Soybean GmUBC13 gene, encoded protein thereof and application of soybean GmUBC13 gene in negative regulation and control of lignin synthesis and phytophthora root rot resistance

Abstract

The invention discloses a soybean GmUBC13 gene, an encoding protein thereof and application of the soybean GmUBC13 gene in negative regulation and control of lignin synthesis and phytophthora root rot resistance, and belongs to the technical field of biology, and the nucleotide sequence of the soybean GmUBC13 gene is shown as SEQ ID NO: 1. The amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO: 2. According to the invention, it is found for the first time that the soybean GmUBC13 gene can negatively regulate lignin biosynthesis and the resistance of soybean to phytophthora root rot, that is, the GmUBC13 gene and the encoded protein thereof are successfully cloned from soybean, and it is confirmed for the first time by systematic functional experiments that the gene encodes an active E2 ubiquitin coupling enzyme. The overexpression of the gene can significantly inhibit the biosynthesis of soybean lignin, and the interference of the expression of the gene can promote the accumulation of lignin, so that the blank about a key negative regulation element in a lignin synthesis regulation network is filled.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a soybean GmUBC13 gene and its encoded protein, and its application in negatively regulating lignin synthesis and combating root rot caused by Mycorrhizal fungi. Background Technology

[0002] Soybeans are an important dual-purpose crop, rich in protein and oil, and a major source of human food and animal feed. In recent years, with changes in the international trade environment, soybeans have become a strategic agricultural product, with their yield and quality holding a prominent position in the national agricultural development strategy. However, soybean production is constrained by many factors, among which pathogen infection is a major limiting factor, seriously affecting soybean yield and quality. Therefore, improving the disease resistance of soybeans is an important way to ensure the profitability of soybean production.

[0003] Lignin is a complex high-molecular-weight organic polymer and one of the main components of plant cell walls, especially abundant in xylem, fibers, and sclerenchyma. It is a key component of the physical and chemical barriers that soybeans use to resist pathogen infection. For example, overexpression of the gene GmC4H, which encodes a key enzyme in the lignin synthesis pathway, significantly improves soybean resistance to Phytophthora root rot; overexpression of PpMYC2 promotes lignin accumulation and improves peach resistance to brown rot; the rice mutant Bsr-k1 increases resistance to rice blast and bacterial wilt by promoting lignin accumulation; GhODO1 interacts with the promoters of lignin genes Gh4CL1 and GhCAD3, activating their expression and increasing lignin accumulation and resistance to Verticillium wilt, fully demonstrating the direct relationship between lignin biosynthesis and plant disease resistance.

[0004] Current research on soybean lignin synthesis and its association with disease resistance largely focuses on verifying the function of positive regulators or key synthases. For example, studies have reported that upregulation of certain transcription factors or enzyme genes can promote lignin accumulation and enhance resistance. However, systematic reports have not been published regarding the existence of key negative regulators in this pathway, or how to release the plant's disease resistance potential by inhibiting such negative regulators, thereby more effectively synergistically increasing lignin content and disease resistance. In particular, no key genes capable of simultaneously negatively regulating lignin synthesis and resistance to Phytophthora root rot have been identified.

[0005] Therefore, we propose the soybean GmUBC13 gene, its encoded protein, and its application in negatively regulating lignin synthesis and combating root rot caused by Mycorrhizal fungi, in order to alleviate or solve the above problems.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background technology of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the soybean GmUBC13 gene, its encoded protein, and its application in the negative regulation of lignin synthesis and resistance to Phytophthora root rot, thus solving the problem of the lack of key negative regulatory factors in the regulation of soybean lignin synthesis and Phytophthora root rot resistance in existing technologies. This invention provides the soybean GmUBC13 gene and its encoded protein, clarifying its dual negative regulatory function, and providing a new target for the breeding of stress-resistant soybean varieties.

[0008] To achieve the above objectives, the present invention provides a soybean GmUBC13 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] The gene encodes a protein whose amino acid sequence is shown in SEQ ID NO: 2.

[0010] A recombinant vector comprising the GmUBC13 gene.

[0011] A recombinant cell containing the soybean GmUBC13 gene, the protein, or the recombinant vector.

[0012] A genetically modified soybean or its hairy root, wherein the genome integrates an exogenous polynucleotide sequence capable of expressing or silencing the protein, or the expression or activity of an endogenous gene encoding the protein is regulated.

[0013] Application of soybean GmUBC13 gene or protein in negative regulation of soybean lignin biosynthesis.

[0014] Application of soybean GmUBC13 gene or protein in reducing soybean resistance to Phytophthora root rot.

[0015] A method for cultivating soybean plants with increased lignin content and / or enhanced resistance to Phytophthora root rot, comprising inhibiting the activity of the GmUBC13 protein or the expression of the gene encoding the protein in the target soybean plant.

[0016] A method for reducing soybean lignin content and / or resistance to Phytophthora root rot, comprising enhancing the activity of the GmUBC13 protein or the expression of the gene encoding the protein in the target soybean plant.

[0017] A protein mutant, which is a variant in which the cysteine ​​at position 89 is replaced by another amino acid based on the amino acid sequence shown in SEQ ID NO: 2, and the mutation causes the protein to lose ubiquitin coupling enzyme activity.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention is the first to demonstrate that GmUBC13, as an E2 ubiquitin coupling enzyme, mainly mediates K48 polyubiquitination modification, degrades lignin synthesis and disease resistance-related target proteins via the 26S proteasome pathway, clarifies its negative regulatory molecular mechanism, and for the first time discovers that the soybean GmUBC13 gene can negatively regulate lignin biosynthesis and soybean resistance to Phytophthora root rot. Specifically, the GmUBC13 gene and its encoded protein were successfully cloned from soybean, and systematic functional experiments confirmed for the first time that this gene encodes an active E2 ubiquitin coupling enzyme. Its overexpression can significantly inhibit soybean lignin biosynthesis, while interfering with its expression can promote lignin accumulation, filling the gap in the lignin synthesis regulatory network regarding key negative regulatory elements.

[0020] This invention utilizes soybean hairy root transformation to overexpress and interfere with the expression of the GmUBC13 gene in soybean hairy roots. It was found that GmUBC13-overexpressing transgenic soybean hairy roots exhibited an enhanced phenotype of increased susceptibility to Phytophthora soybeanis infection, while GmUBC13-interfered transgenic soybean hairy roots exhibited the opposite phenotype. Quantitative proteomics analysis, PRM verification, and lignin content detection experiments all fully demonstrate that GmUBC13 can negatively regulate lignin biosynthesis.

[0021] In this invention, the discovery of soybean E2 ubiquitin coupling enzyme GmUBC13 as a negative regulator of lignin biosynthesis has enriched and improved the soybean lignin biosynthesis pathway to a certain extent, providing an important theoretical basis for cultivating superior soybean varieties with strong stress resistance and has broad application prospects.

[0022] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0023] Figure 1 This is a graph showing the differential expression levels of GmUBC13 in the tissues and organs of the highly resistant soybean variety "Suinong 10" and the highly susceptible soybean variety "Dongnong 50".

[0024] Figure 2 This is a graph showing the differential expression analysis of GmUBC13 induced by the present invention in the highly resistant soybean variety "Suinong 10" and the highly susceptible soybean variety "Dongnong 50" after inoculation with Phytophthora beanus.

[0025] Figure 3 This image shows the screening and detection results of the hairy roots of GmUBC13 overexpressing transgenic soybean according to the present invention.

[0026] Figure 4 This image shows the results of disease resistance identification of the hairy roots of GmUBC13 overexpressing transgenic soybean according to the present invention.

[0027] Figure 5 This is a graph showing the biomass accumulation of Phytophthora soybean in the hairy roots of GmUBC13 overexpressing transgenic soybean according to the present invention.

[0028] Figure 6 The image shows the screening and detection results of the GmUBC13 interference transgenic soybean hairy roots of this invention.

[0029] Figure 7 This is a diagram showing the disease resistance identification results of the GmUBC13 interference transgenic soybean hairy roots of the present invention.

[0030] Figure 8 This is a graph showing the biomass accumulation of Phytophthora soybean in the hairy roots of GmUBC13 interference transgenic soybean according to the present invention.

[0031] Figure 9 This is a graph showing the ubiquitination activity detection results of GmUBC13 of the present invention.

[0032] Figure 10 This is a graph showing the detection results of the ubiquitination type of GmUBC13 in this invention.

[0033] Figure 11 GmUBC13 of the present invention C89A Screening and detection results of transgenic soybean hairy roots overexpressing genetically modified soybean.

[0034] Figure 12 GmUBC13 of the present invention C89A The results of the disease resistance identification of transgenic soybean hairy roots overexpressing the gene are shown in the figure.

[0035] Figure 13 GmUBC13 of the present invention C89A Figure showing the biomass accumulation of Phytophthora soybean in the hairy roots of overexpressing transgenic soybean.

[0036] Figure 14 This is a diagram showing the PRM verification results of the GmUBC13-regulated target protein of the present invention.

[0037] Figure 15 The image shows the detection results of lignin content in the hairy roots of the GmUBC13 transgenic soybean of this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only illustrative and not limiting.

[0039] Example 1: Cloning and sequence analysis of the soybean GmUBC13 gene, specifically including the following steps:

[0040] I. Young leaves of the soybean cultivar "Dongnong 50," which is highly susceptible to Phytophthora root rot, were used as experimental material. Total RNA was extracted from the leaves according to the Invitrogen TRIzol kit manual.

[0041] 2. The extracted total RNA was treated with DNase I; 1 µg of the total RNA treated in step 2 was used for cDNA synthesis. The cDNA synthesis was performed according to the instruction manual of the BD SMART™ RACE cDNA Amplification Kit purchased from BD Biosciences Clontech to obtain cDNA.

[0042] 3. Using the cDNA obtained above as a template, and referring to the TaKaRa PrimeSTAR® HS DNA Polymerase instruction manual, the GmUBC13 gene was amplified using forward primer F1 and reverse primer R1.

[0043] Forward primer F1: 5'-ATGGCATCTCAGGCTAGCCTTC-3'

[0044] Reverse primer R1: 5'-TCACAACATTTCTTGTGACTTCCT-3'

[0045] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 38 cycles; and 72℃ final extension for 10 min. Finally, the PCR products were sequenced using an ABI 3130 sequencer (ABI).

[0046] Sequencing results show that the gene GmUBC13, a negative regulator of soybean lignin biosynthesis, consists of 501 bases, and its nucleotide sequence is shown in SEQ ID No: 1 in the sequence listing.

[0047] Example 2: Expression pattern analysis of the GmUBC13 gene

[0048] I. Differential expression in different tissues and organs:

[0049] Once the seedlings of the highly resistant soybean variety "Suinong 10" and the highly susceptible soybean variety "Dongnong 50" reached the V1 stage, select healthy seedlings of "Dongnong 50" and "Suinong 10" and quickly freeze samples of their roots, stems, leaves and cotyledons in liquid nitrogen. Extract RNA and reverse transcribe it into cDNA. The specific extraction method is the same as in Example 1. Next, using cDNA as a template and forward primer F2 and reverse primer R2 as quantitative primers, the differential expression of GmUBC13 in different tissues and organs of different varieties is analyzed by qRT-PCR.

[0050] Forward primer F2: 5'-CGCTGGACACCTGTTCATACG-3'

[0051] Reverse primer R2: 5'-CGCATCGGCTAACCTTTTT-3'

[0052] The results are as follows Figure 1 As shown, GmUBC13 was most highly expressed in the cotyledons, followed by the roots, stems, and leaves. Furthermore, the relative expression levels in the cotyledons and roots of the highly susceptible soybean variety "Dongnong 50" were significantly higher than those of the highly resistant soybean variety "Suinong 10" (**P<0.01).

[0053] II. Treatment with Phytophthora:

[0054] Phytophthora 1 (the dominant race in Heilongjiang Province) was inoculated into the hypocotyls of Dongnong 50 and Suinong 10. Samples were then taken at corresponding time points, and the samples were flash-frozen in liquid nitrogen. RNA was extracted and reverse transcribed into cDNA, using the same extraction method as in Example 1. Next, using cDNA as a template and F2 as the forward primer and R2 as the reverse primer, the differential expression of GmUBC13 at different time points after infection with Phytophthora 1 in different varieties was analyzed by qRT-PCR.

[0055] The results are as follows Figure 2 As shown, GmUBC13 can be induced to express by Phytophthora in soybean, and the expression level of GmUBC13 differs between resistant and susceptible varieties. The expression level in the highly susceptible soybean variety "Dongnong 50" is significantly higher than that in the highly resistant soybean variety "Suinong 10" (**P<0.01).

[0056] Example 3: Obtaining positive hairy roots of GmUBC13 transgenic soybean

[0057] I. Construction of GmUBC13 plant expression vector

[0058] a. Based on the CDS sequence of GmUBC13 and the vector sequences of plant expression vectors pCAMBIA3301 and pFGC5941, appropriate restriction sites were selected, and forward primers F3 and R3 for the pCAMBIA3301-GmUBC13 recombinant vector and forward primers F4 and R4, forward primers F5 and R5 for the pFGC5941-GmUBC13 recombinant vector were designed to amplify the target fragment.

[0059] Forward primer F3:

[0060] 5'-ACGGGGGACTCTTGACCATGGAAATGGCATCTCAGGCTAGCCTTC-3'

[0061] Reverse primer R3:

[0062] 5'- ACCGTTAATTAACCCCACGTGCAACATTTCTTGTGACTTCCT-3'

[0063] Forward primer F4:

[0064] 5'-TTTCATTTGGAGAGGACACGCTCGAGATGGCATCTCAGGCTAGCCT-3'

[0065] Reverse primer R4:

[0066] 5'-AATCATCGATTGGGCGCGCCCCATGGCATTGGGGTGCCATATCTCTG-3'

[0067] Forward primer F5:

[0068] 5'-TCCCGGGTCTTAATTAACTCTCTAGAATGGCATCTCAGGCTAGCCT-3'

[0069] Reverse primer R5:

[0070] 5'-TTGGTCAATTTGCAGGTATTTGGATCCATTGGGGTGCCATATCTCTG-3'

[0071] b. Using the plasmid pMDTM18-T-GmUBC13 containing the full length of GmUBC13 as a template, the GmUBC13 fragment was amplified according to the above primers. At the same time, the vectors pCAMBIA3301 and pFGC5941 were linearized, and the gel recovery products of the amplified GmUBC13 fragment were homologously ligated with the gel recovery products of the linearized vector.

[0072] c. The above ligation products were sequentially subjected to E. coli Top-10 transformation, bacterial PCR, and sequencing.

[0073] II. Preparation of Agrobacterium K599 competent cells

[0074] a. Add 100 µL of Agrobacterium K599 to 10 mL of YEP liquid medium containing antibiotic (streptomycin), place in a shaker at 28°C, and incubate overnight at 220 rpm.

[0075] b. Take 50 µL of the above bacterial culture and activate it in YEP liquid medium containing antibiotics.

[0076] c. Use a pipette to transfer the activated bacterial solution into a sterilized 1.5 mL EP tube and let it stand on ice for 30 min. Then, place the EP tube in a pre-cooled centrifuge and centrifuge at 8000 rpm for 3 min, and discard the waste liquid.

[0077] d. Take an appropriate amount of pre-cooled CaCl2 (0.05 M) solution on ice and add it to the EP tube, then mix it thoroughly by repeatedly blowing and swishing. Let the EP tube stand on ice for 30 min, then place it in a centrifuge and discard the supernatant after centrifugation.

[0078] e. Add CaCl2 solution to the EP tube and repeatedly pipette, then dispense the Agrobacterium competent cells into the EP tube and store at -80℃.

[0079] III. Transformation of Agrobacterium K599 competent cells

[0080] pCAMBIA3301-GmUBC13 and pFGC5941-GmUBC13 containing recombinant plasmids were transformed into Agrobacterium K599, as follows:

[0081] a. Take 100 µL of K599 competent cells until they partially melt. Add 3 µL of each of the recombinant plasmids containing pCAMBIA3301-GmUBC13 and pFGC5941-GmUBC13 to the competent cells. Gently pipette the mixture to mix.

[0082] b. Place the above competent cells in an ice bath for 5 min, freeze them in liquid nitrogen for 5 min, bathe them in a water bath at 37°C for 5 min, and then place them in an ice bath for 5 min again.

[0083] c. Using a pipette, aspirate 800 µL of YEP liquid culture medium into the competent cells and incubate at 28°C with shaking at 150 rpm for 4 h.

[0084] d. Place the above competent cells in a centrifuge and centrifuge at 5000 rpm for 3 min at room temperature. Then discard part of the supernatant in the centrifuge, and use a pipette to fully lyse and mix the cells. Take 100 μL of the bacterial culture and spread it on YEP solid medium containing 50 mg / L Str. Place the medium in an incubator at 28°C and invert it for 2-4 days.

[0085] e. Once a single colony has grown, pick it up and place it in LB liquid medium containing the corresponding antibiotic. Incubate it in a shaker at 28°C for 2 days, and then perform PCR verification.

[0086] IV. Preparation of culture medium:

[0087] 1 L germination medium (pH: 5.8) content 6BA 1.0 mg B5 Mixed Salt 3.21 g sucrose 20 g Agar 8 g

[0088] V. Preparation method of the infiltration solution:

[0089] 1 L of infection solution content MgCl2 10 mM Acetyleugenol 150 μM

[0090] VI. Preparation method of root induction medium:

[0091] 1 L root induction medium (pH: 5.8) content Carboxybenzyl 250 mg Cephalosporins 250 mg MS salt 2.215 g MES 0.6 g sucrose 20 g

[0092] VII. Germination of soybean seeds:

[0093] Select healthy, plump, and uniformly sized Dongnong 50 soybean seeds and sterilize them with chlorine overnight. Next, in a clean bench, use high-temperature sterilized tweezers to plant the sterilized soybean seeds on the seed culture medium and place them on a light-controlled culture rack at room temperature for 4-6 days.

[0094] 8. Agrobacterium infection:

[0095] a. Take 100 µL of K599 Agrobacterium rhizogenes containing pCAMBIA3301-GmUBC13 and GmUBC13-RNAi recombinant plasmids into 10 mL of YEP medium (containing 50 mg / L Kana) and incubate overnight in a constant temperature shaker at 28°C and 220 rpm.

[0096] b. Take 800 µL of the propagation culture in a clean bench and inoculate it a second time to activate it until the OD600 is about 0.6.

[0097] c. Centrifuge the above bacterial solution at 8,000×g for 15 min, discard the waste liquid, and resuspend the activated K599 Agrobacterium rhizogenes containing the pCAMBIA3301-GmUBC13 and GmUBC13-RNAi recombinant plasmids in the infection solution.

[0098] d. In a clean bench, use a sterilized scalpel to make a concave wound on the outside of the cotyledon, and then arrange the cotyledons evenly on the prepared root induction medium.

[0099] e. Take an appropriate amount of resuspended bacterial cells and apply them to the wound on the cotyledon.

[0100] f. Place the root induction medium on a light-illuminated culture rack and incubate at room temperature for 15-20 days.

[0101] g. Obtain soybean hairy roots.

[0102] Example 4: Detection of positive hairy roots of GmUBC13 overexpressing transgenic soybean

[0103] The obtained GmUBC13-OE and EV (pCAMBIA3301) soybean hairy roots were fluorescently identified using a LUYOR-3415RC excitation light source. Proteins were extracted from the GmUBC13-OE and EV hairy roots and detected by Western blot using Anti-Flag as the antibody. The expression level of GmUBC13 in the GmUBC13-OE hairy roots was detected by qRT-PCR using F2 as the forward primer and R2 as the reverse primer. The results indicate that GmUBC13-overexpressing transgenic soybean positive hairy roots were successfully obtained, as shown in the attached image. Figure 3 As shown.

[0104] Example 5: Disease resistance identification of positive hairy roots of GmUBC13-overexpressing transgenic soybean

[0105] Selected overexpressing transgenic positive hairy roots with uniform growth, cleaned them, and placed them in a pre-washed square iron dish. A suitable thickness of medical gauze was then placed on the dish, moistened with sterilized distilled water. A fixed-size *Phytophthora sojae* race 1 mycelium block (provided by the laboratory for propagation) was placed on the root at the same location. The mycelium was replaced every 24 hours. After 3 days, disease progression was recorded using a Sony camera. The inoculated site was removed with a blade, flash-frozen in liquid nitrogen, and its RNA was extracted and reverse transcribed into cDNA. The extraction method was the same as in Example 1. Next, using the cDNA as a template and forward primer F6 and reverse primer R6 as quantitative primers, the accumulation of *Phytophthora sojae* was detected using qRT-PCR.

[0106] Forward primer F6: 5'-ACTGCACCTTCCAGACCAT-3'

[0107] Reverse primer R6: 5'-CCACCACCTTGATCTTCATG-3'

[0108] As attached Figure 4As shown, 72 h after inoculation with Phytophthora race 1, the inoculation sites of GmUBC13-OE transgenic hairy roots showed more severe disease and turned blackish-brown compared to EV transgenic hairy roots. Simultaneously, biomass accumulation analysis of Phytophthora soybean in the inoculated transgenic hairy roots showed that the biomass accumulation of Phytophthora soybean in GmUBC13-OE transgenic hairy roots was significantly higher than that in EV hairy roots, as shown in the attached figure. Figure 5 As shown in the figure. The results indicate that GmUBC13 is a negative regulator of the response to P. sojae infection.

[0109] Example 6: Detection of positive hairy roots in GmUBC13 transgenic soybean

[0110] Take approximately 15-20 mg of hairy roots with uniform growth and place them in a 1.5 mL EP tube. Add an appropriate amount of EB2 Extraction Buffer, grind, and let stand at room temperature for 10 seconds. Place the tube into a test strip (purchased from ENVIROLOGIX, USA). After the reaction is complete, the appearance of two purple-red control lines in both the detection and control areas of the test strip indicates successful transformation of the transgenic hairy roots. The appearance of only one purple-red control line in the control area indicates failure of the transgenic hairy roots. Using F2 as the forward primer and R2 as the reverse primer, the expression level of GmUBC13 in GmUBC13-RNAi hairy roots is detected by qRT-PCR. (See attached image) Figure 6 As shown, positive hairy roots of GmUBC13 transgenic soybean were successfully obtained.

[0111] Example 7: Disease resistance identification of positive hairy roots of GmUBC13 transgenic soybean.

[0112] Selected interfering transgenic positive hairy roots with uniform growth were inoculated for identification. The inoculation method was the same as in Example 5. The inoculated part was removed with a blade, flash-frozen in liquid nitrogen, and its RNA was extracted and reverse transcribed into cDNA. The extraction method was the same as in Example 1. The accumulation of Phytophthora soybeanis was detected by qRT-PCR. The qRT-PCR detection method was the same as in Example 2.

[0113] The symptoms of disease at the inoculation site of the hairy root of the GmUBC13-RNAi transgenic organism were significantly weaker than those of the EV, as shown in the attached... Figure 7 As shown. Simultaneously, the biomass accumulation of Phytophthora soybean in the transgenic hairy roots after inoculation was analyzed. The results showed that the biomass accumulation of Phytophthora soybean in the GmUBC13-RNAi transgenic hairy roots was significantly lower than that in the EV transgenic hairy roots (…). ** P < 0.01), as shown in the attached document. Figure 8 As shown, the performance of the hairy roots of the GmUBC13-RNAi transgenic plants further demonstrates that GmUBC13 can negatively regulate soybean resistance to Phytophthora soybeanis.

[0114] Example 8: Ubiquitination Analysis of GmUBC13

[0115] I. Construction of the GmUBC13 prokaryotic expression vector

[0116] Based on the CDS sequence of GmUBC13 and the vector sequence of the prokaryotic expression vector pET29b(His), appropriate restriction sites were selected, and forward primer F7 and reverse primer R7 of the GmUBC13-His recombinant vector were designed to amplify the target fragment.

[0117] Forward primer F7: 5'-GGCGATATCGGATCCGAATCCTATGGCATCTCAGGCTAGCCTTC-3'

[0118] Reverse primer R7: 5'-TGCGGCCGCAAGCTTGTCGACCAACATTTCTTGTGACTTCCT-3'

[0119] II. Isolation and purification of GmUBC13-His fusion protein

[0120] The fusion protein GmUBC13-His was purified and recovered using the NOVAGEN protein purification kit. The main solution preparation is as follows:

[0121] a.1×Binding Buffer (100 mL): PH=7.9

[0122] NaCl (0.5 M) 2.922 g Tris-HCl (20 mM) 0.242 g Imidazole (5 mM) 0.0340 g

[0123] b.1×Wash Buffer (100 mL): PH=7.9

[0124] NaCl 0.5 M) 2.922 g Tris-HCl (20 mM) 0.242 g Imidazole (60 mM) 0.409 g

[0125] c.1×Elute Buffer (100 mL):

[0126] NaCl (0.5 M) 2.922 g Tris-HCl (20 mM) 0.242 g Imidazole (1 M) 6.808 g

[0127] III. Preparation of soluble proteins:

[0128] a. Centrifuge at 10,000×g for 10 min to collect bacterial cells, discard the supernatant, and remove as much culture medium as possible. Add 4 mL of ice-cold 1×Binding Buffer to every 100 mL of bacterial cells obtained from the culture medium;

[0129] b. Place the resuspended bacterial solution in a suitable-sized container and sonicate to disrupt it. (Keep the bacterial solution in an ice bath during sonication).

[0130] c. Centrifuge the lysate at 14,000×g for 20 min to remove cell debris, and then filter the supernatant through a 0.45 µm filter membrane to prevent resin blockage after sample loading.

[0131] IV. Preparation and balancing of pre-installed columns:

[0132] a. Remove the pre-packed column cap, aspirate as much of the buffer solution stored on top as possible, and remove the Luer locking sleeve at the bottom of the column;

[0133] b. Add 10 mL of 1×Binding Buffer to the pre-packed column to equilibrate the resin, then allow all the buffer to drain out.

[0134] V. Column chromatography purification of the target protein:

[0135] a. Allow the binding buffer to drain completely before loading the sample;

[0136] b. Wash the column with 10 mL of 1×Binding Buffer;

[0137] c. Wash the column with 10 mL of 1×Wash Buffer;

[0138] d. Elute the bound protein with 5 mL of 1×Elute Buffer;

[0139] e. Store the purified and recovered target protein in a -80°C freezer for later use.

[0140] VI. Ubiquitination Reaction System

[0141] Preparation of reaction system (20 μL):

[0142] name Volume (µL) 10×Reaction Buffer 2 E1 Enzyme (0.5 μg / μL, concentration 4.23 μM) 2 Ubiquitin (freeze-dried powder) 4 (0.588 mM) 10 x Mg2*-ATP 2 E2 Enzyme 2 Substrate (optional)* 1-5 μM <![CDATA[H2O]]> Up to 20 μL

[0143] Reaction conditions: Incubate at 37℃ (30 min-4 h), it is recommended to use a PCR instrument or metal bath.

[0144] The results showed that GmUBC13 possesses ubiquitin-coupled enzyme activity, and cysteine ​​residue at position 89 is the major site of its enzyme activity, as shown in the attached figure. Figure 9 As shown in the attached figure. The results of the ubiquitination modification type indicate that GmUBC13 primarily mediates K48 polyubiquitination modification. Figure 10 As shown, this suggests that GmUBC13-mediated ubiquitination primarily involves substrate degradation via the 26S proteasome pathway.

[0145] Example 9, GmUBC13 C89A Detection of positive hairy roots of transgenic soybean overexpression

[0146] The obtained GmUBC13 was excited using a LUYOR-3415RC excitation source.C89A -OE, GmUBC13-OE, and EV (pCAMBIA3301) soybean hairy roots were identified by fluorescence; GmUBC13 was extracted C89A Proteins from -OE, GmUBC13-OE, and EV hairy roots were detected by Western blot using Anti-Flag as the antibody; GmUBC13 was detected by qRT-PCR using F2 as the forward primer and R2 as the reverse primer. C89A The expression levels of GmUBC13 in hairy roots of -OE and GmUBC13-OE were obtained. The results indicate that GmUBC13 overexpression was successfully obtained. C89A -OE and GmUBC13-OE transgenic soybeans have positive hairy roots, as shown in the attached image. Figure 11 As shown.

[0147] Example 10, GmUBC13 C89A Disease resistance identification of positive hairy roots of transgenic soybean overexpression

[0148] Select overexpressing transgenic positive hairy roots with consistent growth for inoculation identification. The specific inoculation method is the same as in Example 5. Use a blade to remove the inoculated part, quick-freeze it in liquid nitrogen, extract its RNA and reverse transcribe it into cDNA. The extraction method is the same as in Example 1. Use qRT-PCR to detect the accumulation of Phytophthora soybeanis. The qRT-PCR detection method is the same as in Example 2.

[0149] As attached Figure 12 As shown, the disease incidence of hairy roots in the GmUBC13-OE transgenic variety was significantly higher than that in the GmUBC13 variety. C89A -OE transgenic hairy roots showed significantly stronger growth than EV hairy roots. Furthermore, analysis of Phytophthora biomass accumulation in the transgenic hairy roots after inoculation revealed that the biomass accumulation of Phytophthora soybean in GmUBC13-OE transgenic hairy roots was significantly higher than that in GmUBC13. C89A -OE transgenic hairy roots showed significantly higher levels than EV hairy roots, as shown in the attached image. Figure 13 As shown in the figure. The results further indicate that GmUBC13 is a negative regulator of P. sojae infection, and that cysteine ​​at position 89 is the main site of its enzyme activity. Mutation of this site significantly reduced the incidence of disease.

[0150] Example 11: PRM Validation Results of GmUBC13-Regulated Target Proteins

[0151] The tested GmUBC13-OE and GmUBC13 C89A Proteins were extracted from the hairy roots of OE and EV positive transgenic plants and validated using PRM. Results are attached. Figure 14As shown, GmUBC13 can negatively regulate the expression of GmDFR, GmIFS2, GmCYP81E11 and GmPOD.

[0152] Example 12: Determination of lignin content in the hairy roots of GmUBC13 transgenic soybean

[0153] The lignin content of transgenic hairy roots tested with GmUBC13 was determined. The results showed that GmUBC13, as a negative regulator of soybean lignin biosynthesis, could inhibit the accumulation of lignin in soybeans, as shown in the attached figure. Figure 15 As shown.

[0154] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0155] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A soybean GmUBC13 gene, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The protein encoded by the gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that, The vector contains the GmUBC13 gene as described in claim 1.

4. A recombinant cell, characterized in that, The cell contains the soybean GmUBC13 gene of claim 1, the protein of claim 2, or the recombinant vector of claim 3.

5. A genetically modified soybean or its hairy root, characterized in that, Its genome integrates an exogenous polynucleotide sequence capable of expressing or silencing the protein of claim 2, or the expression or activity of an endogenous gene encoding the protein of claim 2 is regulated.

6. The application of the soybean GmUBC13 gene of claim 1 or the protein of claim 2 in the negative regulation of soybean lignin biosynthesis.

7. The application of the soybean GmUBC13 gene of claim 1 or the protein of claim 2 in reducing soybean resistance to Phytophthora root rot.

8. A method for cultivating soybean plants with increased lignin content and / or enhanced resistance to Phytophthora root rot, characterized in that, This includes inhibiting the activity of the GmUBC13 protein of claim 2 or the expression of the gene encoding the protein in the target soybean plant.

9. A method for reducing soybean lignin content and / or resistance to Phytophthora root rot, characterized in that, This includes enhancing the activity of the GmUBC13 protein of claim 2 or the expression of the gene encoding the protein in the target soybean plant.

10. A protein mutant, characterized in that, It is a variant based on the amino acid sequence shown in SEQ ID NO: 2, in which the cysteine ​​at position 89 is replaced by another amino acid, and the mutation causes the protein to lose its ubiquitin coupling enzyme activity.

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