Application of GmPL3 gene in regulating soybean resistance to gray mold

Abstract

The application of the GmPL3 gene in regulating the soybean resistance to gray spot belongs to the technical field of plant disease resistance, and aims to improve the soybean resistance to gray spot and total pectin content, and reduce the pectin lyase activity. The application provides an application of a GmPL3 gene in regulating the soybean resistance to gray spot, pectin lyase activity and total pectin content, wherein the GmPL3 gene is shown as SEQ ID NO. 16. The application can provide a clear gene target, detection method and evaluation system for the creation of soybean varieties resistant to gray spot, gene editing breeding and molecular marker assisted selection.

Description

Technical Field

[0001] This invention belongs to the field of plant disease resistance technology, specifically involving the application of the GmPL3 gene in regulating the resistance of soybean to gray spot disease. Background Technology

[0002] Soybean gray leaf spot (FLS) is an important fungal disease caused by *Cercospora sojina* Hara. It primarily affects soybean leaves, but can also infect stems, pods, and seeds. After infection, leaves develop frog-eye-like lesions, chloroplasts die, and photosynthetic area and efficiency decrease. In severe cases, it leads to premature leaf drop, reduced grain fullness, and affects quality traits such as protein and oil content.

[0003] In my country's major soybean producing areas, the average annual incidence of soybean gray leaf spot can reach 30% to 50%, and in years with an epidemic, the yield reduction of infected varieties can exceed 50%. Another reference paper records that the disease generally causes a yield reduction of 10% to 30%, and in severe cases, it can exceed 50%. Therefore, soybean gray leaf spot has become one of the important diseases restricting soybean yield and quality.

[0004] Currently, the control of soybean gray spot disease mainly relies on chemical agents and the breeding of disease-resistant varieties. Chemical control poses risks of environmental pollution, pesticide costs, and pathogen resistance; traditional disease-resistant breeding is time-consuming, inefficient, and easily affected by pathogen physiological race differentiation and environmental conditions, making it difficult to quickly meet the needs of disease-resistant variety creation.

[0005] Therefore, it is urgent to explore key gene resources related to soybean gray spot resistance, clarify their mechanism of action, establish a reproducible gene editing, molecular detection and disease resistance evaluation system, and create new disease-resistant germplasm to serve the molecular breeding of soybean gray spot resistance. Summary of the Invention

[0006] The purpose of this invention is to improve the resistance of soybean to gray spot disease, the activity of pectin lyase, and the total pectin content.

[0007] This invention provides the application of the GmPL3 gene in regulating soybean resistance to gray leaf spot, pectin lyase activity, and total pectin content, wherein the GmPL3 gene is shown in SEQ ID NO.16.

[0008] This invention provides the application of a recombinant vector containing the GmPL3 gene targeting sequence in improving the resistance of soybean to gray spot disease and the total pectin content, and reducing the activity of pectin lyase. The GmPL3 gene is shown in SEQ ID NO.16; the targeting sequence is shown in SEQ ID NO.1-3.

[0009] Further specifying, the launch carrier is pYLCRISPR / Cas9-MT.

[0010] This invention provides the application of recombinant host cells containing the above-mentioned recombinant vector in improving the resistance of soybean to gray spot disease and the total pectin content, and reducing the activity of pectin lyase, wherein the GmPL3 gene is shown in SEQ ID NO.16.

[0011] Further specifying, the host cell is either Escherichia coli or Agrobacterium.

[0012] This invention provides the application of soybean plants containing the GmPL3 gene knockout in improving soybean resistance to gray spot disease and total pectin content, and reducing pectin lyase activity, wherein the GmPL3 gene is shown in SEQ ID NO.10.

[0013] This invention provides a breeding method for improving soybean resistance to gray leaf spot disease and total pectin content, and reducing pectin lysin activity. The steps of the method are as follows: Step 1: Amplify the sgRNA of the GmPL3 gene, the sgRNA sequence of which is shown in SEQ ID NO.1-3, and then ligate it into a CRISPR vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfect recombinant Agrobacterium into soybean callus and culture to obtain transgenic soybeans.

[0014] To further specify, the primer pair for amplifying sgRNA in step 1 is shown in SEQ ID NO.4-7.

[0015] To further specify, the CRISPR vector in step 1 is pYLCRISPR / Cas9-MT.

[0016] Beneficial effects: The lesion area of ​​GmPL3-edited lines after infection with soybean gray leaf spot was significantly smaller than that of the wild type, indicating that reducing GmPL3 function can improve soybean gray leaf spot resistance. The reduced pectin lyase activity in GmPL3-edited lines improved cell wall pectin metabolism and structural stability, which is beneficial for reducing pathogen invasion and mycelial spread. Attached Figure Description

[0017] Figure 1 The image shows the vector construction diagram and the sequence results of three target sites for GmPL3. Figure 2 The flowchart of the genetic transformation process of soybean GmPL3 plants is shown below: (a) Infection; (b) Co-culture; (c) Recovery; (d) Shoot induction; (e) Screening; (f) Shoot elongation; (g) Rooting; (h) Transgenic plants; Figure 3 Figure 1 shows the positive identification results of GmPL3 gene-edited plants; (a) electrophoresis detection of Bar gene fragments in T0 generation gene-edited plants; (b) electrophoresis detection of Bar gene fragments in T1 generation gene-edited plants; (c) Bar test strip detection in T1 generation gene-edited plants; M represents DNA Marker DL2000; - represents wild-type DN50; 1 represents positive control; 2-23 represent different GmPL3 gene-edited transformed plants; Figure 4 Image showing the target detection results of GmPL3 gene-edited plants; Figure 5 The image shows the PCR amplification results of bacterial culture constructed using the GmPL3 gene overexpression vector; M represents DNA Marker DL5000; 1-8 represent the PCR amplification results of different single colonies of GmPL3. Figure 6 The image shows the PCR identification results of Agrobacterium tumefaciens overexpression vector GmPL3; M represents DNA Marker DL2000; 1-6 represent the PCR amplification results of different single colonies of GmPL3, with a gene size of 1407 bp; Figure 7 The flowchart shows the genetic transformation process of soybean GmPL3 overexpressing plants: 1. Infection, 2. Co-culture, 3. Shoot induction, 4. Screening, 5. Shoot elongation, 6. Rooting, 7. Acclimation. Figure 8 The image shows the PCR identification results of the bar gene in T1 generation overexpressing plants; M represents DNA Marker DL2000; 1 represents water; 2 represents wild-type Dongnong 50; 3 represents the positive control; 4-9 represent the PCR amplification results of GmPL3-OE genetically transformed plants, with a gene size of 269 bp. Figure 9 Image showing the detection results of Bar test strips on T1 generation overexpressing plants; Figure 10 Image showing the results of glufosinate application to T1 generation plants for identification; Figure 11 The graph shows the expression level of the GmPL3 gene in overexpressing plants; ** represents P < 0.01; **** represents P < 0.0001; Figure 12 The results of editing the GmPL3 gene expression level in plants are shown in the figure; Note: *** represents P<0.001; **** represents P<0.0001; Figure 13Figure 1 shows the results of GmPL3-OE1 expression at different time points after infection with the overexpressing strains of gray spot disease; (a) expression level of GmPL3-OE1 at different time points after infection; (b) expression level of GmPL3-OE2 at different time points after infection. Figure 14 Figure 1 shows the results of GmPL3-PL#1 expression levels at different time points after infection with edited strains of gray spot disease; (a) expression level of GmPL3-PL#1 at different time points after infection; (b) expression level of GmPL3-PL#2 at different time points after infection. Figure 15 The following are the results of leaf lesion area measurement: (a) Leaf lesion area at 7 and 14 days of infection, scale bar is 1 cm; (b) Quantitative leaf lesion area at 7 and 14 days of infection; * represents P<0.05; ** represents P<0.01; *** represents P<0.001; ns represents P>0.05; Figure 16 The graph shows the results of pectin lyase activity; ** indicates P < 0.01. Figure 17 Figure 1 shows the results for total pectin and soluble pectin content; (a) total pectin content; (b) soluble pectin content; * represents P<0.05; ** represents P<0.01. Detailed Implementation

[0018] Example 1. Construction of GmPL3 CRISPR / Cas9 editing vector 1. Three CRISPR / Cas9-specific knockout targets were designed for the second exon of the GmPL3 coding region. According to the original manuscript, the target information is as follows: Table 1

[0019] 2. pEGS401 target design The target sequence of the GmPL3 gene for which the CRISPR / Cas9 gene editing vector needs to be constructed was selected. The GmPL3 gene target sequence was designed using the online website CRISPR direct (https: / / crispr.dbcls.jp / ). Adapter primers were added according to the restriction site Bsa I. The primers used are shown in Table 2.

[0020] Table 2 Target Primer Sequences

[0021] 3. Extraction of pEG401 vector plasmid The plasmids were extracted using the EasyPure® Plasmid MiniPrep Kit (EM101) from Beijing TransGen Biotech Co., Ltd., and the eluted DNA was stored at -20°C.

[0022] 4. PCR amplification of tRNA-sgRNA-sgRNA Scaffold-tRNA fragments: To obtain the vector -sgRNA1-Scaffold-tRNA-sgRNA2-Scaffold-tRNA-sgRNA3-, two fragments need to be amplified by PCR: Fragment 1: the first half of sgRNA1-Scaffold-tRNA-sgRNA2; Fragment 2: the second half of sgRNA2-Scaffold-tRNA-sgRNA3; Note that sgRNA2 is split and placed at the junction of the two fragments. Fragment 1 has the sgRNA2 sequence from positions 1 to 12 at its end, and fragment 2 has the sgRNA2 sequence from positions 9 to 20 at its start end. The two sequences overlap at positions 9 to 12. After BsaI digestion, these four bases form complementary sticky ends, allowing the two fragments to ligate precisely and restoring the complete sgRNA2. TGGTCTCG = BsaI recognition site, TGCA = sticky end connecting to the left end of the pGES401 vector, AAC = sticky end connecting to the right end of the pGES401 vector, gttttagagctagaaatagc = universal sequence for forward amplification of the Scaffold region, tgcaccagccgggaatcgaa = universal sequence for reverse amplification of the tRNA / Scaffold region; PCR amplification steps: (1) Use pGES401 empty vector as template.

[0023] (2) Amplify fragment 1: the first half of sgRNA1-Scaffold-tRNA-sgRNA2 using sgRNA-F1 and sgRNA-R1; (3) Fragment 2: the latter half of sgRNA2-Scaffold-tRNA-sgRNA3 was amplified using sgRNA-F2 and sgRNA-R2.

[0024] Fragment 2: 5' end: BsaI + sgRNA2 9-20 nt, 3' end: sgRNA3 reverse complementary sequence + AAAC + BsaI; Table 3 PCR reaction system

[0025] Repeat the mixing of the prepared reaction buffer, briefly centrifuge, and place on the PCR instrument. Run the program according to Table 4: Table 4

[0026] Note: The reaction cycle parameter is set to 35 cycles.

[0027] 5. Purification and recovery of tRNA-sgRNA-sgRNA Scaffold-tRNA fragments The PCR amplified fragments were electrophoresed using the following agarose gel electrophoresis method: (1) Add 0.3 g agarose to 30 mL of 1×TAE buffer, mix well, and heat in a microwave oven for about 1 minute. Once the liquid becomes transparent, it can be taken out. (2) Add 5 µL of nucleic acid dye GelRed solution, mix well and pour into the gel casting plate. Insert the comb teeth at the appropriate position and let it stand at room temperature for about 30 min to solidify. (3) Place the agarose gel in an electrophoresis tank containing 1×TAE buffer, and then perform the sample loading operation. Turn on the power and run at 180 V for 25 min to complete the gel electrophoresis experiment.

[0028] Then, the electrophoresis gel products were purified and recovered using the FastPure® Gel DNA Extraction Mini Kit (DC301) from Nanjing Novizan Co., Ltd. The concentration of the obtained DNA was determined using an ultra-micro spectrophotometer and stored at -20℃.

[0029] 6. Golden Gate One-Step Construction of Carrier The purified target fragment was added to the reaction system along with the pEG401 vector, and Bsa I and T4 ligase were added to achieve a one-step enzyme digestion-ligation process. The Golden Gate reaction system is shown in Table 5, and the procedure is shown in Table 6.

[0030] Table 5 Golden Gate Reaction System

[0031] Table 6 PCR Amplification Procedure

[0032] The final result was a multi-sgRNA tandem editing vector.

[0033] The GmPL3 target sequence was designed using CRISPR direct, and adapter primers were added based on the BsaI restriction site. The tRNA-sgRNA-sgRNA Scaffold-tRNA structural fragment was amplified using the pEG401 plasmid as a template, as shown in the figure. Figure 1 As shown, a multi-sgRNA tandem editing vector was constructed using the Golden Gate one-step method. The constructed product was transformed into DH5α, and after verification by PCR and sequencing, it was transformed into EHA105 Agrobacterium tumefaciens.

[0034] Example 2. Obtaining .GmPL3 edited transgenic soybeans I. Using wild-type soybean Dongnong 50 (DN50) as the recipient, GmPL3 edited plants were obtained through Agrobacterium-mediated transformation of soybean cotyledonary nodes. The basic process is as follows: Figure 2 As shown.

[0035] II. Identification DNA was extracted from the leaves of the edited plant. Primers were designed above and below the target sequence for PCR amplification, followed by sequencing to identify whether editing occurred at the genome level. The required primer sequences are shown in Table 7. Sequencing was compared to determine whether editing occurred at the target site and the type of editing.

[0036] Table 7 Primer sequences

[0037] The results are as follows Figure 3 and Figure 4 As shown.

[0038] III. RT-qPCR Expression Analysis Specific primers were designed using Primer 3 (https: / / primer3.ut.ee / ), and the primer sequences are shown in Table 8. (Soybean) Actin The gene was used as an internal control. The reaction system and procedure are shown in Tables 9 and 10. Quantitative fluorescence analysis was performed on a Roche LightCycler 96. Experimental results were obtained using 2% of the reference gene. -ΔCt Methods for testing T2 generation overexpression and knockout lines GmPL3 The relative expression levels of genes are calculated.

[0039] Table 8 Sequences of RT-qPCR Primers

[0040] Table 9 RT-qPCR reaction system

[0041] Table 10 RT-qPCR reaction procedure

[0042] The results are as follows Figure 12 As shown, the relative expression levels were calculated, and PL#1 / PL#2 / PL#3 / PL#4 decreased by approximately 71.32%, 85.55%, 58.24%, and 41.43%, respectively.

[0043] Example 2. Cloning of the GmPL3 gene and construction of an overexpression vector The CDS sequence of GmPL3 was obtained from the Phytozome 13 database, and amplification primers with the stop codon removed were designed using Primer 3. RNA was extracted from fresh leaves of the RIL6013 parent, and cDNA was obtained by reverse transcription. The cDNA was then used as a template for PCR amplification, yielding a GmPL3 coding region fragment of approximately 1407 bp.

[0044] Table 11

[0045] The GmPL3 coding region fragment recovered from the gel was directionally cloned into the overexpression vector pTF101 via homologous recombination, and transformed into E. coli DH5α. Positive identification results were as follows: Figure 5 As shown, single clones were selected for colony PCR and sequencing verification. After successful sequencing, plasmids were extracted and transformed into Agrobacterium tumefaciens EHA105. Positive identification results are shown below. Figure 6 As shown, it is used for subsequent soybean genetic transformation.

[0046] Complete nucleotide sequence of SEQ ID NO.16GmPL3:

[0047] SEQ ID NO.17 GmPL3 encodes the following amino acid sequence: MRIPSTLVEVLLVLALFCCSPWTVWSSTTLCQQTNEEVRPHSVSITEFGAVGDGVTLNTKAFQNAIFYLNSFADKGGAKLFVPAGRWLTGSFDLISHLTLSLDKDAVILGSTNPEDW PVVDPLPSYGRGRELPGGRHKSLIYGHNLTDVIITGNNGTIDGQGSIWWNRFWNRSLDYTRPHLVELMNSTGVLISNLTFLNSPFWTIHPVYCSQVTVQNVRILAPHDSPNTDGIDPD SSDNVCIEDCYISTGDDLIAIKSGWDEYGIAYGRPSTNIIIHRLVGRTQTSGIAIGSEMSGGVSEVHAEDIQFYDSYNAIRIKTSPGRGYVRNIYVSNVTLANVDIAITFTGLYGE HPDDAYNPNALPVIEKITIKDVVGENIKTAGLIEGIEGDNFVNICLSNIILNVTSNYPWNCSYVKGYSDLVQPEACEPLKERIFPGHCSDCYYLTNQIQSSNSQNRAGQTSGDIRG*.

[0048] Example 3. Obtaining GmPL3 overexpressing transgenic soybeans I. Using wild-type soybean Dongnong 50 (DN50) as the recipient, GmPL3 overexpression was obtained via Agrobacterium-mediated soybean cotyledon node transformation. The basic procedure is as follows: Figure 7 As shown.

[0049] II. Screening of positive plants and identification of expression levels 1. Bar test strip detection Place an appropriate amount of leaves in a 1.5 mL centrifuge tube, add 0.2 mL of distilled water, crush the leaves with a grinder, insert the Bar test strip into the tube, and let it stand for 5-8 minutes to observe the results. Positive plants will show a purple-red band in both the test area (T) and the control area (C) of the test strip. If only the control area (C) shows a purple-red band, the plant is negative.

[0050] 2. PCR amplification of the Bar gene fragment DNA was extracted from leaves of young soybean plants that had been overexpressed and edited, using the instructions for the Novizan FastPurePlant DNA Isolation Mini Kit (DC104).

[0051] Based on the herbicide resistance Bar gene sequence, specific detection primers were screened and synthesized using Primer 5 primer design software. Specific primer information is shown in Table 12. During the PCR reaction, DN50 was used as a template control for PCR amplification. The reaction system and procedure are shown in Tables 13 and 14. Finally, the size of the target band was checked by agarose gel electrophoresis.

[0052] Table 12 Bar Gene amplification primer sequences

[0053] Table 13 PCR Reaction System

[0054] Table 14 PCR Amplification Procedure

[0055] Note: The reaction cycle parameter is set to 20 cycles.

[0056] The results are as follows Figure 8-11 As shown.

[0057] Table 15

[0058] Example 4. Expression pattern of GmPL3 under gray spot disease infection I. The mixed gray spot pathogen was first propagated on PDA medium, and then on sorghum medium was used for secondary propagation. The sorghum was boiled for about 1 hour and then sterilized. The original inoculum blocks were inoculated and cultured at 26℃ for about 30 days, shaking daily. After the sorghum grains had fully propagated, they were spread out to a depth of about 1 cm, covered with damp gauze, and kept at room temperature in the dark for 48 hours to maintain humidity and promote spore growth. The spore suspension was prepared by washing with purified water and filtering three times. After adding 50 g / L of solid sucrose, the suspension was sprayed on soybean leaves.

[0059] Inoculation can be carried out by spraying after the soybean has grown its third trifoliate compound leaf, and samples should be taken at time points such as 0 h, 6 h, 24 h, 48 h or 0 d, 7 d, 14 d for the determination of expression level, lesion area and physiological indicators.

[0060] II. Expression pattern of GmPL3 under gray spot disease infection The relative expression levels of GmPL3 in the T2 generation overexpression lines GmPL3-OE1, GmPL3-OE2, and wild-type were measured at 0 h, 6 h, 24 h, and 48 h after infection with *Gnaphalium affine*. The results showed significant differences in GmPL3 expression levels at all four time points after infection, reaching a peak at 24 h, indicating that *Gnaphalium affine* infection can further induce upregulation of GmPL3 expression in the overexpression lines.

[0061] The relative expression levels of GmPL3 in the T2 generation edited lines GmPL3-PL#1, GmPL3-PL#2, and wild type were determined at 0 h, 6 h, 24 h, and 48 h after infection with *Gnaphalium affine*. The results showed... Figure 14 As shown, the expression level of GmPL3 in the edited lines remained at a very low level for 6–48 h after infection, indicating that the GmPL3 transcriptional response of the plants to infection by gray spot fungus was significantly suppressed after the GmPL3 gene was knocked out.

[0062] Example 5. Disease resistance phenotype analysis Table 16

[0063] Lesion area measurement: After infection with gray leaf spot, leaves were observed to show lesions of varying degrees. The lesion area was measured from leaves of the T2 generation (WT, GmPL3-OE1, and GmPL3-PL#1) at 7 and 14 days after infection. The results are as follows... Figure 15 As shown in (a), the overexpressing strain GmPL3-OE1 had the largest lesion area, while the edited strain GmPL3-PL#1 had the mildest lesion severity, and the lesion area gradually increased with the duration of infection. The quantified lesion area is shown in Figure (a). Figure 15 As shown in (b) of the results, the assay results indicate that the GmPL3 gene is a key gene regulating gray spot disease infection, and overexpression or knockout of this gene significantly increases or decreases the lesion area.

[0064] Pectin lyase activity assay: The GmPL3 gene encodes an active pectin lyase that catalyzes the β-elimination degradation of pectin. Therefore, this study measured the pectin lyase activity of wild-type DN50 and GmPL3 genetically transformed lines. The results are as follows: Figure 16 As shown, compared with wild-type DN50, the pectin lyase activity in the GmPL3 edited lines was significantly reduced, with the enzyme activity of the GmPL3-PL#1 line being approximately 0.89 times that of DN50; while the overexpression line GmPL3-OE1 showed the opposite trend, with the enzyme activity of the GmPL3-OE1 line being approximately 1.11 times that of DN50.

[0065] Determination of total pectin and soluble pectin content: The total pectin and soluble pectin contents of wild-type DN50 and T2 generation GmPL3 genetically transformed lines were determined. The results are as follows: Figure 17 As shown, compared to wild-type DN50, the total pectin content in the GmPL3 edited lines showed an increasing trend, rising by approximately 5.7% compared to the control, while the soluble pectin content decreased significantly, decreasing by approximately 8.6% compared to the control. The GmPL3 overexpression lines, however, showed the opposite trend.

Claims

1. The application of the GmPL3 gene in regulating soybean resistance to gray leaf spot, pectin lyase activity, and total pectin content, characterized in that... The GmPL3 gene is shown in SEQ ID NO.

16.

2. The application of a recombinant vector containing the GmPL3 gene targeting sequence in improving soybean resistance to gray leaf spot and total pectin content, and reducing pectin lyase activity, characterized in that... The GmPL3 gene is shown in SEQ ID NO.16; the targeting sequence is shown in SEQ ID NO.1-3.

3. The application according to claim 2, characterized in that, The launch carrier is pYLCRISPR / Cas9-MT.

4. The application of recombinant host cells containing the recombinant vector of claim 2 in improving soybean resistance to gray leaf spot and total pectin content, and reducing pectin lyase activity, characterized in that, The GmPL3 gene is shown in SEQ ID NO.

16.

5. The application according to claim 4, characterized in that, The host cells are Escherichia coli or Agrobacterium.

6. The application of soybean plants containing the GmPL3 gene knockout in improving soybean resistance to gray leaf spot disease and total pectin content, and reducing pectin lyase activity, characterized in that... The GmPL3 gene is shown in SEQ ID NO.

16.

7. A breeding method for improving soybean resistance to gray leaf spot disease and total pectin content while reducing pectin lyase activity, characterized in that, The steps of the method are as follows: Step 1: Amplify the sgRNA of the GmPL3 gene, the sgRNA sequence of which is shown in SEQ ID NO.1-3, and then ligate it into a CRISPR vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfect recombinant Agrobacterium into soybean callus and culture to obtain transgenic soybeans.

8. The breeding method according to claim 7, characterized in that, The primer pair for amplifying sgRNA in step 1 is shown in SEQ ID NO. 4-7.

9. The breeding method according to claim 7, characterized in that, In step 1, the CRISPR vector is pYLCRISPR / Cas9-MT.

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