T6SS effect protein and application thereof in promotion of soybean root nodule formation and yield increase

Abstract

The invention discloses a T6SS effect protein and application of the T6SS effect protein in promotion of soybean root nodule formation and yield increase. The amino acid sequence of the effect protein is SEQ ID No.1. The problems that an existing rhizobium inoculant is low in nodulation efficiency and unclear in growth-promoting bacterium action mechanism are solved, recombinant microorganisms expressing genes of the T6SS effect protein are constructed, and the recombinant microorganisms and soybean rhizobium are co-inoculated to rhizosphere, so that (1) the number of effective root nodules of soybeans is increased to more than two times; (2) the activity of nitrogenase is improved by at least two times; and (3) the yield is increased by more than 1.5 times under the greenhouse condition.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to a T6SS effector protein and its application in promoting soybean nodule formation and increasing yield. Background Art

[0002] As an important economic and oilseed crop, soybeans are a major source of edible vegetable oil and plant protein, with enormous production potential worldwide. As a nitrogen-intensive crop, soybeans derive their nitrogen from three primary sources: soil, fertilizer, and symbiotic nitrogen fixation. Symbiotic nitrogen fixation can provide 70% of the total nitrogen required for soybean growth, and even up to 85% of the nitrogen required for soybean seeds. Therefore, symbiotic nitrogen fixation is a key factor in determining high soybean yields.

[0003] Although numerous studies have explored the effects of rhizobium inoculation on legume growth, nitrogen fixation efficiency, and stress tolerance, the application of a single rhizobium inoculant in various soil environments still faces numerous limitations. For example, factors such as poor environmental adaptability, suppression of the host immune response, and competition among bacteria can ultimately lead to unstable inoculation responses and failure to achieve the desired results.

[0004] Therefore, the strategy of co-inoculating rhizobia and beneficial microorganisms is expected to improve its application effect. The present invention selects Pseudomonas fluorescens as a synergistic strain based on its unique triple biological characteristics: strong rhizosphere colonization ability, immune regulation function and nutritional synergistic effect. Although the co-inoculation of Pseudomonas and rhizobia has been reported, there are two major unsolved problems in the existing technology: 1. The molecular mechanism is unclear: existing research only stays at phenotypic observation, lacks analysis of key effector proteins and their action pathways; 2. Insufficient broad spectrum: known nodulation-promoting strains are mostly targeted at specific rhizobia and cannot be applied across strains. Summary of the Invention

[0005] Based on the above reasons, the present invention proposes a T6SS effector protein and its application in promoting soybean nodule formation and increasing yield. Specifically, to achieve the objectives of the present invention, the present invention intends to adopt the following technical solutions:

[0006] On the one hand, the present invention relates to a T6SS effector protein, whose amino acid sequence is SEQ ID No. 1, specifically: MTVEYHFPVSSTATPARVDGVVIGVLLDVPGADAPVVAFPGCPGETGLVARTTTPLAREDIGAQVALMFEAGDLTRPLVIGRIQRLPQTATPAVAHLDGERLEFTAEREIVLRCGKASITLTREGKVLIRGTYLSNRSSGVNRIKGGSVQIN.

[0007] Another aspect of the present invention relates to a recombinant microorganism, characterized in that it comprises a nucleotide sequence encoding a protein of SEQ ID NO: 2, specifically:

[0008] ATGACCGTTGAATATCATTTCCCCGTTTCCTCTACCGCCACGCCCGCGCGTGGATGGCGTCGTGATTGGGGTGCTGCTGGATGTGCCCGGGGCAGATGCCCCGGTGGTGGCCTTCCCTGGCTGCCCCGGCGAAACGGGACTCGTCGCGCGCACCACCACCCCGCTCGCCCGTGAAGACATCGGCGCCCAGGTCGCACTGATGTTCGAGGCCGGCGATTTGACCCGGCC GCTGGTGATCGGTCGCATCCAGCGCTGCCGCAAACCGCCACGCCAGCCGTCGCCCACCTGGACGGCGAGCGCTTGGAGTTCACCGCAGAGCGGGAAATCGTCCTGCGCTGCGGTAAGGCGAGCATTACCCTCACGCGTGAGGGCAAGGTGCTGATCCGTGGGACCTATCTTTCGAACCGGTCATCCGGCGTGAACCGCATCAAGGGCGGTTCGGTGCAGATCAACTAG.

[0009] In a preferred embodiment of the present invention, the recombinant microorganism is obtained by introducing the gene of the effector protein into a microorganism of the genus Pseudomonas, Burkholderia or Bacillus.

[0010] Another aspect of the present invention also relates to the use of the above-mentioned T6SS effector protein in promoting soybean nodule number, nitrogenase activity and / or leghemoglobin content.

[0011] Another aspect of the present invention relates to a method for increasing soybean yield, comprising applying the above-mentioned recombinant microorganism during the nodulation stage.

[0012] The present invention solves the problems of low nodulation efficiency and unclear mechanism of action of growth-promoting bacteria in existing rhizobium inoculants. By constructing a recombinant microorganism expressing the gene of the effector protein and co-inoculating it with soybean rhizobia in the rhizosphere, the following results are achieved: (1) the number of effective soybean nodules is increased by more than two times; (2) the nitrogenase activity is increased by at least two times; and (3) the yield under greenhouse conditions is increased by more than 1.5 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1:T6SS effector protein TepN can increase the number of rhizobia nodules and nitrogenase activity;

[0014] Figure 2 :Heterologous expression of the T6SS effector protein TepN enables Pseudomonas to promote rhizobium nodulation;

[0015] Figure 3 :The T6SS effector protein TepN has universal effects;

[0016] Figure 4 :T6SS effector protein TepN can increase soybean yield under greenhouse conditions. DETAILED DESCRIPTION

[0017] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0018] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0019] Example 1:

[0020] Soybeans were co-inoculated with wild-type Pseudomonas fluorescens and a mutant strain lacking the T6SS effector protein TepN, along with a rhizobium strain. The specific procedures are as follows: Soybean growth medium preparation: A mixture of perlite and vermiculite in a 2:1 ratio was used as the soybean growth medium. The mixed medium was dispensed into 700 mL fungus bags. Each bag was then filled with 400 mL of nitrogen-free nutrient solution and secured with cotton string. The filled bags were autoclaved at 121°C for 2 hours. Soybean seed pretreatment: In a biosafety cabinet, soybean seeds were shaken and rinsed 2-3 times with sterile distilled water. Next, the seeds were shaken with 75% ethanol for 30 seconds and then rinsed four times with sterile distilled water. The seeds were then treated with 50% sodium hypochlorite for 3 minutes, with continuous shaking. Finally, the seeds were shaken and rinsed six times with sterile distilled water. After processing, use sterilized tweezers to place the seeds into sterilized fungus bags, two per bag, and tie with cotton thread approximately 2 cm from the bag opening. Soybean planting and cultivation: Transfer the planted fungus bags to an artificial climate chamber. The climate chamber environmental parameters are set as follows: daytime temperature 25°C, daily light duration 16 hours; nighttime temperature 21°C; air relative humidity maintained at 80%. After the soybean seedlings have grown for one week, screen the seedlings in each fungus bag, remove the one in poor condition, retain one, and untie the fungus bag to allow the seedlings to continue growing. Rhizobium and Pseudomonas fluorescens co-inoculation treatment: Strain activation and culture: Rhizobium: The rhizobium strain is streaked on TY solid culture medium for activation, and a single colony is selected and inoculated into TY liquid culture medium. Culture is incubated at 30°C with shaking at 200 rpm until the bacterial solution reaches logarithmic growth. Pseudomonas fluorescens: After activating Pseudomonas fluorescens by streaking on LB solid medium, pick a single colony and inoculate it into LB liquid medium. Also culture at 30°C and 200 rpm with shaking until the logarithmic growth phase. The cultured rhizobia and Pseudomonas fluorescens bacterial suspensions were centrifuged at 5000 rpm for 5 minutes to collect the cells. The collected cells were washed twice with sterile distilled water, resuspended, and diluted to OD 600≈0.2. When the first two true leaves of the soybean seedlings are fully expanded, the fungus inoculation is carried out. A mixed bacterial solution is prepared according to the volume ratio of Pseudomonas fluorescens to Rhizobium at 6:1, and 10 mL of the mixed bacterial solution is applied to each fungus bag. At the same time, a control treatment is set up: the Blank group is irrigated with 10 mL of sterile distilled water; the Rhizobium group is only irrigated with Rhizobium solution. To ensure the stability of nutrition and water during the growth period of soybeans, all plants are supplemented with 50 mL of sterile nitrogen-free nutrient solution every 5 days. After 28 days of cultivation, the soybean plants are removed, the nodules formed on the roots are counted and counted, and the nitrogenase activity of the nodules is determined by the acetylene reduction method, and the content of nodule leghemoglobin is determined by spectrophotometry. The specific experimental method is as follows: the collected soybean plants are removed from the substrate, cleaned, and dried with absorbent paper, and photographed to record the morphology. The number of nodules on each soybean plant was counted, and one nodule was randomly broken open to identify a valid nodule and photographed. A 0.2 g sample of fresh soybean nodules was placed in a 20 mL penicillin vial, which was capped. A microinjector was used to inject 200 μL of acetylene gas into the vial, and the vial was sealed with film to prevent leakage. After the penicillin vial was allowed to react at room temperature for 1–2 h, 100 μL of the gas in the vial was aspirated using a microinjector for gas chromatography analysis. Nitrogenase activity was calculated based on the measured ethylene concentration. Fresh soybean nodules were ground into a powder in liquid nitrogen and mixed with phosphate buffer (0.1 M, pH 6.8) at 4°C. The amount of phosphate buffer used was approximately four times the nodule volume. The mixture was centrifuged at 100 g for 15 min at 4°C. The pellet was discarded, and the supernatant was centrifuged at 21,460 g for 20 min at 4°C. The resulting supernatant was measured spectrophotometrically at 540 nm. A standard curve was prepared using bovine hemoglobin as the standard protein, and the leghemoglobin content was calculated based on the fresh weight.

[0021] Among them, Q8 is the treatment of single inoculation with rhizobia; WT-Q8 is the treatment of co-inoculation with wild-type Pseudomonas fluorescens and rhizobia; ΔtepN is the treatment of co-inoculation with Pseudomonas fluorescens that lacks the tepN gene and rhizobia. Figure 1 The experimental results showed that the mutant strain lacking the T6SS effector protein TepN significantly reduced the number of nodules and nitrogenase activity (see Figure 1 AC). Furthermore, leghemoglobin content was measured (see Figure D). The results showed that leghemoglobin content in soybean roots was significantly reduced in strains lacking the T6SS effector protein TepN, and leghemoglobin gene expression was also significantly decreased compared to the wild-type strain, further demonstrating the role of the T6SS effector protein TepN in promoting nitrogen fixation in soybeans.

[0022] In order to further verify the direct role of T6SS effector protein TepN in promoting rhizobium nodulation, some Pseudomonas that originally did not have the ability to promote rhizobium nodulation were selected, and the TepN protein was heterologously expressed in them, with the empty expression vector as a control. The specific method is: by double-enzyme digestion of the expression plasmid pBBRMCS-5, and the amplified tepN gene was subjected to the same double-enzyme digestion treatment, the two were connected with ligase, and transformed into Escherichia coli competent cells, and then the positive clones were verified by colony PCR, plasmid extraction was performed, and the correctness of the recombinant vector was verified by small-scale enzyme digestion. Pick a fresh wild-type strain of Pseudomonas and inoculate it into 3mL of LB culture medium; transfer it to another 5mL of LB liquid at a ratio of 1:100, and shake at 30℃ and 200rpm to OD 600 About 0.6; 4℃, 4500rpm centrifugation for 3min to collect bacteria; wash the bacteria repeatedly 2-3 times with 300mM sucrose that was pre-cooled after sterilization; resuspend the bacteria with 300mM sucrose for the last time, divide it into packages, and prepare the competent cells for electroporation immediately; add the recombinant plasmid pBBRMCS-5-tepN and the empty plasmid pBBRMCS-5 to the competent cells respectively, after ice bath for 5min, transfer all to the electroporation cup, add 800μL of antibiotic-free LB liquid to recover after electroporation, collect the bacteria after 2h and spread them on the LB solid plate containing the corresponding antibiotics, and place them at 30℃ for culture. After the electroporated colonies grow, inoculate them with LB containing relevant antibiotics. This bacteria is the heterologous expression and control strain. According to the above-mentioned co-inoculation method, soybeans were co-inoculated with heterologous expression-related strains and rhizobia-related strains. The experimental results showed that heterologous expression of TepN protein can enable these pseudomonads to acquire the ability to promote rhizobia nodulation and nitrogen fixation (see Figure 2 AC).

[0023] Next, we selected two other rhizobia that matched Huang 13 in soybeans and co-inoculated soybeans with Pseudomonas fluorescens at a ratio of 1:6 according to the above rhizobia treatment method. Similarly, after 28 days of cultivation, we took out the soybean plants, counted the nodules formed on the roots, and measured the nitrogenase activity of the nodules using the acetylene reduction method. The results showed that the T6SS effector protein TepN can also promote nodulation, indicating that it has universality ( Figure 3 AC).

[0024] In order to detect the potential of T6SS effector protein TepN to increase soybean yield, soybeans were co-inoculated with fluorescent Pseudomonas and rhizobia in sterilized substrates and natural soils. The natural soil was selected from 8 plots in Caoxinzhuang, Yangling Demonstration Zone, Shaanxi Province, which is the natural planting soil for soybeans. During the experiment, the soybean plants were treated strictly according to the standard sterilization process. After 8 weeks of continuous cultivation in a greenhouse environment, the number of beans per plant in different treatment groups was counted; after the soybeans matured, the 100-grain weight of the beans was measured, which was used as a yield evaluation indicator. The experimental results showed that under both sterilized substrate and natural soil planting conditions, the number of beans per plant in the wild-type fluorescent Pseudomonas treatment group was significantly higher than that in the TepN mutant treatment group; during the soybean maturity period, the 100-grain weight of the beans in the wild-type fluorescent Pseudomonas treatment group also increased significantly. This shows that in a greenhouse planting environment, TepN has the potential to significantly increase soybean yield ( Figure 4 AE).

[0025] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A T6SS effector protein, whose amino acid sequence is SEQ ID No. 1, specifically: MTVEYHFPVSSTATPARVDGVVIGVLLDVPGADAPVVAFPGCPGETGLVARTTTPLAREDIG AQVALMFEAGDLTRPLVIGRIQRLPQTATPAVAHLDGERLEFTAEREIVLRCGKASITLTREGK VLIRGTYLSNRSSGVNRIKGGSVQIN.

2. A recombinant microorganism, characterized in that The nucleotide sequence encoding the T6SS effector protein is SEQ ID NO:

2. 3 . The recombinant microorganism according to claim 2 , wherein the recombinant microorganism is obtained by introducing the tepN gene into a microorganism of the genus Pseudomonas, Burkholderia or Bacillus.

4. Use of the T6SS effector protein according to claim 1 in promoting the number of soybean nodules.

5. Use of the T6SS effector protein according to claim 1 in promoting soybean nitrogenase activity.

6. Use of the T6SS effector protein according to claim 1 in promoting the content of soybean leghemoglobin.

7. Use of the T6SS effector protein according to claim 1 in simultaneously promoting soybean nodule number, nitrogenase activity and leghemoglobin content.

8. A method for increasing soybean yield, comprising administering the recombinant microorganism according to claim 2 or 3 during the nodulation stage.

Images