGCCG-3' (SEQ ID NO. 8).

[0063] Table 4 PCR amplification system for identification of TaSUS21 overexpression positive plants

[0064]

[0065] Table 5 PCR amplification procedure for identification of TaSUS21 overexpression positive plants

[0066]

[0067] (3) Detection of gene expression in TaSUS21 overexpressing plants and determination of carotenoid content in grains

[0068] Through PCR identification and screening of overexpression positive plants, a total of 17 T0 generation overexpression positive lines were obtained, and after additional generations, T2 generation overexpression lines were obtained. Using TaSUS-F3 and TaSUS-R3 primers, real-time fluorescence quantitative PCR was used to detect the relative expression level of TaSUS21 gene in T2 generation overexpression plants ( Figure 5 ). The PCR amplification system and amplification program are shown in Table 6 and Table 7 respectively.

[0069] The forward primer of the primer pair is the upstream primer TaSUS-F3 whose nucleotide sequence is shown as SEQ ID NO.9, and the reverse primer of the primer pair is the downstream primer TaSUS-R3 whose nucleotide sequence is shown as SEQ ID NO.10.

[0070] TaSUS-F3: 5'-ATGAAGAGGTGCTTCCAAAGG-3' (SEQ ID NO. 9);

[0071] TaSUS-R3: 5'-GCTTCCTGAGTCGAACAAATAAT-3' (SEQ ID NO. 10).

[0072] Table 6 Fluorescence quantitative PCR amplification system for detecting the expression of TaSUS21 gene

[0073]

[0074] Table 7 Fluorescence quantitative PCR amplification program for detecting the expression of TaSUS21 gene

[0075]

[0076] from Figure 5 It can be seen that the expression level of the target gene TaSUS21 in the T2 generation overexpression positive lines (SUS-OE-5, SUS-OE-6 and SUS-OE-18) is significantly higher than that in the wild-type wheat Fielder, which is about 30-70 times the gene expression level in Fielder plants.

[0077] Subsequently, the carotenoid content in the grains of wild-type wheat Fielder and T2 generation overexpression positive lines was determined ( Figure 6). It can be seen that compared with the wild-type Fielder, the carotenoid content in the grains of the T2 generation overexpression positive strains was significantly reduced, indicating that the TaSUS21 gene negatively regulates the carotenoid content in wheat grains. Therefore, the use of TaSUS21 gene-edited materials can obtain new wheat germplasm with high carotenoid content.

[0078] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A gene related to carotenoid content in wheat grains TaSUS21 , characterized in that, The genomic nucleotide sequence thereof is shown in SEQ ID NO.

1.

2. A gene related to carotenoid content in wheat grains according to claim 1 TaSUS21 , characterized in that, The full-length cDNA sequence is: 1) the nucleotide sequence shown in SEQ ID NO.2; or, 2) A nucleotide sequence with equivalent function derived from the nucleotide sequence shown in SEQ ID NO.

2.

3. A gene related to carotenoid content in wheat grains according to claim 2 TaSUS21 , characterized in that, Its full-length CDS sequence is: 1) the nucleic acid sequence shown in SEQ ID NO.3; or, 2) A nucleic acid sequence with equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.

3.

4. Using a gene related to carotenoid content in wheat grains according to any one of claims 1 to 3 TaSUS21 Constructed recombinant plant overexpression vector.

5. A recombinant overexpression strain constructed by the recombinant plant overexpression vector according to claim 4.

6. A gene related to carotenoid content in wheat grains according to any one of claims 1 to 3 TaSUS21 Application in the preparation of transgenic plant cells.

7. The gene related to carotenoid content in wheat grains according to any one of claims 1 to 3 TaSUS21 Application of genes in wheat breeding.

8. A gene related to carotenoid content in wheat grains according to claim 1 TaSUS21 The encoded protein is characterized in that Its amino acid sequence is: 1) the amino acid sequence shown in SEQ ID NO.4; or, 2) Add, delete or replace one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO.4 to obtain the sequence of active fragments or conservative variants.

9. The gene related to carotenoid content in wheat grains according to claim 8 TaSUS21 Application of the encoded protein in wheat breeding.

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