Medicago truncatula ap2 / erf transcription factor erm1 and applications thereof

Abstract

The application discloses a sequence of Medicago truncatula AP2 / ERF transcription factor ERM1 and application of the sequence in improving mycorrhizal colonization efficiency of leguminous plants and promoting mycorrhizal symbiosis, and relates to the technical field of plant genetic engineering. ERM1 The gene has a significant promoting effect on the symbiosis of alfalfa and arbuscular mycorrhizal fungi, and the application has important significance for clarifying the biological function of ERM1 in the symbiosis of leguminous plants and mycorrhizal fungi, thereby regulating the symbiosis between plants and mycorrhizal fungi, and provides a research foundation and scientific basis for improving the symbiosis efficiency of leguminous crops by using biological strengthening means.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant genetic engineering technology, and particularly relates to application of AP2 / ERF transcription factor ERM1 in promoting plant and cluster root symbiosis. BACKGROUND

[0002] Arbuscular mycorrhiza (AM) is an important and universal mutual symbiosis formed by arbuscular mycorrhizal fungi (AMF) of Glomeromycota and plants (Redecker et al., 2000), and nearly 90% of terrestrial plants can interact with AM fungi to form a symbiotic system. During the mycorrhizal symbiosis, efficient exchange of nutrients occurs between plants and fungi: through symbiosis with AM fungi, plants expand the root absorption surface area to obtain more mineral nutrients such as phosphorus and nitrogen from the soil (Parniske, 2008; Smith et al., 2009); at the same time, plants transfer nearly 20% of the photosynthetic fixed carbon source to AM fungi for their growth needs (Smith and Read, 2008; Wang et al., 2017). The hyphae of AM fungi highly branch after penetrating the epidermal cells of plants to reach the internal cortical cells, forming arbuscules, thereby providing a large interface for nutrient exchange. Studies have shown that the main form of carbon source provided by plants (Medicago truncatula) for symbiotic AM fungi (arbuscular mycorrhizal fungi) is fatty acids, and fatty acid molecules can be directly transferred to AM fungi through the ABCG transporter protein STR of plants (Jiang et al., 2017; Luginbuehl et al., 2017). Therefore, studying the molecular mechanism of fatty acid nutrient transport and efficient utilization during the mycorrhizal symbiosis process has important significance for maintaining the carbon and nitrogen balance of the ecological system and the development of agricultural production (Oldroyd and Downie, 2008; Parniske, 2008).

[0003] Transcriptional Factors (TFs) are DNA-binding proteins that specifically recognize and bind to cis-acting elements in the promoter region of genes, thereby activating or inhibiting the transcription and expression of downstream genes under specific spatiotemporal conditions. AP2 / ERF transcription factors are a family of plant-specific transcription factors that have a very conserved DNA-binding domain and can specifically bind to ERE, GCC-box and other cis-elements in the promoter sequence of target genes. They are involved in the processes of plant growth and development, biotic / non-biotic stress response gene expression and signal transduction. In the symbiosis of alfalfa-arbuscular mycorrhizal fungi, AP2 / ERF transcription factor WRI5a is reported to directly regulate the expression of STR as a key factor for bidirectional exchange of carbon and phosphorus nutrition, affecting the expression of genes related to the entire fatty acid metabolic pathway and phosphorus transport (Jiang et al., 2018). Although there are multiple AP2 transcription factor encoding genes that are significantly up-regulated by AM symbiosis induction in the published alfalfa chip data, including ERM1 (AP2 / ERF transcription factor required for mycorrhization 1), their functions and related molecular mechanisms in symbiosis are still unknown.

[0004] Therefore, mining and identifying AP2 / ERF transcription factors related to mycorrhizal fungal symbiosis from the excellent legume model plant Medicago truncatula and analyzing their regulatory functions in the symbiotic process not only enrich people's scientific theoretical understanding of plant-AM fungal symbiosis, but also promote agricultural variety improvement, ecological environment construction and soil optimization in production practice, which has important significance. SUMMARY

[0005] In order to make up for the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a Medicago truncatula AP2 / ERF transcription factor ERM1 and its application in improving the mycorrhizal colonization efficiency of legume plants and promoting mycorrhizal symbiosis.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] A Medicago truncatula AP2 / ERF transcription factor ERM1, the DNA sequence of the coding gene of which is the nucleotide sequence shown in SEQ ID NO. 1, and the protein sequence of which is the amino acid sequence shown in SEQ ID NO. 2.

[0008] The application of the Medicago truncatula AP2 / ERF transcription factor ERM1 in improving the mycorrhizal colonization efficiency of legume plants and promoting mycorrhizal symbiosis.

[0009] The application of the AP2 / ERF transcription factor ERM1 of Medicago truncatula in promoting symbiosis of legume plants and mycorrhizal fungi.

[0010] The legume plants include alfalfa, Lotus japonicus, soybean, pea, peanut, bean, mung bean, adzuki bean, broad bean, cowpea, Chinese milk vetch, liquorice and Astragalus.

[0011] The mycorrhizal fungi are fungi, in particular mycorrhizal fungi, and the mycorrhizal roots include natural root systems and hairy roots of the legume plants.

[0012] The application has the following advantages and effects relative to the prior art:

[0013] The application clones the cDNA sequence of the AP2 / ERF transcription factor ERM1 of Medicago truncatula, although the AP2 / ERF gene family to which the gene ERM1 belongs has some members cloned and reported in plants such as Arabidopsis and alfalfa, but the function research of the AP2 / ERF gene family in symbiosis of legume plants and arbuscular mycorrhizal fungi is not very clear. The ERM1 gene of the application has a significant promoting effect on symbiosis of alfalfa and arbuscular mycorrhizal fungi, which has important significance for elucidating the biological function of the ERM1 gene in symbiosis of legume plants and mycorrhizal fungi, thereby regulating symbiosis between plants and mycorrhizal fungi, and provides a research basis and scientific basis for improving symbiosis efficiency of legume crops by using biological strengthening means. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Expression of ERM1 at 3, 7, 14, 21, 28 and 35 days after infection of the mycorrhizal fungi R.irregularis;

[0015] Figure 2 Cellular localization analysis of ERM1 protein;

[0016] Figure 3 Protein structure pattern diagram of ERM1;

[0017] Figure 4 Symbiotic phenotype analysis of ERM1-OE transgenic plants;

[0018] Figure 5 Symbiotic phenotype analysis of ERM1-RNAi mutants. DETAILED DESCRIPTION

[0019] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Unless otherwise defined, the experimental methods in the following examples, which are not specified with specific conditions, are generally in accordance with the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or the conditions suggested by the manufacturer. The various common chemical reagents used in the examples are commercially available products. The primer synthesis and sequencing work are completed by Shenguo (Shanghai) Biotechnology Co., Ltd.

[0020] All the professional and scientific terms used in the present application have the same meanings as those familiar to the skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred implementation methods and materials described in the present application are only for demonstration.

[0021] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Unless otherwise defined, the experimental methods in the following examples, which are not specified with specific conditions, are generally in accordance with the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or the conditions suggested by the manufacturer. The various common chemical reagents used in the examples are commercially available products. The primer synthesis and sequencing work are completed by Shenguo (Shanghai) Biotechnology Co., Ltd.

[0022] Example 1. Response of Medicago truncatula ERM1 gene to arbuscular mycorrhiza

[0023] The sand:perlite ratio of 1:1 hole disc containing or not containing mycorrhizal fungi was prepared, and wild type A17 ecotype alfalfa material (seed obtained from Shanghai Institute of Plant Physiology and Ecology) was planted. At different time periods (3, 7, 14, 21, 28, 35, 42 days) after inoculation with mycorrhizal fungi R. irregularis for infection, the infection efficiency of mycorrhiza was observed under a microscope by ink staining, and the samples of mycorrhizal infection group and non-infection control group were collected, crushed in liquid nitrogen, and total RNA was extracted using Trizol reagent, and cDNA first strand was synthesized using Takara D6110A kit of Takara reagent company, and the specific details are described in the instruction manual. The obtained cDNA fragments were used as templates, M. truncatula Elongation factor 1 (MtEF-1) was used as an internal reference gene, and real-time fluorescent quantitative PCR amplification was performed using the synthesized qRT-PCR forward and reverse primers to detect the mRNA expression of MtEF-1 and alfalfa AP2 / ERF transcription factor coding gene ERM1 (Medtr6g012970).

[0024] The qRT-PCR primer sequences used are as follows:

[0025] EF1-qRT-F: 5'-CTTTGCTTGGTGCTGTTTAGATGG-3';

[0026] EF1-qRT-R: 5'-ATTCCAAAGGCGGCTGCATA-3';

[0027] ERM1-qRT-F: 5'-CCTGATTTTCGGTTTCTCGACG-3';

[0028] ERM1-qRT-R: 5'-TGCTCTTCCATTTCCTCAGCA-3'.

[0029] The PCR reaction system is as follows:

[0030]

[0031]

[0032] The PCR amplification conditions are as follows:

[0033] The results show that with the increase of mycorrhizal infection efficiency at different time points, the expression amount of the alfalfa AP2 / ERF transcription factor coding gene ERM1 is strongly induced (see Figure 1 ). The above results show that the alfalfa AP2 / ERF transcription factor ERM1 plays an important role in the process of mycorrhizal symbiosis.

[0034] Example 2. Cloning of Medicago truncatula ERM1 gene and construction of RNAi and overexpression vectors

[0035] In the present application, the cDNA of wild-type R108 leaf of alfalfa is used as a template, the high-fidelity DNA polymerase Hieff Gold High Fidelity DNA Polymerase of Yisen Biotechnology Co., Ltd. is used to design and synthesize PCR forward and reverse primers to amplify the full-length sequence 1236bp of the ORF of the ERM1 gene. After the target fragment is cloned, the Hieff OneStep Cloning Kit kit is used to insert the ERM1 gene sequence into the intermediate vector of pENTR / SD / D-Topo and pDONOR-Zeo (purchased from Invitrogen Co., Ltd.) by recombination exchange method, and the positive clones are screened after the E. coli DH5α is transformed, the plasmid is extracted and sequenced, and the results are shown in SEQ ID NO. 1.

[0036] The PCR primer sequences used are as follows:

[0037] MtERM1-CDS-F: 5'-ATGGCAAGGAAGAGAAAGGTTTCTGAAGCA-3';

[0038] MtERM1-CDS-R: 5'-CCTAATTGAATGAACCATTTTCAAGTAAGTT-3';

[0039] The PCR reaction system is as follows:

[0040]

[0041]

[0042] The PCR amplification conditions are as follows:

[0043]

[0044] Example 3. ERM1 protein is located in the nucleus

[0045] The CDA sequence of ERM1 connected to pDONOR-Zeo is constructed into the pGWB441 vector through the LR reaction, and the correct 35S::ERM1-EYFP plasmid is obtained. The transient expression system of tobacco leaf infected by GV3101 agrobacterium is used to analyze the location of ERM1 protein. The fluorescence confocal microscope observation results are shown in Figure 2 As shown in A, the YFP location signal in the nucleus of the epidermal cells of tobacco leaves can be detected.

[0046] The Medicago truncatula wild type R108 seeds are polished with sandpaper and then surface sterilized, and then germinated on a 1% agar plate. At the same time, the constructed ERM1-pGWB441 vector plasmid and the empty EV plasmid are transformed into the Agrobacterium tumefaciens strain by electroporation, and cultured at 28°C for 2 days. Single colonies are inoculated into 5ml TY medium containing the corresponding antibiotics, and cultured at 28°C overnight. The next day, the agrobacterium is inoculated into 100ml TY medium containing the corresponding antibiotics at a ratio of 1:50, and cultured for 10-16h until OD600=0.6-0.8. The bacterial cells are collected by centrifugation at 4000rpm, and then resuspended in 5ml TY medium without antibiotics for standby. The 12-18h germinated alfalfa seedlings are cut off 3-5mm from the root tip, and then immersed in the above resuspended bacterial solution. The soaked seedlings are transferred into FP medium in turn, and cultured vertically and under light at 22°C. After 7 days, the roots grown near the radicle are completely cut off, and then the seedlings are transferred into Mod FP medium in turn. The seedlings are cultured vertically and under light at 22°C for 3-4 weeks.

[0047] The EV and 35S::ERM1-EYFP transgenic materials are obtained by alfalfa hairy root transformation, and the hairy root materials are observed by fluorescence confocal microscope, as shown in Figure 2 B, ERM1 is also located in the nucleus in alfalfa root cells.

[0048] As shown in Figure 3 The protein domain analysis of ERM1 protein showed that ERM1 protein contains an AP2 domain, belonging to AP2 / ERF class transcription factors. The above results showed that ERM1 gene encodes AP2 / ERF class transcription factors.

[0049] Example 4. Overexpression of ERM1 significantly increases the level of mycorrhizal symbiosis

[0050] The ERM1 gene was constructed from the pENTR intermediate vector to the pK7WG2R plant expression vector through the Gateway LR reaction, and the positive clones were screened to extract the plasmid for sequencing comparison. The pK7WG2R-ERM1 vector plasmid with correct sequencing was obtained. The overexpression material of ERM1 was obtained by transforming alfalfa hairy roots, which was recorded as ERM1-OE. The homozygous strain obtained by transforming the pBGWFS7 empty vector was recorded as Empty Vector, abbreviated as EV.

[0051] After 6 weeks of mycorrhizal infection, the colonization rate of cluster fungi was observed and counted under a microscope by ink staining, and the hyphal infection was observed by WGA staining. As shown in Figure 4 A, the mycorrhizal infection efficiency of ERM1-OE transgenic plants 42 days after infection with R. irregularis was significantly higher than that of empty control EV-OE, as shown in Figure 4 B, the root cells of ERM1-OE transgenic plants showed more green fluorescent signal of hyphae 42 days after infection with R. irregularis than the empty control plants. The above results showed that ERM1 (Medtr2g014300) played an important role in the positive regulation of symbiosis. Overexpression of ERM1 could significantly promote the process of mycorrhizal symbiosis.

[0052] Example 5. RNAi of ERM1 inhibits mycorrhizal symbiosis

[0053] The designed ERM1-RNAi-1 and ERM1-RNAi-2 primers were used to clone the RNAi target sequence of ERM1 (as shown in Figure 5 A) using ERM1 cDNA as a template, and then constructed into the pENTR / SD / D-Topo intermediate vector, and then constructed into the pK7GWIWGIIR vector through the LR reaction.

[0054] The PCR primer sequences of ERM1-RNAi-1 and ERM1-RNAi-2 used are as follows:

[0055] RNAi-1-F: 5'-AAGAAGGAGCCCTTCACCATGGCAAGGAAGAGAAAGG-3';

[0056] RNAi-1-R: 5'-GTCGGCGCGCCCACCCTTATTTCGACGAAGAAAAGGAA-3';

[0057] RNAi-2-F: 5'-AAGAAGGAGCCCTTCACCAGAGGAGCGTAGGGAGCAAC-3';

[0058] RNAi-2-R: 5'-GTCGGCGCGCCCACCCTTAGGTGAGTAGTAACTAGTTG-3';

[0059] The pK7GWIWGIIR-ERM1 expression vector was then used to obtain RNAi transformed root material of the corresponding gene in wild type A17 by Agrobacterium rhizogenes mediated alfalfa hairy root transformation. As shown in Figure 5 B and Figure 5 C, after 6 weeks of mycorrhizal infection, the colonization rate of the cluster fungus was observed under a microscope by ink staining, and the hyphal infection was observed by WGA staining. The results were similar to those of erm1. The root cells of the ERM1-RNAi mutant plants could not form a complete cluster mycorrhizal structure, and the colonization rate of the branches, the colonization rate of the hyphae in the roots, and the total colonization rate were significantly lower than those of wild type A17. The above phenotype analysis results show that ERM1 does indeed play an important regulatory function in the symbiotic process, affecting the colonization of the fungus in the plant roots and the formation and development of the cluster roots. In summary, the inventions illustrated in the present application can be appropriately implemented without the presence of any elements, limitations not specifically disclosed herein. Therefore, for example, the terms "comprise / comprising" and the like should be understood as open-ended and non-limiting. In addition, the terms and expressions used in the present application are used as descriptive terms and not as limiting terms, and such terms and expressions are not intended to exclude any equivalent features shown and described from the scope claimed by the present application, but it should be recognized that various modifications can be made within the scope claimed by the present application. Therefore, it should be understood that although the present application has been specifically disclosed by preferred embodiments and optional features, those skilled in the art can employ modifications and variations of the present application embodied therein, and such modifications and variations are considered to be within the scope of the present application.

Claims

1. The application of the alfalfa AP2 / ERF transcription factor ERM1 in promoting mycorrhizal symbiosis between alfalfa and arbuscular mycorrhizal fungi, characterized in that, The nucleotide sequence of ERM1 is shown in SEQ ID NO.1; the protein sequence of ERM1 is shown in SEQ ID NO.2.

Images