SbLAC14 gene separated from sorghum as well as encoding protein and application of SbLAC14 gene

Abstract

The invention discloses an SbLAC14 gene separated from sorghum as well as an encoding protein and application thereof, and belongs to the field of plant genetic engineering and molecular biology. The laccase gene SbLAC14 with a coding region nucleotide sequence as shown in SEQ ID NO.2 is separated from sorghum for the first time through multi-omics conjoint analysis. Functional verification experiment results show that overexpression of the SbLAC14 gene can improve the content of polymeric proanthocyanidins of wild type arabidopsis thaliana, deepen the color of seed coats, and also can completely recover the polymerization defect of proanthocyanidins of a tt10 mutant and the superficial type of the color of the seed coats, which proves that the SbLAC14 gene has the biological function of catalyzing flavane-3-alcohol monomer polymerization to form proanthocyanidins. The invention provides a gene resource and an operation target with important application value for plant procyanidine synthesis regulation and control, and can be used for tannin metabolic engineering and variety improvement.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, and in particular to an SbLAC14 gene isolated from sorghum, its encoded protein, and its applications. Background Technology

[0002] sorghum ( Sorghum bicolor Sorghum (L.) Moench is the world's fifth largest cereal crop, characterized by drought resistance and tolerance to poor soil, and is widely cultivated in tropical and subtropical regions. Sorghum is one of the main raw materials for baijiu (Chinese liquor) brewing. The tannin content in sorghum grains is one of the key factors affecting the flavor and quality of baijiu. Tannins, especially condensed tannins (also known as proanthocyanidins), are a class of polyphenolic compounds polymerized from flavan-3-ol monomers (such as catechins and epicatechin).

[0003] Plant laccases (LACs) belong to the polycopper oxidase family and play a crucial role in lignin synthesis, stress response, and the polymerization of secondary metabolites in plants. However, in sorghum, although transcription factors Tannin1 and Tannin2, which regulate tannin synthesis, have been identified, the key enzyme genes responsible for catalyzing the final polymerization of flavan-3-ol monomers into polymerized proanthocyanidins, particularly members of the laccase gene family, have not yet been cloned and functionally verified. Therefore, identifying and utilizing key genes regulating proanthocyanidin polymerization in sorghum is of great significance for elucidating the molecular mechanisms of tannin synthesis and for genetically improving sorghum quality traits. Summary of the Invention

[0004] The purpose of this invention is to provide an SbLAC14 gene isolated from sorghum, its encoded protein, and its applications, in order to solve the problems existing in the prior art. This invention demonstrates that the SbLAC14 gene has the biological function of catalyzing the polymerization of flavan-3-ol monomers to form proanthocyanidins, providing a directly usable gene resource and operational target for molecular breeding of sorghum and other plants.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an application of the SbLAC14 protein in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content; The amino acid sequence of the SbLAC14 protein is shown in SEQ ID NO.1.

[0006] Furthermore, the expression level of the SbLAC14 protein was upregulated in plants, promoting the synthesis of proanthocyanidins in the plants; The plant in question is either sorghum or Arabidopsis thaliana.

[0007] The present invention also provides an application of the gene encoding the above-mentioned SbLAC14 protein in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0008] Furthermore, the expression level of the encoding gene is upregulated in plants to promote the synthesis of proanthocyanidins in the plants; The plant in question is either sorghum or Arabidopsis thaliana.

[0009] The present invention also provides an application of an overexpression vector, wherein the overexpression vector contains the gene encoding the SbLAC14 protein; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; The application is any one of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content.

[0010] The present invention also provides the use of engineered bacteria comprising the above-described overexpression vector in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content.

[0011] The present invention also provides a method for promoting the synthesis of proanthocyanidins in plants, comprising the step of upregulating the expression level of the gene encoding the SbLAC14 protein in plants to promote the synthesis of proanthocyanidins in said plants; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0012] Furthermore, the plant is sorghum or Arabidopsis thaliana.

[0013] This invention also provides a breeding method for transgenic plants with increased proanthocyanidin content, comprising the following steps: The gene encoding the SbLAC14 protein is overexpressed in plant cells, the plant cells are then cultured, and the plant cells are used to regenerate plants, thus obtaining transgenic plants with increased proanthocyanidin content. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0014] Furthermore, the plant is sorghum or Arabidopsis thaliana.

[0015] The present invention discloses the following technical effects: This invention, through multi-omics joint analysis, isolated the laccase gene SbLAC14, whose coding nucleotide sequence is shown in SEQ ID NO. 2, from sorghum. Functional verification experiments showed that overexpression of the SbLAC14 gene can increase the content of polymerized proanthocyanidins in wild-type Arabidopsis thaliana, deepen the seed coat color, and completely restore the proanthocyanidin polymerization defect and the light seed coat color phenotype of the tt10 mutant, proving that the SbLAC14 gene has the biological function of catalyzing the polymerization of flavan-3-ol monomers to form proanthocyanidins. This invention provides an important supplement to the improvement of the tannin metabolism pathway in sorghum, and provides directly usable gene resources and manipulation targets for molecular breeding of sorghum and other plants. Molecular markers developed based on this gene can be used to assist in screening sorghum germplasm with different tannin contents; by regulating the expression of this gene through genetic engineering, crop quality can be precisely improved to meet the needs of different industries such as food processing and feed production, and it has extremely high scientific research value and broad industrial application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This diagram illustrates the tissue-specific and temporal expression patterns of SbLAC14. A represents the tissue specificity of SbLAC14; B represents the expression dynamics of SbLAC14 during seed development after pollination; S1 represents 5 days, S2 represents 10 days, S3 represents 15 days, S4 represents 20 days, S5 represents 25 days, S6 represents 30 days, S7 represents 35 days, and S8 represents 40 days. Figure 2Figure 1 shows the results of SbLAC14 gene function verification in four Arabidopsis thaliana lines: wild-type (WT), wild-type overexpressing SbLAC14 (OE-LAC14), tt10 mutant (tt10), and tt10 mutant overexpressing SbLAC14 (OE-LAC14 / tt10). In the figure, A represents the seed phenotype of the four Arabidopsis thaliana lines; I-IV represent unstained wild-type seeds (I), overexpressing SbLAC14 wild-type seeds (II), tt10 mutant seeds (III), and overexpressing SbLAC14 tt10 mutant seeds (IV); V-VIII represent DMACA-stained wild-type seeds (V), overexpressing SbLAC14 wild-type seeds (VI), tt10 mutant seeds (VII), and overexpressing SbLAC14 tt10 mutant seeds (VIII). Scale bar: 400. μm; BE was a quantitative analysis of the contents of soluble proanthocyanidins (B), insoluble proanthocyanidins (C), catechins (D) and epicatechins (E) in mature seeds of four Arabidopsis thaliana varieties; data are expressed as mean ± standard deviation, n=3, and different lowercase letters indicate significant differences at the p<0.05 level; Figure 3 The relative expression levels of the SbLAC14 gene in four Arabidopsis thaliana strains were presented: wild-type (WT), wild-type overexpressing SbLAC14 (OE-LAC14), tt10 mutant (tt10), and tt10 mutant overexpressing SbLAC14 (OE-LAC14 / tt10). Data are expressed as mean ± standard deviation, n=3. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention first identified a highly expressed laccase gene in the seed coat through transcriptome analysis, named SbLAC14, and then analyzed the expression dynamics of this gene during seed development after pollination. Figure 1 Phylogenetic analysis showed that it belongs to a specific laccase subfamily. Transforming the SbLAC14 gene into the Arabidopsis thaliana laccase loss-of-function mutant tt10 revealed that this gene could completely restore the seed coat color lightening phenotype of the tt10 mutant caused by proanthocyanidin polymerization defects, while simultaneously restoring the content of polymerized proanthocyanidins and reducing the content of flavan-3-ol monomers. Figure 2 This functional verification experiment directly demonstrates for the first time that the SbLAC14 gene isolated in this invention has the biological function of catalyzing the polymerization of flavan-3-ol monomers to form proanthocyanidins. Therefore, the sorghum SbLAC14 gene provided by this invention is a novel gene resource with important application value, which can be used for tannin metabolism engineering and variety improvement in plants (especially sorghum).

[0024] Example 1: Cloning and Sequence Analysis of the SbLAC14 Gene in Sorghum 1. Materials Using the high-tannin sorghum variety Tx623 as material, its developing seeds were collected 20 days after flowering, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0025] 2. Total RNA extraction and cDNA synthesis Total RNA was extracted from sorghum seeds using the Trizol method. First-strand cDNA was synthesized using 1 μg of total RNA as a template via a reverse transcription kit and stored at -20°C for later use.

[0026] 3. Cloning of the SbLAC14 gene Specific primers were designed based on the sequence information (gene ID: Sobic.004G236000) in the sorghum genome database. The primer sequences are shown in SEQ ID NO.3-4.

[0027] Forward primer: 5'-ACCGGGTAGTTTAGGCACTG-3', SEQ ID NO.3; Reverse primer: 5'-CATCCATGGCTCATGCCTTTG-3', SEQ ID NO.4.

[0028] Using the cDNA synthesized in step 1.2 as a template, PCR amplification was performed.

[0029] The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 20 sec, 35 cycles; 72℃ final extension for 10 min.

[0030] The PCR product was recovered, ligated into the T vector, transformed into E. coli DH5α, and positive clones were selected for sequencing.

[0031] 4. Sequence Analysis Sequencing results showed that the obtained cDNA fragment was 2090 bp in length, containing a complete open reading frame, and was named SbLAC14. The nucleotide sequence of its coding region, shown in SEQ ID NO.2, is 1773 bp in length. The protein encoded by this gene consists of 590 amino acids and is also named SbLAC14, with its amino acid sequence shown in SEQ ID NO.1. BLAST alignment analysis showed that SbLAC14 belongs to the typical laccase family and contains a conserved copper ion-binding domain.

[0032] SEQ ID NO.1: MPLGQRSKMGGVAKMPAAGRLSWLLLLGVVLAFGVAASPAQAARNTHYDFVIKETKVTRLCQEKTILAVNGQFPGPTIYARKDDVVIVNVYNQGNKNITLHWHGVDQPRNPWSDGPEYITQCPIQPGANFTYKIIFTEEEGTLWWHAHSDFDRATVHGAIVIHPKRGSVYPYTKPHKEIPIILGEWWNADVEQVLLESQRTGGDVQLSDANTINGQPGDFAPCSKNDTFRMFVEHDKTYLLRVINAGLTNEMFFAIAGHRLTVVGTDGRYLKPFTVDYIMISPGQTMNMLLEANRPTNGSANSCYYMAARPFFTNIGLPVNDKNTTAILEYTDVSPSVGPPDSPYLPVINDTAAATAYTVQLRSLVTKEHPIDVPMEVDEHMLVTISINTLPCGANETCEGPGNGRLAASLNNVSFMPPTIDILDAYYYSISGVYEPDFPNKPPFFFNFTASDPPVEFQLTKRGTKVKVVEYGTVVEVVFQDTAILGAESHPMHLHGFSFYVVGRGFGNFDKDKDPTTYNLVDPPYQNTVSVPTGGWAAIRFRASNPGVWYMHCHFDRHTAWGMDTVFIVKNGKTPGAQMMPRPPNMPMC。

[0033] SEQ ID NO.2:

[0034] Example 2 Functional verification of the SbLAC14 gene 1. Construction of plant overexpression vectors The correctly sequenced coding region of the SbLAC14 gene (SEQ ID NO.2) from Example 1 was constructed into the plant expression vector pCAMBIA3301 containing the 35S promoter via enzyme digestion and ligation, yielding the recombinant plasmid 35S::SbLAC14. This recombinant plasmid was then transformed into Agrobacterium GV3101 for plant genetic transformation.

[0035] 2. Genetic transformation and positive identification in Arabidopsis thaliana Agrobacterium containing 35S::SbLAC14 was transformed into wild-type Arabidopsis thaliana Col-0 (WT) and the clear seed coat mutant tt10 using the inflorescence immersion method. T0 generation seeds were harvested and screened on MS medium containing kanamycin to obtain resistant seedlings. The expression level of SbLAC14 in T1 generation plants was detected by qRT-PCR. Individual plants with high expression levels were selected for seed collection and propagation to T3 generation to obtain homozygous overexpression lines (OE-LAC14#1-OE-LAC14#3 and OE-LAC14 / tt10#1-OE-LAC14 / tt10#3). qRT-PCR results showed that the SbLAC14 gene was significantly highly expressed in the transgenic lines compared to the wild-type or tt10 mutant. Figure 3 ).

[0036] 3. Phenotypic and Metabolite Analysis of Transgenic Arabidopsis thaliana The mature seeds of the T3 generation homozygous line were observed, and the results were as follows: Figure 2 As shown in sections I-IV of A. The tt10 mutant seeds are light brown, while the seeds of the OE-LAC14 / tt10 line completely revert to the dark brown color consistent with the wild-type Col-0. DMACA staining specifically makes proanthocyanidins appear deep blue, and the results show that the staining depth of the OE-LAC14 / tt10 line seeds is similar to that of the wild type, while the tt10 mutant stains very lightly (…). Figure 2 (VIII of A in the middle).

[0037] Further UPLC-MS / MS was used to quantitatively analyze flavan-3-ol monomers (catechin and epicatechin) and polymeric proanthocyanidins in mature seeds. The results showed that, compared with the wild type, the tt10 mutant accumulated significantly higher levels of catechin and epicatechin (approximately 2-fold), while the content of polymeric proanthocyanidins decreased by approximately 50%. In the OE-LAC14 / tt10 line, the contents of both monomers recovered to levels similar to the wild type, and the content of polymeric proanthocyanidins also recovered to 95%-102% of the wild type. Figure 2(BE). These results clearly demonstrate that the SbLAC14 gene cloned from sorghum, which encodes a laccase that catalyzes the polymerization of flavan-3-ol monomers to form proanthocyanidins, can completely compensate for the functional defects of the Arabidopsis tt10 mutant.

[0038] In summary, this invention is the first to isolate and verify the function of the sorghum SbLAC14 gene, clarifying the crucial role of its encoded protein in catalyzing proanthocyanidin polymerization. The isolation and functional analysis of this new gene provide an important supplement to the understanding of sorghum tannin metabolism pathways and offer directly usable gene resources and manipulative targets for molecular breeding of sorghum and other plants. Molecular markers developed based on this gene can be used to assist in screening sorghum germplasm with different tannin contents; by regulating the expression of this gene through genetic engineering, crop quality can be precisely improved to meet the needs of various industries such as food processing and feed production, demonstrating extremely high scientific research value and broad prospects for industrial application.

[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An application of an SbLAC14 protein in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content; The amino acid sequence of the SbLAC14 protein is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, Upregulating the expression level of the SbLAC14 protein in plants promotes the synthesis of proanthocyanidins in the plants; The plant in question is either sorghum or Arabidopsis thaliana.

3. The use of the gene encoding the SbLAC14 protein as described in claim 1 in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. The application as described in claim 3, characterized in that, Upregulate the expression level of the encoding gene in plants to promote the synthesis of proanthocyanidins in the plants; The plant in question is either sorghum or Arabidopsis thaliana.

5. An application of an overexpression vector, characterized in that, The overexpression vector contains the gene encoding the SbLAC14 protein; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; The application is any one of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content.

6. The use of an engineered bacterium comprising the overexpression vector of claim 5 in any of the following: (1) Application in regulating the synthesis of plant proanthocyanidins; (2) Application in the cultivation of transgenic plants with increased proanthocyanidin content.

7. A method for promoting the synthesis of proanthocyanidins in plants, characterized in that, This includes the step of upregulating the expression of the gene encoding the SbLAC14 protein in plants to promote the synthesis of proanthocyanidins in the plants; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

8. The method as described in claim 7, characterized in that, The plant in question is either sorghum or Arabidopsis thaliana.

9. A breeding method for a transgenic plant with increased proanthocyanidin content, characterized in that, Includes the following steps: The gene encoding the SbLAC14 protein is overexpressed in plant cells, the plant cells are then cultured, and the plant cells are used to regenerate plants, thus obtaining transgenic plants with increased proanthocyanidin content. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

10. The breeding method as described in claim 9, characterized in that, The plant in question is either sorghum or Arabidopsis thaliana.

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