Use of erf15 gene in regulating plant arbuscular mycorrhizal symbiosis

Abstract

The application discloses application of an ERF15 gene in regulating and controlling plant arbuscular mycorrhizal symbiosis and belongs to the technical field of biotechnology.The nucleotide sequence of the protein coding region of the ERF15 gene is shown in SEQ ID NO.1 or has at least 70% homology with the sequence shown in SEQ ID NO.1 and the coded protein is functionally equivalent.The application discloses, for the first time, positive regulation of the ERF15 gene in the plant arbuscular mycorrhizal symbiosis, analyzes the molecular mechanism of the ERF15 in promoting phosphorus absorption through the mycorrhizal symbiosis pathway, and provides gene resources and a new idea for plant molecular stress resistance genetic breeding and the like.The application has very important practical value for improving the absorption and utilization efficiency of plants on phosphorus elements, reducing the use of phosphorus fertilizer, and improving the utilization of infertile soil and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of ERF15 gene in regulating plant arbuscular mycorrhizal symbiosis and improving plant phosphorus uptake. BACKGROUND

[0002] Phosphorus is one of the essential macroelements for plant growth and development, and is also an indispensable element for high yield and high efficiency in modern horticultural production. However, due to its low solubility and mobility in soil, it is a limiting ion in many environments. Studies have shown that phosphorus deficiency leads to yield loss of various crops ranging from 25% to 60%. To solve this problem, quick-acting phosphorus fertilizer is regularly applied in agricultural production, but only 10% to 25% of the phosphorus is utilized by plants, resulting in a low input-output ratio, and excessive use can damage the soil environment and cause soil eutrophication.

[0003] To cope with the limitation of phosphorus, plants have evolved a set of adaptive strategies, i.e., forming a mutually beneficial symbiotic relationship with arbuscular mycorrhizal fungi, known as arbuscular mycorrhizal symbiosis. Studies have shown that in low-phosphorus or even phosphorus-deficient soil environments, the contribution rate of mycorrhizal symbiosis to plant phosphorus uptake can reach 50% to 75%, and even as high as 90%. Therefore, clarifying the regulation mechanism of arbuscular mycorrhizal symbiosis is crucial for reducing the use of chemical fertilizers and improving crop production efficiency.

[0004] Strigolactones (SLs) are a new type of plant hormone produced in plant roots. When phosphorus or nitrogen is deficient, the biosynthesis and secretion of strigolactones are strongly induced, which can promote the germination of arbuscular mycorrhizal fungal spores, branching and elongation of hyphae, thereby increasing the chances of contact with the host. Currently, several enzymes have been confirmed to be involved in the biosynthesis of strigolactones, including β-carotene isomerase D27 (Dwarf 27), carotenoid cleavage dioxygenase (CCD7) (MAX3 / RMS5 / HTD1 / D17) and CCD8 (MAX4 / RMS1 / DAD1 / D10), and cytochrome P450 monooxygenase (Cytochrome P450, MAX1). Although the role of strigolactones in arbuscular mycorrhizal symbiosis has been widely reported, the regulation mechanism of strigolactones remains to be further explored.

[0005] The colonization of plant roots by arbuscular mycorrhizal fungi is accompanied by dramatic local and systemic transcriptional changes. Recent studies have shown that the expression of genes of a series of transcription factors such as GRAS family, AP2 / ERF family and MYB family is significantly up-regulated in the symbiotic process of arbuscular mycorrhizal fungi with crops such as alfalfa, Lotus japonicus, tomato and sunflower. These transcription factors are not only specifically regulated by arbuscular mycorrhizae, but also participate in the hormone or other molecular signal pathways and regulate the resistance of plants to abiotic stress. AP2 / ERF transcription factors are a large class of transcription factors mainly existing in plants, which are mainly divided into four subfamilies. Studies have reported that the AP2 subfamily is closely related to the synthesis of fatty acids. The ERF subfamily genes mainly participate in various hormone signal pathways and play an important role in the growth and development of plants and stress resistance. In addition, previous studies have also pointed out that the ERF family genes respond to mycorrhizal symbiosis (Identification and expression analysis of GRAS transcription factor genes involved in the control of arbuscular mycorrhizal development in tomato. Front. Plant Sci. 2019, 10, 268.), but the specific mechanism is still unclear and needs further study.

[0006] Tomato (Solanum lycopersicum L.) belongs to Solanaceae crops and is one of the most widely planted vegetables in the world. Due to its small genome, relatively short growth cycle, high genetic diversity, easy transgenic and cultivation, and the completion of whole genome sequencing and the development of molecular genetics research, tomato has become one of the classic model plants in the field of plant research. Therefore, it is of important theoretical and practical significance to explore the molecular mechanism of plant symbiosis with arbuscular mycorrhizal fungi and to improve the absorption of plants to phosphorus by taking tomato as the object. SUMMARY

[0007] The purpose of the present application is to explore the genes involved in the regulation of arbuscular mycorrhizal symbiotic mechanism from the tomato genome, and to apply them to the cultivation of transgenic plants by using biological technology means, so as to promote the symbiotic effect of plants and arbuscular mycorrhizal fungi and improve the absorption of plants to nutrients, especially phosphorus.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The application provides application of an ERF15 gene in regulating plant arbuscular mycorrhizal symbiosis, wherein a nucleotide sequence of a protein coding region of the ERF15 gene is shown as SEQ ID NO. 1 or has at least 70 % homology with the sequence shown as SEQ ID NO. 1 and the coded protein is functionally equivalent.

[0010] The CDS region of the ERF15 (Ethylene-responsive transcription factor 15) gene has a length of 714 bp, and the coded protein is composed of 237 amino acid residues, and the sequence is shown as SEQ ID NO. 2.

[0011] Further, the plant can be but is not limited to tomato.

[0012] Further, the arbuscular mycorrhizal fungus can be but is not limited to Rhizophagus intraradices.

[0013] Further, the ERF15 gene is functionally deficient, and the colonization of the arbuscular mycorrhizal fungus in the plant root system is inhibited.

[0014] The application obtains an ERF15 gene functionally deficient mutant through a gene editing technology and a tissue culture technology, and compared with a wild type plant, the colonization rate of the arbuscular mycorrhizal fungus in the mutant is significantly reduced, and then the absorption of the root system to phosphorus is affected, which indicates that the ERF15 gene plays an important role in the symbiosis of the plant and the arbuscular mycorrhizal fungus.

[0015] The plant hormone strigolactone (SL) plays an important role in the process of plant arbuscular mycorrhizal symbiosis, and carotenoid cleavage dioxygenase 7 (CCD7) and carotenoid cleavage dioxygenase 8 (CCD8) are key enzymes for the synthesis of SL, and mechanism research shows that the protein coded by the ERF15 gene acts on the promoters of the strigolactone synthesis genes CCD7 and CCD8, and activates gene transcription. Specifically, the protein coded by the ERF15 gene can directly bind to special elements on the promoters of CCD7 and CCD8, and promote the transcription of the downstream CCD7 and CCD8 genes. It is shown that the ERF15 gene positively regulates the synthesis of the plant hormone strigolactone.

[0016] Further, the application comprises: up-regulating the expression of the ERF15 gene in the plant body by using a biological technical means, thereby promoting the symbiosis of the plant and the arbuscular mycorrhizal fungus, and then improving the absorption of the plant root system to phosphorus.

[0017] The application has the beneficial effects:

[0018] The application discloses a positive regulation role of ERF15 gene in plant arbuscular mycorrhizal symbiosis for the first time, analyzes a molecular mechanism of ERF15 in promoting phosphorus absorption through a mycorrhizal symbiotic pathway, and proves the use of ERF15 gene in promoting plant arbuscular mycorrhizal symbiosis and improving phosphorus absorption of plants. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a schematic diagram of a gene editing site and amino acid coding change of a T2 generation homozygous mutant plant of SlERF15 gene knockout mediated by CRISPR / Cas9.

[0020] Figure 2 Figure 2 is a trypan blue staining diagram (A) and root colonization rate (B) of an erf15 deletion mutant plant inoculated with arbuscular mycorrhizal fungal spores for 20 days.

[0021] Figure 3 is a content change of strigolactones (SL) in roots of the erf15 deletion mutant plant inoculated (AM) or not inoculated (NM) with arbuscular mycorrhizal fungal spores for 10 days.

[0022] Figure 3 Figure 3 is a content change of strigolactones (SL) in roots of the erf15 deletion mutant plant inoculated (AM) or not inoculated (NM) with arbuscular mycorrhizal fungal spores for 10 days.

[0023] Figure 3 is a content change of strigolactones (SL) in roots of the erf15 deletion mutant plant inoculated (AM) or not inoculated (NM) with arbuscular mycorrhizal fungal spores for 10 days.

[0024] Figure 4 Figure 4 is a luciferase reporter gene detection ratio of SlERF15 protein on transcription of strigolactone synthesis genes SlCCD7 (A) and SlCCD8 (B).

[0025] Figure 5Changes in plant biomass (A) and phosphorus content (B) of erf15 loss-of-function mutant plants inoculated (AM) or not inoculated (NM) with arbuscular mycorrhizal fungal spores 40 days after inoculation;

[0026] where NM represents non-inoculated arbuscular mycorrhizal fungal spores; AM represents inoculated arbuscular mycorrhizal fungal spores. Statistical analysis of data was performed by analysis of variance (ANOVA), and the significance of differences in data was analyzed using the Tukey test, and different letters in the figure represent significant differences in the mean value at P < 0.05. DETAILED DESCRIPTION

[0027] The application will be further described below in conjunction with specific examples, but the application is not limited to the following examples.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used in the experiments can be obtained through commercial channels.

[0029] Example 1: Construction and identification of tomato SlERF15 gene mutant vector

[0030] First, find the full-length DNA sequence of tomato SlERF15 gene (Solyc06g054630) on the SGN website https: / / solgenomics.net / , and input it into the website http: / / crispr.hzau.edu.cn / CRISPR2 / , find a 20bp base sequence before the PAM structure with high onscore score, GC content >40%, and located in the protein coding region as the specific targeting sgRNA of the protein coding region of the gene, as shown in SEQ ID NO. 3.

[0031] After annealing the synthesized target sequence, it is connected to the BbsI site of the AtU6-sgRNA-AtUBQ-Cas9 vector, and then the newly obtained AtU6-sgRNA(SlERF15)-AtUBQ-Cas9 fragment is connected to the Hind III / KpnI site of the pCAMBIA1301 vector, to construct a tomato SlERF15 gene CRISPR expression vector. The above recombinant plasmid is sequenced, and the sequencing is correct for subsequent experiments.

[0032] Example 2: Construction and detection of tomato SlERF15 gene transgenic material

[0033] The gene editing vector pCAMBIA1301:AtU6-sgRNA(SlERF15)-AtUBQ-Cas9 is transformed into Agrobacterium tumefaciens strain EHA105, and tomato cotyledon infection is performed. Through induction of callus, resistance induction differentiation and rooting culture, a tissue culture seedling T0 generation is obtained. A small amount of T0 generation plant leaf is taken to extract genomic DNA, which is used as a template for PCR amplification of the sgRNA-containing DNA fragment. Then, the PCR product is sequenced and compared. Plants with base mutations, deletions or insertions in the sgRNA sequence are self-crossed to obtain T0 generation seeds. The verification primers are as follows:

[0034] Verification primer before: 5'-TCATCATAATAATCCTCTAATTCAATATCC-3' (SEQ ID NO. 4);

[0035] Verification primer after: 5'-GCCCATTTGGACCACCCTA-3' (SEQ ID NO. 5).

[0036] The T0 generation seeds are sown to obtain T1 generation plants. The sgRNA sequence base editing and the presence or absence of Cas9 in the T1 generation plants are detected using the above method. Plants without Cas9 but with sgRNA variation are selected for breeding and self-crossing to obtain one strain of T1 generation homozygous mutant plants without exogenous gene Cas9 and stable inheritance. The gene editing site is as shown in Figure 1 .

[0037] The above strain is further self-crossed and bred. After sowing the seeds, T2 generation plants without exogenous gene Cas9 and with sgRNA variation are obtained.

[0038] The following examples are all based on T2 generation homozygous strains (erf15#1, erf15#3) as materials for experiments.

[0039] Example 3: Phenotypic identification of tomato erf15 mutant plants inoculated with arbuscular mycorrhizal fungal spores

[0040] The arbuscular mycorrhizal fungus (AMF) used in this experiment is Rhizophagus intraradices UT126a (formerly Glomus intraradices) from INVAM. The fungus spores are propagated using corn plants. After three months of low-phosphorus cultivation, the corn root system is harvested and cut into pieces, and mixed with the substrate. Store at 20°C for use.

[0041] Wild-type tomatoes (Condine Red, CR) (WT) and mutant plants (erf15) are divided into two groups, the experimental group and the control group.

[0042] When tomato seedlings grow to three leaves one heart, select the uniform growth of seedlings in a total volume of 600 ml of sterile culture medium (poor soil: sand: vermiculite = 1:1:1, high temperature sterilization), using "sandwich method" to inoculate fungal spores (AM; ~ 400 spores / plant) or not inoculated with fungal spores (NM).

[0043] After transplanting, place it in a day and night temperature of 25℃ / 20℃, light period of 12h / 12h (day / night), relative humidity of 75%, light intensity of 600μmol·m -2 -1 Artificial climate chamber cultivation, every two days irrigation of a phosphorus deficiency Hoagland nutrient solution (1mM KH2PO4 replaced by 1mM KCl).

[0044] After 20 days of treatment of arbuscular mycorrhizal fungal spore inoculation, root colonization phenotype observation and root length colonization rate statistics were carried out.

[0045] Root arbuscular mycorrhizal fungal colonization phenotype observation adopts trypan blue staining method, the specific method is as follows:

[0046] (1) Wash the root system of the plant inoculated with arbuscular mycorrhizal fungal spores for 20 days, cut into 1cm long root segments, and immerse in 10% KOH solution;

[0047] (2) 95℃ water bath for 30-40min (depending on the tenderness of the root), take out and cool;

[0048] (3) Immersed in 2% HCl solution for acidification for 5min, remove the liquid;

[0049] (4) Place the sample in 0.05% trypan blue reagent (trypan blue is dissolved in lactic acid glycerol reagent, w / v), 95℃ water bath for 10min, cool, and pour off the liquid;

[0050] (5) Place the sample in lactic acid glycerol reagent (lactic acid: glycerol: water = 8:1:1) at room temperature to decolorize, time≥24h;

[0051] (6) Randomly select root segments and place them on a grid line slide, observe under a 20x microscope (Leica Microsystems, Germany), and count according to the grid line intersection, and count >300 root segments for each treatment.

[0052] The results of root arbuscular mycorrhizal fungal colonization phenotype and root length colonization rate statistics are as follows Figure 2 .

[0053] ​The results showed that the number of mycelium, arbuscule and vesicle in erf15 mutant plants were significantly reduced compared with WT, indicating that the colonization of arbuscular mycorrhizal fungi in roots was inhibited, suggesting that SlERF15 gene can promote the symbiosis of arbuscular mycorrhizal fungi and tomato.

[0054] Example 4: Effect of tomato SlERF15 gene on the content of plant hormones after inoculation of arbuscular mycorrhizal fungal spores

[0055] Strigolactones (SL) is a new type of hormone, as a signal molecule between fungi and plants, promotes the branching of AMF hyphae, promotes the germination of spores, and ultimately realizes the plant mycorrhizal symbiosis. In view of the important role of SL in arbuscular mycorrhizal symbiosis, we determined the SL content in the roots of WT and erf15 mutant plants after inoculation (AM) and non-inoculation (NM) treatment.

[0056] Tomato root samples were taken 10 days after inoculation with fungal spores, and the content of SL was extracted and determined, and the specific method was as follows:

[0057] (1) The root system of the plant was washed, 0.5 g was weighed and ground in liquid nitrogen, and then transferred to a 2 mL test tube;

[0058] (2) Add 0.5 mL of 40% acetone (acetone: water = 2:3, v / v), shake vortex and mix well;

[0059] (3) 4℃, 8000rpm, 10min, remove the supernatant;

[0060] (4) Add 0.5 mL of 50% acetone, shake vortex and mix well, then put it in 4℃ shaker overnight;

[0061] (5) 4℃, 8000rpm, 5min, collect the supernatant solution in a new test tube;

[0062] (6) Resuspend the precipitate with 0.5 mL of 50% acetone, shake vortex and mix well, then put it in 4℃ shaker for 1h;

[0063] (7) 4℃, 8000rpm, 5min, collect the supernatant solution again and mix well;

[0064] (8) After evaporating acetone at 35℃, the extract was obtained;

[0065] (9) Dissolve the extract in 50% methanol (methanol: water = 1:1, v / v);

[0066] (10) Take 0.1 mL of supernatant for determination, and determine the content of three strigolactones by LC-MS / MS method (Agilent Technologies, California, America). The results of SL content determination are shown inFigure 3 .

[0067] The experimental results show that after 10 days of fungal spore inoculation, the content of strigolactones in the root system of wild type plants increases obviously, but the upward trend in the erf15 deletion mutant is obviously inhibited, indicating that the SlERF15 gene can promote the synthesis of strigolactones.

[0068] Example 5: Verification of the positive regulation of tomato SlERF15 protein on the transcription of strigolactone synthesis genes

[0069] (1) Construction of pGreenII 0029 62-SK vector containing SlERF15 gene: The CDS sequence of SlERF15 was obtained by PCR amplification using tomato cDNA as the template, as shown in SEQ ID NO. 1. The primers are as follows:

[0070] SlERF15-SK-F: 5'-cgctctagaactagtggatccATGGAAAACAATTCTACTCATGATCAG-3' (SEQ ID NO. 6);

[0071] SlERF15-SK-R: 5'-tgatttcagcgaattggtaccAAAGTTCCATAGGAAGGATTGTTGA-3' (SEQ ID NO. 7);

[0072] The above vector was double digested with BamH I and Kpn I restriction enzymes, and then homologous recombination and transformation of E. coli DH5a competent cells were performed.

[0073] (2) Construction of pGreen II 0800-LUC vector containing SlCCD7 (Solyc01g090660) and SlCCD8 (Solyc08g066650) gene promoter sequences: The tomato genomic DNA was used as the template, and the enzyme digestion site was Xho I and Smal. The primers used are as follows:

[0074] pSlCCD7-LUC-F:

[0075] 5'-ggtaccgggccccccctcgagTTGGTCAATACAAATTAAACCCCA-3' (SEQ ID NO. 8);

[0076] pSlCCD7-LUC-R:

[0077] 5'-agaactagtggatcccccgggACAATTGGTCCATGATTATAATTCACA-3' (SEQ ID NO. 9);

[0078] pSlCCD8-LUC-F:

[0079] 5'-ggtaccgggccccccctcgagTGAATATAGCTCACTTATACATAAATTATACTC C-3' (SEQ ID NO. 10);

[0080] pSlCCD8-LUC-R:

[0081] 5'-agaactagtggatccccgggATGGCCGTGGTGCGTTGT-3' (SEQ ID NO. 11).

[0082] (3) Electroporate the correctly sequenced plasmid into Agrobacterium GV3101:psoup competent cells to obtain Agrobacterium strains that can be used for dual-luciferase reporter gene detection experiments.

[0083] (4) The two Agrobacterium cultures were resuspended in an infection solution containing 150 μM acetylsuccinone and adjusted to the optimal concentration (OD). 600 =0.75), and activated in a 28℃ incubator for 3 hours. The mixture of SlERF15-SK:pSlCCD7-LUC / pSlCCD8-LUC at a ratio of 10:1 was then used to co-infect leaves of *Nicotiana benthamiana*. Three days later, the activities of firefly luciferase and *Reniformis luciferase* were detected using a Dual-Luciferase Reporter Assay System from Promega, USA. The fluorescence ratio of the two was calculated in relation to the value of the pGreenII 0029 62-SK empty vector with either SlCCD7 or SlCCD8 promoter (set to 1). The experimental results are shown in […]. Figure 4 .

[0084] Experimental results showed that, compared with the empty vector, the expression of SlERF15 protein activated the promoters of SlCCD7 and SlCCD8 genes, and the LUC / REN ratio was relatively high, indicating that SlERF15 promoted the transcription of SlCCD7 and SlCCD8 genes.

[0085] Example 6: Effects of the tomato SlERF15 gene on plant biomass and phosphorus uptake after inoculation with arbuscular mycorrhizal fungal spores

[0086] Mycorrhizal symbiosis can improve the absorption of phosphorus and promote the accumulation of plant biomass. We measured the biomass and phosphorus content of WT and erf15 mutant plants after inoculation (AM) and non-inoculation (NM) treatment.

[0087] Tomato plant samples were taken 30 days after inoculation with fungal spores, and the biomass and phosphorus content were determined as follows:

[0088] (1) The roots of the plants treated for 30 days were washed clean and dried, and the roots, stems and leaves were collected. They were placed in an oven at 105°C for 30 min and dried at 65°C until constant weight, and weighed;

[0089] (2) The dried samples were ground and mixed, and 0.1 g was taken in a digestion test tube, and 2 mL of concentrated H2SO4 was added for carbonization for 30 min;

[0090] (3) Digestion at 150°C for 1 h, with the addition of 30% H2O2 several times during the process, until the solution was colorless, and then continue to digest for 30 min;

[0091] (4) After cooling, the solution was filtered into a 50 mL test tube, and the volume was adjusted with double distilled water and mixed well;

[0092] (5) Take 5 mL of the solution in a new 50 mL test tube, add appropriate amount of double distilled water, adjust the pH to 7-8, and dilute with double distilled water to 45 mL for determination. The phosphorus content was determined by molybdenum-antimony anti-absorption spectrophotometry (standard number: NY / T 2017-2011).

[0093] Add 5 mL of molybdenum-antimony anti-coloring agent to each sample to 50 mL, and develop color in the dark for 30 min, and measure the absorbance value: take 200 μL of the developed solution to measure the absorbance value at 700 nm wavelength.

[0094] Calculation formula:

[0095]

[0096] Where c = the concentration calculated according to the formula mg / L; V3 = the total volume of the developed solution mL; V2 = the total volume of digestion mL; V1 = the volume added to the developed solution mL; m = the weight of digestion g.

[0097] The experimental results Figure 5 ) show that mycorrhizal symbiosis can promote the absorption of phosphorus in wild-type plants, increase the accumulation of phosphorus content and biomass, but the absence of SlERF15 gene hinders mycorrhizal symbiosis and weakens the effect of mycorrhizal symbiosis, resulting in significantly lower phosphorus content and biomass than wild-type plants.

[0098] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that such changes and modifications be included within the scope of the application as defined by the appended claims.

Claims

1. Use of an ERF15 gene in modulating plant arbuscular mycorrhizal symbiosis, characterized in that, The nucleotide sequence of the protein coding region of the ERF15 gene is shown as SEQ ID NO. 1, and the application comprises: up-regulating the expression of the ERF15 gene in the plant body by using biological technical means, thereby promoting the symbiosis of the plant and the arbuscular mycorrhizae, and further improving the absorption of the plant root system to phosphorus; and the plant is tomato.

2. Use according to claim 1, wherein The amino acid sequence of the protein encoded by the ERF15 gene is shown as SEQ ID NO.

2.

3. The use according to claim 1, wherein The arbuscular mycorrhizal fungus is Rhizophagus intraradices.

4. The use according to claim 1, wherein The ERF15 gene positively regulates the synthesis of the plant hormone strigolactone.

5. The use according to claim 4, wherein the compound is ###0002### The protein encoded by the ERF15 gene acts on the promoters of strigolactone synthesis genes CCD7 and CCD8, and activates gene transcription.

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