Ceramide synthetase coding gene GmCerS1 for regulating and controlling effective branching number of soybeans and application of ceramide synthetase coding gene GmCerS1

Abstract

The invention belongs to the technical field of plant genetic engineering and the technical field of soybean breeding, and particularly relates to a ceramide synthetase encoding gene GmCerS1 for regulating and controlling the effective branching number of soybeans and application of the ceramide synthetase encoding gene GmCerS1. The invention discloses the ceramide synthetase encoding gene GmCerS1 for regulating and controlling the effective branching number of soybeans for the first time, the nucleotide sequence of the ceramide synthetase encoding gene GmCerS1 is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No.2. The gene is located in an endoplasmic reticulum, and formation and development of lateral branches are promoted by up-regulating expression of genes such as cellulose synthetase, GA20 oxidase and auxin transporter. The effective branch number and pod number of an excessive transgenic plant obtained through genetic transformation are obviously more than those of a wild type. The ceramide synthetase coding gene GmCerS1 for regulating and controlling the soybean effective branch number is disclosed for the first time, a soybean branch regulation and control gene bank is greatly enriched, and a new gene resource is provided for improving soybean effective branch number breeding.

Description

Technical Field

[0001] The present invention belongs to the technical fields of plant genetic engineering and soybean breeding, and particularly relates to a ceramide synthase encoding gene GmCerS1 for regulating the number of effective soybean branches and an application thereof. Background Art

[0002] Soybean (Glycine max) originated in China and is the country's primary source of vegetable oil and protein, as well as an important dual-purpose grain, oil, and feed crop. Currently, China is the world's largest soybean importer, with a high degree of external dependence. Therefore, increasing soybean production capacity is an urgent and critical task for ensuring my country's food security. The number of effective branches, a key factor in determining soybean plant architecture, directly affects soybean yield. Previous studies, using genome-wide association studies (GWAS), identified the major controlling gene for effective branch number in soybean, Dt2. The study found that Dt2 negatively regulates effective branch number in soybeans. Knocking out the Dt2 gene using CRISPR / Cas9 gene editing technology significantly increased the number of effective branches and plot yield. Conversely, strains overexpressing Dt2 had significantly reduced effective branch number and decreased yield (Liang et al., 2022).

[0003] Lateral branch development in plants is regulated by multiple factors, including genetic factors, environmental factors, and endogenous hormones. Each soybean compound leaf can produce lateral branches or axillary inflorescences (pods) in the axil. From a developmental biology perspective, lateral branches and axillary inflorescences / pods are both derived from axillary meristems (AxMs). However, the molecular switch that determines the development of soybean axillary meristems into branches or inflorescences remains unclear. Therefore, the fate of axillary meristem cells is a key factor in the establishment of soybean branches.

[0004] Lipid metabolism plays an important role in plant growth and development. Fatty acids and lipids not only provide structural integrity and energy for various metabolic processes in cell expansion, but also act as signal transduction mediators, exerting effects inside and outside the cell. For example, exogenous application of C24:0 can induce large amounts of ethylene production and promote the growth of Arabidopsis root hair cells; knocking out two phospholipid flippases (ALA4 / 5) reduced cell size at various stages of vegetative growth, indicating that lipid homeostasis is important for cell expansion during vegetative growth (Davis et al. 2020); cotton lipid transfer protein 4 (GhLTP4) promotes fiber cell elongation by increasing ceramide (Cers) content and activating the auxin response pathway; in cotton ovule culture, exogenous application of sphingolipids (Sph) can offset the effects of sphingolipid synthesis inhibitors (Myriocin) on cotton fiber elongation, thereby promoting fiber elongation (Chen et al. 2021). Therefore, focusing on plant sphingolipids to explore plant growth and development is of great significance.

[0005] Sphingolipids are a highly conserved class of lipids. Discovered in the brain over a century ago by J.L.T. Hudichum, sphingolipids are characterized by a long-chain (scathe-like) backbone. Sphingolipids are essential components of the lipid bilayer and, along with cholesterol, form the most important element of lipid rafts. They are closely involved in membrane dynamics, including vesicle trafficking. Ceramide is a central molecule in all sphingomyelin metabolism. Its formation begins with the condensation of two common cellular metabolites, serine and palmitoyl-CoA, in the endoplasmic reticulum (ER). The enzyme catalyzing this rate-limiting step is serine palmitoyltransferase (SPT). L-serine and palmitoyl-CoA are synthesized into 3-ketodihydrosphingosine by serine palmitoyltransferase (SPT). The acyl group at the 2-amino position of the reaction product, 3-ketodihydrosphingosine, is then hydrolyzed by ceramidase (a ketoreductase acting through coenzyme I), yielding dihydrosphingosine (also known as sphingosine). Dihydroceramide (also known as ceramide) is regenerated by ceramide synthase (CerS).

[0006] The N-acylation of dihydrosphingosine is catalyzed by six different ceramide synthases (CerS), each with a different acyl-CoA carbon chain length preference. This acyl chain specificity is determined by an 11-residue sequence located in a loop between the last two transmembrane domains of CerS, resulting in the production of the respective (dihydro)ceramide. In humans, there are six CerS-encoding genes, CerS1 through CerS6, each with distinct acyl chain length specificity and tissue distribution. Dihydroceramide is synthesized by the introduction of a double bond from DES. In the de novo synthesis pathway, dihydrosphingosine can also be converted to dihydrosphingosine phosphate by the action of sphingosine kinase (SphK); dihydroceramide can also be converted to dihydrosphingomyelin by the action of sphingomyelin synthase (SMS). Dihydroceramide is an intermediate in de novo ceramide synthesis and plays a role in mitochondrial reactive oxygen species production and autophagy (Luttgeharm et al. 2016).

[0007] Existing studies have shown that all CerS enzymes are associated with the endoplasmic reticulum (ER) and contain a crucial TRAM-Lag1p-CLN8 (TLC) domain, which is the CerS catalytic domain required for ceramide synthesis. Ceramide, the product of ceramide synthase (CerS), is primarily used to synthesize one of the two major glycosphingolipids found in plants: glucosylceramide (GlcCer) and glycosylinositol phosphoceramide (GIPC). Overexpression of the ceramide synthases LOH1 and LOH3 in Arabidopsis thaliana results in higher biomass than wild-type plants, in part due to increased cell division, suggesting that overproduction of very-long-chain fatty acid / trihydroxy LCB ceramides promotes cell division and growth (Luttgeharm et al., 2015).

[0008] However, the function of ceramide synthase in regulating soybean branch development has not been reported. Therefore, elucidating the molecular mechanism of the soybean GmCerS1 gene in the formation of the effective number of soybean branches is important for breeding new high-yield soybean varieties. Summary of the Invention

[0009] The present invention conducted sequence cloning and analysis of the GmCerS1 gene. Sequencing results revealed a 936-bp CDS sequence (SEQ ID No. 1) encoding 311 amino acids (SEQ ID No. 2). Comparative analysis of homologous proteins revealed that the GmCerS1 protein sequence possesses five transmembrane domains and a TRAM-Lag1p-CLN8 (TLC) domain. Further expression pattern analysis revealed that GmCerS1 was expressed at highest levels in roots, with relatively high expression also observed in young stems. Subcellular localization confirmed that GmCerS1 was localized to the endoplasmic reticulum. Furthermore, an overexpression vector pTF101-eGFP was constructed, and Agrobacterium-mediated genetic transformation was performed using "William 82" as the receptor to generate transgenic lines overexpressing the ceramide synthase-encoding gene GmCerS1. Statistical analysis of the effective branch and pod numbers of the overexpressing transgenic lines and wild-type lines revealed that the GmCerS1-overexpressing transgenic lines promoted soybean branch formation and increased the number of effective branches and pods. Further RNA-seq sequencing was performed on the lateral branches of wild-type and overexpressing transgenic soybean plants at the R2 stage (full flowering period), and KEGG enrichment analysis was performed on the differentially expressed genes between the GmCerS1 overexpressing transgenic materials and the wild type. The results showed that they were mainly enriched in metabolic pathways such as phenylpropanoid metabolism, wax synthesis, and anthocyanin synthesis; further analysis showed that in the GmCerS1 overexpressing transgenic materials, the expression levels of genes such as cellulose synthase, GA20 oxidase, and auxin transporter were upregulated, indicating that GmCerS1 may promote the formation and development of soybean lateral branches by upregulating the expression of cellulose synthase, GA20 oxidase and auxin transporter genes.

[0010] In view of this, the present invention aims to provide a ceramide synthase encoding gene GmCerS1 for regulating the number of effective branches of soybean and its application.

[0011] In order to achieve the above object, the present invention provides the following technical solutions:

[0012] The present invention provides an isolated ceramide synthase encoding gene GmCerS1, the nucleotide sequence of the ceramide synthase encoding gene GmCerS1 is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0013] The present invention also provides the use of the ceramide synthase encoding gene GmCerS1 in increasing the number of effective branches of soybean.

[0014] Furthermore, in the application, the ceramide synthase encoding gene GmCerS1 promotes the formation and development of soybean lateral branches by upregulating the expression of cellulose synthase, GA20 oxidase and auxin transporter genes.

[0015] The present invention also provides a method for increasing the number of effective branches and pods in soybeans by using the ceramide synthase encoding gene GmCerS1, the method comprising:

[0016] S1. Clone the ceramide synthase encoding gene GmCerS1 into a recombinant expression vector;

[0017] S2. The recombinant expression vector is transformed into soybean by Agrobacterium-mediated method;

[0018] S3. Screening and identification of transgenic plants overexpressing GmCerS1;

[0019] S4. Statistical analysis confirmed that the number of effective branches and pods of the transgenic plants increased significantly.

[0020] Furthermore, the recombinant expression vector is a pTF101-eGFP vector, and the ceramide synthase encoding gene GmCerS1 is in an expressible state in the vector.

[0021] Furthermore, the screening and identification steps include:

[0022] S1. Identify positive transgenic lines by PCR;

[0023] S2. qRT-PCR analysis of the expression level of the GmCerS1 gene in positive transgenic lines;

[0024] S3. Select transgenic lines with significantly upregulated expression levels for subsequent phenotypic analysis.

[0025] The present invention also provides a protein encoded by the ceramide synthase encoding gene GmCerS1. The amino acid sequence of the protein is shown in SEQ ID No. 2, and comprises five transmembrane structures and a TRAM-Lag1p-CLN8 domain.

[0026] The present invention also provides a recombinant expression vector containing the ceramide synthase encoding gene GmCerS1.

[0027] The present invention also provides the use of the ceramide synthase encoding gene GmCerS1, the protein encoded by the ceramide synthase encoding gene GmCerS1, and a recombinant expression vector containing the ceramide synthase encoding gene GmCerS1 in improving the effective branch number of soybeans.

[0028] Beneficial effects of the present invention: The present invention provides a ceramide synthase encoding gene GmCerS1 for regulating the number of effective branches of soybean, the nucleotide sequence of which is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.2. The gene is located in the endoplasmic reticulum and promotes the formation and development of lateral branches by upregulating the expression of genes such as cellulose synthase, GA20 oxidase, and auxin transporter. The number of effective branches and pods of the over-transgenic plants obtained by genetic transformation is significantly higher than that of the wild type. The ceramide synthase encoding gene GmCerS1 for increasing the number of effective branches of soybean disclosed by the present invention is disclosed for the first time, which greatly enriches the soybean branch regulation gene library and provides a new gene resource for breeding to improve the number of effective branches of soybean. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure shows the sequence characteristics of GmCerS1; A is the phylogenetic tree analysis; B is the amino acid sequence alignment analysis.

[0030] Figure 2 Analysis of GmCerS1 expression pattern; A is the SoyMD website information; B is the qRT-PCR identification of GmCerS tissue expression.

[0031] Figure 3 Subcellular localization of GmCerS1.

[0032] Figure 4 Identification of GmCerS1 overexpressing transgenic lines; Figure A shows the agarose gel electrophoresis identification of GmCerS1 constitutively overexpressing transgenic plants; Figure B shows the expression level identification of GmCerS1 overexpressing transgenic lines using qRT-PCR.

[0033] Figure 5 Analysis of the effective branch number of GmCerS1 transgenic plants and wild type in the R1 stage; Figure A is the phenotypic identification of WT and GmCerS1 transgenic soybean plants; Figure B is the statistical analysis.

[0034] Figure 6 Analysis of pod numbers of GmCerS1 transgenic plants and wild type plants in the R2 stage.

[0035] Figure 7 Figure 4 shows the differential gene fold analysis between GmCerS1 overexpressing transgenic plants and wild-type controls.

[0036] Figure 8 KEGG enrichment analysis of differentially expressed genes between GmCerS1 overexpressing transgenic plants and wild-type controls. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above-mentioned contents of the present invention are encompassed within the scope that the present invention is intended to protect. It should be noted that, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental reagents in the examples, unless otherwise specified, can all be obtained commercially. The experimental methods in the examples, unless otherwise specified, are all conventional methods.

[0038] Example 1 Cloning and sequence characterization of GmCerS1

[0039] GmCerS1 was subjected to sequence cloning analysis, phylogenetic analysis, and protein sequence alignment. Using the cDNA of soybean "William82" leaves as a template, the CDS sequence of GmCerS was amplified. The sequencing results showed that the gene CDS sequence was 936 bp long (SEQ ID No. 1), encoding 311 amino acids (SEQ ID No. 2), which was consistent with the gene size predicted by the database. The accession number was Glyma.16G091200, and the ORF was 2234 bp long, containing 5 introns and 6 exons. There are a total of 6 ceramide synthase-encoding genes in soybean. Phylogenetic analysis was performed with 3 ceramide synthase-encoding genes in Arabidopsis thaliana. The results showed that the genetic distance between the GmCerS protein and the Arabidopsis thaliana ceramide synthase-encoding gene (LOH1) was close, so it was named GmCerS1 ( Figure 1 A). Sequence alignment with homologous proteins from Arabidopsis and plants of the same genus showed that the GmCerS1 protein sequence has five transmembrane structures and a TRAM-Lag1p-CLN8 (TLC) domain ( Figure 1 B).

[0040] Example 2 Analysis of GmCerS1 expression pattern

[0041] According to the expression profile characteristics of Glyma.16G091200 predicted by the SoyMD website, GmCerS1 is expressed in both the main stem and the side branches of soybean ( Figure 2 A). RNA was extracted from different soybean tissues, and the expression of GmCerS1 in different tissues was analyzed by qRT-PCR. It was found that its expression level was highest in roots, but it was also highly expressed in young stems ( Figure 2B). To investigate the subcellular localization of GmCerS1 protein, the pCAMBIA1300-GmCerS1-eGFP vector carrying a green fluorescent marker (eGFP) was transformed into Agrobacterium GV3101 and then injected into tobacco for transient expression. The results showed that GmCerS1 was localized in the endoplasmic reticulum ( Figure 3 ).

[0042] Example 3 Screening and identification of GmCerS1 transgenic lines

[0043] The overexpression vector pTF101-eGFP was constructed and transformed with "William 82" as the recipient by Agrobacterium-mediated method. After PCR identification, 3 positive strains were obtained ( Figure 4 A). qRT-PCR was used to identify the expression of positive plants. The results showed that the expression of the three positive overexpression lines was upregulated, among which the GmCerS1-OE-8 line had the highest expression, which was 6 times that of the WT ( Figure 4 B).

[0044] Example 4 Phenotypic Identification

[0045] (1) GmCerS1 regulates the number of effective branches in soybean

[0046] The statistical analysis of the effective branch numbers of GmCerS1 overexpressing transgenic plants and wild type controls showed that the effective branch numbers of GmCerS1-OE-7 and GmCerS1-OE-8 overexpressing transgenic plants were significantly higher than those of the wild type ( Figure 5 A and 5B).

[0047] (2) GmCerS1 increased soybean pod number by increasing the number of effective branches

[0048] The pod numbers of GmCerS1 overexpressing transgenic plants and wild-type controls were then counted. The results showed that GmCerS1-OE-7 and GmCerS1-OE-8 overexpressing transgenic plants increased the number of soybean pods by increasing the number of effective branches ( Figure 6 A and 6B).

[0049] Example 5 Transcriptome Data Analysis

[0050] To explore the molecular mechanism by which GmCerS1 regulates branch development, RNA-seq sequencing was performed on the lateral branches of wild-type and overexpressing transgenic soybean plants at the R2 stage (full flowering stage). The number of genes with a 1-2-fold expression difference between GmCerS1 overexpressing transgenic materials and wild-type treatments and materials was the largest, followed by genes with a 2-4-fold difference, and fewer genes with a 4-fold difference or more ( Figure 7 A and 7B). KEGG enrichment analysis was performed on differentially expressed genes ( Figure 8 A and 8B), and found that they were mainly enriched in metabolic pathways such as phenylpropanoid metabolism, wax synthesis, and anthocyanin synthesis; further analysis showed that in GmCerS1 overexpressing transgenic materials, the expression levels of genes such as cellulose synthase, GA20 oxidase, and auxin transporter were upregulated, indicating that GmCerS1 may promote the formation and development of soybean lateral branches by upregulating the expression of cellulose synthase, GA20 oxidase and auxin transporter genes.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An isolated ceramide synthase encoding gene GmCerS1, characterized in that: The nucleotide sequence of the ceramide synthase encoding gene GmCerS1 is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

2. Use of the ceramide synthase encoding gene GmCerS1 according to claim 1 in increasing the number of effective branches in soybean.

3. The use according to claim 2, characterized in that The ceramide synthase encoding gene GmCerS1 promotes the formation and development of soybean lateral branches by upregulating the expression of cellulose synthase, GA20 oxidase and auxin transporter genes.

4. A method for increasing the number of effective branches and pods in soybean using the ceramide synthase encoding gene GmCerS1 according to claim 1, characterized in that: The method comprises: S1. Clone the ceramide synthase encoding gene GmCerS1 into a recombinant expression vector; S2. The recombinant expression vector is transformed into soybean by Agrobacterium-mediated method; S3. Screening and identification of transgenic plants overexpressing GmCerS1; S4. Statistical analysis confirmed that the number of effective branches and pods of the transgenic plants increased significantly.

5. The method according to claim 4, characterized in that The recombinant expression vector is a pTF101-eGFP vector, and the ceramide synthase encoding gene GmCerS1 is in an expressible state in the vector.

6. The method according to claim 4, characterized in that The screening and identification steps include: S1. Identify positive transgenic lines by PCR; S2. qRT-PCR analysis of the expression level of the GmCerS1 gene in positive transgenic lines; S3. Select transgenic lines with significantly upregulated expression levels for subsequent phenotypic analysis.

7. The protein encoded by the ceramide synthase encoding gene GmCerS1 according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID No. 2, which comprises five transmembrane structures and a TRAM-Lag1p-CLN8 domain.

8. A recombinant expression vector containing the ceramide synthase encoding gene GmCerS1 according to claim 1.

9. Use of the ceramide synthase encoding gene GmCerS1 according to claim 1, the protein according to claim 7, and the vector according to claim 8 in improving the effective branch number of soybean.

Images