Application of soybean GmCHR36 gene in regulation and control of soybean lateral root development and Cd resistance

Abstract

The invention discloses application of a soybean GmCHR36 gene in regulation and control of soybean lateral root development and Cd resistance. It is found that the soybean GmCHR36 gene can regulate and control soybean root system development and Cd resistance, gene editing is utilized to knock out GmCHR36 to obtain a knockout mutant, and compared with a wild plant, the mutant has the advantages that the number of lateral roots is reduced, and the lateral roots are shortened; under the stress of Cd, the cadmium poisoning degree of soybeans is reduced, the Cd concentration of grains is reduced, and the resistance to Cd is improved. Therefore, the GmCHR36 gene is a regulatory factor of soybean lateral root development and Cd resistance, and can provide a new gene target for soybean genetic breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and specifically relates to soybeans. GmCHR36 Application of genes in regulating soybean lateral root development and Cd resistance. Background Technology

[0002] Soybeans Glycine max Soybeans (L.) Merr. are annual herbaceous plants belonging to the genus *Glycine* in the legume family. They are an important food crop, commonly used for various soy products, soybean oil extraction, soy sauce brewing, protein extraction, and animal feed production. Therefore, safe soybean production is a crucial factor affecting feed supply and food security in the livestock industry. However, some regions currently face Cd contamination in their soil. Thus, breeding soybean varieties that can grow normally in Cd-contaminated soil (Cd resistance) and have Cd concentrations in grains below safe limits (low accumulation) is essential for sustainable development. However, soybean Cd resistance and accumulation involve physiological processes such as transmembrane transport, intercellular transport, chelate synthesis, vacuolar retention, and organ distribution, making it a complex trait controlled by multiple genes.

[0003] Roots are the first organs in plants to come into contact with heavy metal ions. Under cadmium stress, the taproot and lateral roots of soybeans are inhibited (Wang et al Epigenetic mechanisms are involved in the inheritance and adaptation of plant tolerance to cadmium stress. The interaction of DNA methylation, histone modification, chromatin remodeling, and RNA-related silencing can regulate gene expression without altering the DNA sequence, thereby inducing a multi-level epigenetic response to cadmium stress in plants (Ma et al., 2022). Cadmium stress-induced chromatin alterations (including methylation, acetylation, phosphorylation, and ubiquitination) affect gene expression by changing chromatin structure (Niekerk et al., 2021). Histone modification plays a key role in the regulation of gene expression under cadmium stress. Histones are proteins that wrap around DNA and undergo various chemical modifications such as methylation, acetylation, phosphorylation, and ubiquitination. Immunofluorescence studies of cadmium stress in broad bean root cells showed significantly increased levels of DNA damage and H4 histone acetylation (Nouairi et al., 2019), indicating that histone acetylation plays an important role in responding to oxidative DNA damage and regulating the cell cycle and transcription.

[0004] Snf2 protein, as the core ATPase subunit of the chromatin remodeling complex, precisely regulates gene expression programs. Snf2 protein provides energy through ATP hydrolysis, driving nucleosome sliding, expulsion, or histone variant replacement, thereby regulating chromatin DNA accessibility and influencing various intranuclear processes, including transcription, DNA replication, and repair. There are 66 genes encoding Snf2 protein in the soybean genome (Wang,et al. (2023) GmCHR36 It belongs to the Snf2 family, but its function has not been studied. Therefore, it is of great significance to study the effect of this gene on soybean Cd resistance and accumulation. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide soybean... GmCHR36 Application of genes or related biological materials in regulating soybean lateral root development and / or Cd resistance.

[0006] Another object of the present invention is to provide a method for regulating soybean lateral root development and / or Cd resistance.

[0007] Another object of the present invention is to provide a soybean GmCHR36 Gene mutants.

[0008] Another object of the present invention is to provide the soybean. GmCHR36 Application of gene mutants.

[0009] The objective of this invention is achieved through the following technical solution: soybeans GmCHR36 The application of genes or related biological materials in regulating soybean lateral root development and / or Cd resistance, wherein the soybean GmCHR36 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The soybeans mentioned GmCHR36 Gene-related biological materials are any one or more combinations of the following biological materials: (A) Contains the above-mentioned soybeans GmCHR36 Gene expression cassettes; (B) Contains the above-mentioned soybeans GmCHR36 Recombinant gene expression vectors; (C) A recombinant expression vector containing the expression cassette described in (A); (D) Contains the above-mentioned soybeans GmCHR36 Recombinant bacteria; (E) Recombinant bacteria containing the expression cassette described in (A); (F) Recombinant bacteria containing the recombinant expression vector described in (B) or (C).

[0010] The soybeans mentioned GmCHR36 The gene annotation number in the phytozome database (https: / / phytozome-next.jgi.doe.gov / ) is: Glyma.11G154800 The nucleotide sequence of [ ] is shown in SEQ ID NO.1.

[0011] The regulation of soybean lateral root development and Cd resistance is achieved through the following methods: (a) Knockout soybeans GmCHR36 Gene( GmCHR36 Gene mutations can reduce the number (amount) of soybean lateral roots, shorten the length of soybean lateral roots, and improve soybean's resistance to Cd (reduce Cd accumulation in soybeans). (b) Soybean overexpression GmCHR36 Genes that increase the number (quantity) of soybean lateral roots, increase the length of soybean lateral roots, and reduce soybean resistance to Cd (increase Cd accumulation in soybeans).

[0012] In method (a), the knockout GmCHR36 Gene editing was achieved using CRISPR-Cas9 technology: Soybean genes were constructed using CRISPR-Cas9 editing technology. GmCHR36 Gene knockout vectors enable soybeans GmCHR36 The loss of gene function reduces the number and length of soybean lateral roots, thereby increasing soybean's resistance to Cd (reducing the Cd concentration (content) in soybean seeds).

[0013] In method (a), the knockout GmCHR36 Gene selection is achieved through the following methods: GmCHR36 The coding region of the gene contains a deletion of 261 bases from position 94 to position 355, and a deletion of all bases after position 461 (bases 462-4032 are deleted).

[0014] In method (b), the overexpression is preferably achieved by: [the soybean...] GmCHR36 The gene is integrated into the soybean genome, making the soybean GmCHR36 Gene expression in soybeans (regulating soybean) GmCHR36 (Activation of genes) to increase the number (quantity) and length of soybean lateral roots, thereby reducing soybean resistance to Cd.

[0015] The present invention also provides the above-mentioned soybeans. GmCHR36 Application of genes or related biological materials in soybean genetic breeding (preparation of transgenic soybeans, improvement of soybean varieties).

[0016] A method for regulating soybean lateral root development and / or Cd resistance by knocking out soybeans GmCHR36 Genes, reducing soybean GmCHR36 The biological functions of the gene or its encoded protein reduce the number (amount) of soybean lateral roots, shorten the length of soybean lateral roots, and improve soybean resistance to Cd (reduce Cd accumulation in soybean).

[0017] A type of soybean GmCHR36 Gene mutants, in GmCHR36The coding region of the gene contains a deletion of 261 bases from position 94 to position 355, and a deletion of all bases after position 461 (bases 462-4032 are deleted).

[0018] Contains the soybean GmCHR36 Expression cassettes of gene mutants, recombinant expression vectors, or recombinant bacteria.

[0019] The soybeans mentioned GmCHR36 Gene mutants, through mutation of the GmCHR36 gene (knockout of the GmCHR36 gene), shorten the length of soybean lateral roots, reduce the number of lateral roots, and improve resistance to Cd (reduce Cd accumulation in soybeans).

[0020] The soybean variety mentioned is W82.

[0021] The soybeans mentioned GmCHR36 Gene mutants and / or soybeans containing the above GmCHR36 The application of gene mutant expression cassettes, recombinant expression vectors, or recombinant bacteria in reducing the number (quantity) of soybean lateral roots, shortening the length of soybean lateral roots, improving soybean resistance to Cd (reducing Cd accumulation in soybeans), and soybean genetic breeding (preparing transgenic soybeans and improving soybean varieties).

[0022] The present invention has the following advantages and effects compared with the prior art: 1. This invention provides soybeans GmCHR36 Application of genes and their encoded proteins in regulating soybean lateral root development and Cd resistance: This invention discovers the application of soybean genes and their encoded proteins in regulating soybean lateral root development and Cd resistance. GmCHR36 Genes can regulate soybean root development and Cd resistance by reducing soybean... GmCHR36 The biological functions of genes or their encoded proteins can enhance Cd resistance in soybeans, providing new gene targets for soybean genetic breeding.

[0023] 2. This invention utilizes gene editing knockout. GmCHR36 A knockout mutant was obtained. Compared with the WT mutant, the number of lateral roots was reduced and the lateral roots were shorter. Under Cd stress, the mutant plants were stunted, the leaf bases turned red, and the leaves curled throughout the plant compared with the WT mutant. The Cd concentration in the grains was slightly lower. Therefore, GmCHR36 Genes are regulatory factors in soybean lateral root development and Cd resistance, enhancing... GmCHR36 Gene expression can be used to improve soybean lateral root development, while silencing or reducing it. GmCHR36 Gene expression can improve Cd accumulation in soybean seeds and enhance soybean's resistance to Cd. Attached Figure Description

[0024] Figure 1 As described in the embodiments of the present invention GmCHR36 Map showing the location of the knockout target in gene knockout mutants.

[0025] Figure 2 As described in the embodiments of the present invention GmCHR36 T0 sequencing results of gene knockout mutant lines.

[0026] Figure 3 As described in the embodiments of the present invention GmCHR36 Photographs showing the differences between T1 generation homozygous knockout mutant plants under normal conditions and under Cd stress; where a shows the root phenotype of WT (W82) and knockout mutant after one week of growth on blue germination paper; b shows the phenotype of WT (W82) and knockout mutant after hydroponics in Cd (9 μM) solution; c shows the growth status of WT (W82) and knockout mutant after 15 days of cultivation in Cd-contaminated soil.

[0027] Figure 4 This is a schematic diagram of Cd poisoning levels in soybeans in an embodiment of the present invention; where A, E, and E represent Cd poisoning levels 1-5 in soybean germplasm resources, respectively. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0029] Example 1 GmCHR36 Construction and phenotypic observation of knockout mutant plants 1.1 GmCHR36 Obtaining homozygous knockout mutant plants The knockout mutant in this embodiment is based on the commercially available soybean variety W82 (Williams 82), and was knocked out using gene editing (CRISPR-Cas9) technology. GmCHR36The gene (nucleotide sequence as shown in SEQ ID NO.1, protein sequence as shown in SEQ ID NO.2), with the knockout mutant material being developed by Wuhan Aidijing Biotechnology Co., Ltd., was used. Homozygous knockout mutants were detected using both polypropylene gel electrophoresis and sequencing. After obtaining T0 generation transgenic soybean seedlings, DNA was extracted from soybean leaves, and the fragment was amplified using primers provided by the company and sequenced. The mutation type of the transgenic soybean seedlings was analyzed based on the sequencing peak diagram. T0 generation seeds were harvested and labeled with the mutation type. In the T1 generation, primers were designed based on the mutation type determined in the T0 generation to amplify the target fragment, and polypropylene gel electrophoresis was used for detection. Homozygous plants were identified based on the control bands, and 1-2 plants with homozygous bands were selected and sent to the company for sequencing to ensure that the correct homozygous plants were selected. Homozygous T1 generation can be used for Cd treatment to investigate traits. Among them: soybeans GmCHR36 Gene (SEQ ID NO.1): soybeans GmCHR36 The protein sequence encoded by the gene (SEQ ID NO.2):

[0030] The specific steps for T1 generation identification are as follows: (1) DNA was extracted from soybean leaves using the CTAB method: Genomic DNA was extracted from soybean leaves using the CTAB method (Reference: Doyle J, Doyle JL, Doyle J, et al. A rapid DNA isolation procedure for small amounts of fresh leaf tissue[J]. 1987.). In brief, an appropriate amount of soybean leaf material (from any stage of T0, T1, or T2 generation of green soybean leaves) was ground in liquid nitrogen, then incubated with 2% CTAB extraction solution at 65°C; subsequently, extraction was performed using chloroform / isoamyl alcohol (24:1, v / v), the supernatant was collected, and DNA was precipitated with pre-cooled isoamyl alcohol; finally, the precipitate was washed with 70% ethanol, dried, and dissolved in sterile water for later use or stored at -20°C.

[0031] (2) PCR reaction The PCR amplification system was GenStar's 2× Taq dye premixed PCR system (10 μL). The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0032] There are four target sites for knockout: T1: 5′-CTCGGGGAAGGGTATGTCGG TGG-3′ (SEQ ID NO.3); T2: 5′-AATCTTCGGCGAAGGGGGCG AGG-3′ (SEQ ID NO. 4); T3: 5′-GGCGCACTTGCGAGTGACCA TGG-3′ (SEQ ID NO.5); T4: 5′-GTGCGTTGAAGCGCTTCCTA GGG-3′ (SEQ ID NO. 6); The detection primer sequences are as follows: F: 5′-CAGCGACTGGTAAAGGTAGA-3′ (SEQ ID NO.7); R: 5′-AGAATTCGAAGGCGAGAAAGAA-3′ (SEQ ID NO. 8).

[0033] Table 1. PCR reaction system

[0034] Table 2. PCR reaction procedure

[0035] Note: The Tm annealing temperature and time can be adjusted according to the size of the amplified product.

[0036] (3) Electrophoresis detection PCR products were separated by non-denaturing polyacrylamide gel electrophoresis and developed using silver nitrate staining (Reference: Bassam, BJ, Caetano-Anolles, G., Gresshoff, PM, 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 80–83. https: / / doi.org / 10.1016 / 0003-2697(91)90120-I). The appropriate gel concentration (e.g., 6% or 10%) was selected based on the expected PCR product fragment size. After loading, electrophoresis was performed at a constant voltage of 300 V for 1–2 hours until the bands were clearly separated.

[0037] GmCHR36 The knockout target location of gene knockout mutants is as follows: Figure 1 As shown, GmCHR36 T0 sequencing results of gene knockout mutant lines are as follows Figure 2 As shown, two homozygous mutant lines were screened in the T1 generation and named... GmCHR36-1 and GmCHR36-2 All of them are in GmCHR36 The coding region of the gene contains a deletion of 261 bases from position 94 to position 355, and a deletion of all bases after position 461 (bases 462-4032 are deleted).

[0038] 1.2 GmCHR36 Phenotypic observation of knockout homozygous mutants Select plump soybeans free of insect holes, specifically W82 (WT) and homozygous knockout mutants. GmCHR36-1 (Note: GmCHR36-2 (This experiment was not conducted due to the small number of seeds.) Seedlings were raised in the substrate for one week, and their characteristics were investigated under hydroponic and Cd-contaminated soil conditions. The specific steps are as follows: 1.2.1 Soybean seedlings were raised for one week in a light incubator (12 h light, 12 h dark, 25 ℃, 80% humidity). Soybean seedlings with uniform growth (WT, 12 h light, 12 h dark, 25 ℃, 80% humidity) were then selected. GmCHR36-1 The roots were rinsed clean with tap water, and the lateral roots were spread out with tweezers and photographed. ImageJ and Rhizo Vision Explorer software were used to analyze the images to obtain the taproot length and the number of lateral roots. Three replicates were performed for each variety. Results are shown below. Figure 3As shown in a: it can be seen that, compared with WT, the knockout mutant has a reduced number of lateral roots and a shorter lateral root length.

[0039] 1.2.2 Soybean seedlings (WT, 1.5-1.5 kg) that have been raised for one week and are of uniform growth are transplanted. GmCHR36-1 Transfer the solution to a 6 L gray plastic container and add Hogrange nutrient solution at a ratio of 1:1000 (by volume): 5 mM Ca(NO3)2, 5 mM KNO3, 1 mM KH2PO4, 1 mM MgSO4, 50 μM H3BO3, 4.5 mM MnCl2, 3.8 μM ZnSO4, 0.3 mM CuSO4, 0.1 mM (NH4)6Mo7O 24 The cells were cultured with 50 μM Fe-EDTA and two treatments: a control (CK) and a treatment with Cd (9 μM CdCl2). Each variety was tested in triplicate. Phenotypic characteristics were observed and photographed after 7 days of culture. Results are as follows: Figure 3 As shown in Figure b: it can be seen that under Cd stress, the mutant... GmCHR36-1 Compared to WT, the plant is shorter, has reddish leaf bases, and the entire plant's leaves are curled. The mutant... GmCHR36-1 The number of lateral roots decreases and the length of lateral roots becomes shorter.

[0040] 1.2.3 Select soybean seedlings (WT, 1 week after seedling establishment) that are of uniform growth. GmCHR36-1 Plants were transplanted into Cd-contaminated soil (CdCl2 was added directly to the soil, resulting in a Cd concentration of 22.025 mg / kg), with three plants per pot and three replicates per variety. The Cd poisoning phenotype of the plants was observed regularly. After the phenotype stabilized (15 days), plant growth was photographed and the Cd poisoning level was analyzed. Seeds were harvested after plant maturity, and a sufficient quantity was ground and sent to Guangzhou Hongcheng Testing Co., Ltd. for testing of total Cd concentration. The difference in total seed concentration between homozygous mutants and wild-type plants was compared. Based on the Cd poisoning grading criteria of Boggess et al. (Reference: Boggess SF, Willavize S, Koeppe D E. Differential response of soybean varieties to soilcadmium 1[J]. Agronomy Journal, 1978, 70(5): 756-760) and combined with the characteristics of soybean germplasm resources in our laboratory, the Cd poisoning grading is divided into a 1-5 level standard (AE represents Cd poisoning grading 1-5 for soybean germplasm resources). The Cd poisoning grading evaluation is as follows: Figure 4 The details are as follows: (1) Grade 1 leaves are red only at the base, and the leaves are not deformed (not bent, not drooping) and do not turn green; (2) The redness at the base of the second grade spreads along the veins to the front and edge, and the single leaf does not deform (if 1 to 2 leaves of the whole plant droop, it can still be judged as 2), and does not fade; (3) In the third grade, the midrib of one-third of the leaflets of the whole plant is slightly bent or drooping, the veins are red, and the leaves do not turn green. (4) Grade 4 leaves have prominent red veins, and the veins of 1 / 3 to 2 / 3 of the leaves of the whole plant bend downwards. If the leaves are slightly fading in green or if the leaves are tightly clenched, wrinkled, or shriveled (but there are still undeformed leaves at the top), they are all judged as Grade 4. (5) Level 5: The whole plant leaves are tightly clenched, wrinkled, and crumpled. Some leaves change from green to light yellow and the coloring is uniform. The leaves are brittle and easily broken. The plant is short or the whole plant leaves are deformed. 2 / 3 of the leaves are clenched, wrinkled, and faded.

[0041] The results are as follows Figure 3 As shown in c and Table 3: it can be seen that mutations occurred under soil cultivation conditions. Body GmCHR36-1 The plants are relatively shorter than those in WT plants. Figure 3 c); knockout mutant GmCHR36-1 Compared with WT, the Cd concentration in the grains decreased (Table 3); analysis showed that the mutant under soil cultivation conditions... GmCHR36-1 The cadmium poisoning level is grade 2, while the WT poisoning level is grade 3. (Explanation) GmCHR36 Genes are regulatory factors for soybean lateral root development and Cd resistance, and can provide new gene targets for soybean genetic breeding.

[0042] Table 3

[0043] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Soybeans GmCHR36 The application of genes or related biological materials in regulating soybean lateral root development and / or Cd resistance, characterized by: The soybean GmCHR36 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The soybeans mentioned GmCHR36 Gene-related biological materials are any one or more combinations of the following biological materials: (A) Contains the above-mentioned soybeans GmCHR36 Gene expression cassettes; (B) Contains the above-mentioned soybeans GmCHR36 Recombinant gene expression vectors; (C) A recombinant expression vector containing the expression cassette described in (A); (D) Contains the above-mentioned soybeans GmCHR36 Recombinant bacteria; (E) Recombinant bacteria containing the expression cassette described in (A); (F) Recombinant bacteria containing the recombinant expression vector described in (B) or (C).

2. The application according to claim 1, characterized in that: The soybeans mentioned GmCHR36 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The regulation of soybean lateral root development and Cd resistance is achieved through the following methods: (a) Knockout soybeans GmCHR36 Genes that reduce the number and length of soybean lateral roots and improve soybean resistance to Cd; (b) Soybean overexpression GmCHR36 Genes that increase the number and length of soybean lateral roots and reduce soybean resistance to Cd.

4. The application according to claim 3, characterized in that: In method (a), the knockout GmCHR36 Gene editing is achieved using CRISPR-Cas9 technology.

5. The application according to claim 4, characterized in that: In method (a), the knockout GmCHR36 Genes are achieved through the following methods: GmCHR36 The coding region of the gene contains a deletion of 261 bases from position 94 to position 355, and all bases from position 462 to position 4032 after position 461 are deleted.

6. The soybean as described in claim 1 GmCHR36 Application of genes or related biological materials in soybean genetic breeding.

7. A method for regulating soybean lateral root development and / or Cd resistance, characterized in that: By knocking out soybeans GmCHR36 Genes, reducing soybean GmCHR36 The biological functions of the gene or its encoded protein reduce the number of soybean lateral roots, shorten the length of soybean lateral roots, and improve soybean resistance to Cd. The soybeans mentioned GmCHR36 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. A type of soybean GmCHR36 Gene mutants, characterized by: exist GmCHR36 The coding region of the gene contains a deletion of 261 bases from position 94 to position 355, and all bases from position 462 to position 4032 after position 461 are deleted.

9. Containing the soybean of claim 8 GmCHR36 Expression cassettes of gene mutants, recombinant expression vectors, or recombinant bacteria.

10. The soybean according to claim 8 GmCHR36 Gene mutants and / or those containing soybean as described in claim 9 GmCHR36 The application of gene mutant expression cassettes, recombinant expression vectors, or recombinant bacteria in reducing the number of soybean lateral roots, shortening the length of soybean lateral roots, improving soybean resistance to Cd, and in soybean genetic breeding.

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