Rhizobium inducible promoter LjPUB7pro and application thereof

Abstract

The invention belongs to the field of plant biotechnology and genetic engineering, and particularly relates to a rhizobium inducible promoter LjPUB7pro and application thereof. The invention provides a brand new rhizobium inducible promoter LjPUB7pro, and the nucleotide sequence of the rhizobium inducible promoter LjPUB7pro is shown as SEQ ID NO. 1; according to the invention, a lotus corniculatus public database and RNAseq data are utilized to analyze that the promoter can be specifically expressed in lotus corniculatus, the promoter LjPUB7pro is transferred into lotus corniculatus through an agrobacterium-mediated method, under the condition of rhizobium induction, the expression quantity of a reporter gene GUS in the root can be driven to be obviously improved, and a substantive technical effect is achieved. The LjPUB7pro promoter provides a new effective regulation and control element for root specific expression, has great research significance in leguminous crop nitrogen fixation mechanism research and agricultural production, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a rhizobium-induced promoter LjPUB7pro and its application. Background Art

[0002] Rhizobia are special bacteria that can live in the soil or in root nodules formed on the roots of leguminous plants. In root nodules, they form a symbiotic relationship with leguminous plants. The plants provide energy to rhizobia through photosynthesis, and rhizobia fix atmospheric nitrogen in the root nodules and convert it into a form that plants can utilize to support the plant's metabolism. The complex process by which rhizobia produce nitrogen for leguminous plants is called biological nitrogen fixation. In addition, nitrogen is an essential macronutrient for plant growth and development, so agricultural production highly depends on industrial nitrogen fertilizers. However, the production of nitrogen fertilizers requires a large amount of fossil energy, and excessive application of nitrogen fertilizers will cause soil compaction and degradation and water pollution, affecting the sustainable development of agriculture.

[0003] Biological nitrogen fixation is the largest natural source of biologically available nitrogen in nature. Leguminous plants and rhizobia can interact to form a unique organ, namely the symbiotic root nodule. Symbiotic nitrogen fixation in root nodules is an important pathway for the reduction of nitrogen gas to ammonia that can be absorbed and utilized by plants in the earth's ecosystem, contributing more than 60% of the terrestrial biological nitrogen fixation, and is of great significance for maintaining primary production and carbon sequestration in agricultural and natural ecosystems.

[0004] In addition, studies have found that the initiation and development of root nodules are both regulated by transcription factors. However, there are few reports on transcription factors that are specifically expressed in soybean root nodules and participate in the regulation of symbiotic nitrogen fixation. Promoters can regulate gene transcription and expression. Inducible promoters can receive external induction signals, quickly and effectively initiate gene expression, respond quickly to external signals, and can also quickly relieve stress and stop expression. During the process of establishing symbiotic nitrogen fixation between rhizobia and leguminous plants, a fine-tuned and complex nodulation signal transduction chain is formed, accompanied by the induced expression of a series of genes, especially at the initial stage of the symbiotic signal between leguminous plants and rhizobia. Therefore, studying the tissue-specific promoters during this process and whether they are induced to express has an important role in studying the expression and biological functions of target genes, and at the same time helps to analyze the symbiotic nitrogen fixation mechanism, and has important theoretical significance and application value for agricultural breeding improvement.

[0005] In the transcriptome data of the present invention, a gene LjPUB7 that is specifically expressed in roots and significantly induced by rhizobia was screened. The LjPUB7 gene is a gene homologous to Medicago truncatula, and its function has not been reported in Lotus japonicus. Through research, it is speculated that this gene may play an important role in nodule symbiotic nitrogen fixation. At the same time, the promoter is an important part of gene expression regulation. Therefore, studying the promoter of LjPUB7 is of great significance for analyzing the molecular mechanism of symbiotic nitrogen fixation. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a rhizobia-induced promoter LjPUB7pro and its application; the rhizobia-induced promoter LjPUB7pro is a creative research discovery of the inventor, and there is no relevant literature report on the promoter LjPUB7pro. This promoter can be specifically expressed in roots and can drive the expression of foreign genes under the induction of rhizobia, thereby improving the transcription effect.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a rhizobia-induced promoter LjPUB7pro, and the nucleotide sequence of the rhizobia-induced promoter LjPUB7pro is as shown in SEQ ID NO.1, or a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion or addition of at least one nucleotide and having the same function.

[0009] The present invention provides a primer pair for amplifying the rhizobia-induced promoter LjPUB7pro in the above technical solution. The nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer of the primer pair is as shown in SEQ ID NO.3.

[0010] The present invention also provides the application of the above LjPUB7pro promoter or an element containing the LjPUB7pro promoter in the specific induction expression in plant roots; the element is any one of the following 1) to 4):

[0011] 1) Expression cassette;

[0012] 2) Expression vector;

[0013] 3) Recombinant microorganism;

[0014] 4) Transgenic plant or cell;

[0015] Preferably, the expression cassette (5' to 3') comprises a promoter region (composed of the DNA molecule of the promoter LjPUB7pro), a transcription initiation region, a target gene region, a transcription termination region, and optionally a translation termination region.

[0016] Preferably, the expression vector is a vector containing the above DNA molecule or expression cassette constructed using a plant expression vector; wherein the plant expression vector includes DX2181G.

[0017] Preferably, the recombinant microorganism can be a microorganism containing the above DNA molecule, or the above expression cassette, or the above expression vector. The microorganism can be yeast, bacteria, or fungi, and the bacteria can be Agrobacterium (including Agrobacterium tumefaciens LBA4404) or Escherichia coli.

[0018] Preferably, the transgenic plant is a recipient plant transformed with the DNA molecule of the promoter LjPUB7pro and its progeny, specifically including seeds, callus, whole plants, or cells.

[0019] Preferably, the transgenic plant includes leguminous plants; the leguminous plants include one or more of Lotus japonicus, Medicago sativa, Glycine max, Arachis hypogaea, Phaseolus vulgaris, Vigna unguiculata, and Cicer arietinum; more preferably Lotus japonicus.

[0020] The present invention provides a method for driving and / or enhancing the expression of a target gene using the inducible promoter LjPUB7pro described in the above technical solution, comprising the following steps:

[0021] Introduce the promoter LjPUB7pro or an element containing the promoter LjPUB7pro and the target gene into the plant genome to obtain a transgenic plant, thereby achieving the driving and / or enhancing of the expression of the target gene.

[0022] Preferably, construct a recombinant expression vector with the promoter LjPUB7pro or an element containing the promoter LjPUB7pro and the target gene, transfer the recombinant expression vector into Agrobacterium, and use the Agrobacterium-mediated transformation method to infect the explants of the plant with Agrobacterium to obtain transgenic plants. Treat with rhizobia to drive and / or enhance the expression of the target gene.

[0023] Among them, the target gene can be an endogenous gene of the plant or an exogenous gene (such as the GUS gene). The driving of the expression of the target gene can be to drive the expression of the target gene under the induction condition of rhizobia. The expression of the target gene can be to initiate the expression of the target gene in specific tissues.

[0024] The specific tissue includes any one or more of roots, stems, leaves, flowers or pods; the plant includes leguminous plants; the leguminous plants include Lotus corniculatus, Glycine max, Medicago sativa; the Agrobacterium includes Agrobacterium tumefaciens LBA4404.

[0025] Beneficial effects:

[0026] The present invention provides a novel rhizobium-inducible promoter LjPUB7pro, and the nucleotide sequence of the promoter LjPUB7pro is shown as SEQ ID NO.1; meanwhile, a primer pair for amplifying the rhizobium-inducible promoter LjPUB7pro in the above technical solution is provided, and the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO.2; the nucleotide sequence of the downstream primer of the primer pair is shown as SEQ ID NO.3.

[0027] The promoter LjPUB7pro provided by the present invention is a root-specific promoter, which can be specifically expressed in roots, providing a new and effective regulatory element for root-specific expression; moreover, this promoter has the characteristics of rhizobium-inducible expression. By constructing a recombinant vector with the promoter and the reporter gene GUS and transferring it into Lotus corniculatus, under the condition of rhizobium induction, it can drive the specific expression of the GUS gene in roots, which has great research significance for the study of the nitrogen fixation mechanism of leguminous crops and agricultural production, and has a broad application prospect. Description of the drawings

[0028] Figure 1The LjPUB7 gene is mainly expressed in roots and specifically induced by rhizobia; Figure A shows the expression pattern of LjPUB7 in different tissues and organs of Lotus japonicus, including leaf, immature flower, mature flower, pod, seed, and root; Figures B and C show the expression of LjPUB7 under different microbial stimuli. In Figure B, MAFF303099_mock and AM_mock are controls, and the others are inoculated with M. loti MAFF303099 for 3 days and 12 days, and with AM arbuscular mycorrhiza for 15 days and 27 days in sequence; in Figure C, mock_microbial is the untreated control, and the others are inoculated with M. loti R7A, S. fredii HH103, B. elkanii USDA6, P. syringae pv tomato DC3000, and R. solanacearum JS763 for 3 days; the original data are from (Mun et al 2016) (A), (Handa et al 2015) (B), and (Kelly et al 2018) (C); dpi is the time after inoculation with rhizobia (day post inoculation).

[0029] Figure 2 LjPUB7 is mainly expressed in the early stage of rhizobia inoculation; Figure A shows the spatio-temporal expression of LjPUB7 in roots of wild-type Lotus japonicus after rhizobia inoculation. The original data are from ( et al 2009), where dpi is the time after inoculation with rhizobia (day post in-oculation), root_nodC_3dpi is inoculated with NodC mutant rhizobia for 3 days, and root_nodule is the root tissue containing nodules; Figure B shows the expression of LjPUB7 in roots, root hairs, nodule primordia, and nodules of wild-type Lotus japonicus after rhizobia inoculation. The original data are from (Mun et al 2016); Figure C shows the expression of LjPUB7 in different nodulation symbiosis-defective mutants after rhizobia inoculation. Among them, rootSZ is the Root Susceptible Zone, that is, the area of root tissue that is more sensitive to rhizobia. The original data are from ( etal 2009).

[0030] Figure 3To verify the expression of LjPUB7 gene by qRT-PCR; after wild-type Lotus japonicus was inoculated with rhizobia at different times (1 day, 3 days, 5 days, 7 days) or not, the spatio-temporal expression of LjPUB7 (A, B) and the marker gene LjNIN (C) related to nodule symbiosis in roots was detected by qRT-PCR.

[0031] Figure 4 To analyze the expression of LjPUB7 in roots at different times after inoculation with rhizobia by histochemical staining (GUS staining); roots without inoculation (A) and inoculated with rhizobia at different times: 1 dpi (B), 2 dpi (C), 3 dpi (D), 5 dpi (E) and 7 dpi (F) were taken for GUS staining and photography after hairy root transformation; where bars = 50 μm. Specific implementation manners

[0032] The present invention provides a rhizobia-inducible promoter LjPUB7pro and its application, and the nucleotide sequence of the LjPUB7pro gene is shown as SEQ ID NO.1:

[0033]

[0034] The present invention provides a primer pair for amplifying the inducible promoter LjPUB7pro. The nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer of the primer pair is as shown in SEQ ID NO.3:

[0035] LjPUB7pro-F: 5’-GGGTTAGGAGATAAGAGCAAAGAGGGAAAT-3’;

[0036] LjPUB7pro-R: 5’-TGTGGTTGTTTGTTCTTACCTTATGAGG-3’.

[0037] The present invention also provides the application of the above LjPUB7pro promoter or an element containing the LjPUB7pro promoter in specifically inducing expression in plant roots. The element is any one of the following 1) to 4): 1) an expression cassette; 2) an expression vector; 3) a recombinant microorganism; 4) a transgenic plant or cell.

[0038] In the present invention, the expression cassette (5' to 3') may include a promoter region (composed of the DNA molecule), a transcription initiation region, a target gene region, a transcription termination region, and optionally a translation termination region. The expression vector is a vector containing the above DNA molecule or the above expression cassette constructed by using a plant expression vector (DX2181G). The recombinant microorganism may be a microorganism containing the above DNA molecule or the above expression cassette or the above recombinant vector. The microorganism may be yeast, bacteria, or fungi. The bacteria may be Agrobacterium (such as Agrobacterium tumefaciens LBA4404) or Escherichia coli. The transgenic plant is a plant that contains the DNA molecule transformed into a recipient plant and its progeny, specifically including seeds, callus, whole plants, and cells. The transgenic plants include leguminous plants; leguminous plants include one or more of Lotus japonicus, Medicago sativa, Glycine max, Arachis hypogaea, Phaseolus vulgaris, Vigna unguiculata, and Cicer arietinum, and more preferably Lotus japonicus.

[0039] The present invention provides a method for driving and / or enhancing the expression of a target gene by using the inducible promoter LjPUB7pro as described in the above technical solution, including the following steps:

[0040] Introduce the promoter LjPUB7pro or an element containing the promoter LjPUB7pro and the target gene described in the above technical solution into the plant genome to obtain a transgenic plant, and drive and / or enhance the expression of the target gene.

[0041] In the present invention, a recombinant expression vector is constructed by combining the promoter LjPUB7pro or an element containing the promoter LjPUB7pro with a target gene. The recombinant expression vector is transferred into Agrobacterium, and the Agrobacterium is used to infect explants of a plant by the Agrobacterium-mediated transformation method to obtain transgenic plants. The transgenic plants are treated with rhizobia to drive and / or enhance the expression of the target gene. The target gene can be an endogenous gene of the plant or an exogenous gene (such as the GUS gene). The driving of the target gene expression can be to drive the expression of the target gene under the induction condition of rhizobia. The expression of the target gene can be to initiate the expression of the target gene in a specific tissue. The specific tissue includes any one or more of roots, stems, leaves, flowers or pods. The plant includes leguminous plants. The leguminous plants include Lotus japonicus, Glycine max, Medicago sativa.

[0042] To further illustrate the present invention, the applications provided by the present invention will be described in detail below in combination with examples, but they should not be construed as limiting the protection scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all conventional commercially available products unless otherwise specified.

[0043] Example 1:

[0044] Analysis of the expression pattern of the Lotus japonicus LjPUB7 gene

[0045] By searching and analyzing the RNAseq data in the Lotus japonicus public database (Lotus Base) and the SRA database of NCBI, the expression of the LjPUB7 gene in different tissues and after treatment with rhizobia is analyzed. Figure 1The LjPUB7 gene is mainly expressed in roots and is specifically induced by rhizobia; in Figure A, the expression pattern of LjPUB7 in different tissues and organs of Lotus japonicus is shown, including leaf, immature flower, mature flower, pod, seed, and root; in Figures B and C, the expression of LjPUB7 under different microbial stimulations is shown. In Figure B, MAFF303099_mock and AM_mock are controls, and the others are inoculated with M. loti MAFF303099 for 3 days and 12 days, and with AM arbuscular mycorrhiza for 15 days and 27 days in sequence; in Figure C, mock_microbial is the untreated control, and the others are inoculated with M. loti R7A, S. fredii HH103, B. elkanii USDA6, P. syringae pv tomato DC3000, and R. solanacearum JS763 for 3 days. The original data sources are (Mun et al 2016) (Figure A), (Handa et al 2015) (Figure B), and (Kelly et al 2018) (Figure C); dpi is the time after inoculation with rhizobia (day postinoculation).

[0046] The results showed that LjPUB7 was mainly expressed in the underground part (roots), while the expression levels were relatively low in the aboveground parts (leaves, flowers, pods, seeds) ( Figure 1 in Figure A). After inoculation with rhizobia, the transcriptional level of LjPUB7 was significantly upregulated, but there was no change in the transcriptional level of LjPUB7 when inoculated with arbuscular mycorrhiza (A. mycorrhizae), the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, or Ralstonia solanacearum JS763 ( Figure 1 in Figures B and C), indicating that LjPUB7 is induced by rhizobia and specifically participates in the plant-rhizobia interaction.

[0047] It is worth noting that not only M. loti R7A can induce the upregulation of LjPUB7 expression, but S. fredii HH103 can also induce the expression of LjPUB7, and the induction levels of the two are comparable ( Figure 1C). R7A has strong host specificity and is a rhizobium that perfectly matches Lotus japonicus, enabling Lotus japonicus to produce normal infection threads and nodules. HH103, on the other hand, is a broad-spectrum rhizobium that can only produce white non-nitrogen-fixing nodules when inoculated on Lotus japonicus (Jiménez-Guerrero et al 2020). The expression level of LjPUB7 is induced by rhizobia but is not related to whether the nodules can fix nitrogen normally, indicating that this gene is more likely to be involved in the early stage of nodule symbiosis and not in the later nodule development and nitrogen fixation process. Bradyrhizobium elkanii USDA61 is a soybean rhizobium that cannot nodulate Lotus japonicus, but it can also induce the transcriptional expression of LjPUB7, although the induction level is lower than that of R7A and HH103( Figure 1 C). The pathogens DC3000 and R. solanacearum JS763 cannot induce the transcriptional expression of LjPUB7. These results suggest that LjPUB7 specifically participates in the interaction between Lotus japonicus and rhizobia and there is a complex regulatory pattern.

[0048] Example 2:

[0049] The LjPUB7 gene of Lotus japonicus is mainly expressed in the early stage of symbiosis

[0050] Further analysis was carried out through the RNAseq data in the public database of Lotus japonicus (Lotus Base) and the SRA database of NCBI; Figure 2 It is shown that LjPUB7 is mainly expressed in the early stage after inoculation with rhizobia; Figure A shows the spatio-temporal expression of LjPUB7 in roots of wild-type Lotus japonicus after inoculation with rhizobia. The original data is from ( et al 2009), where dpi is the time after inoculation with rhizobia (day post in-oculation), root_nodC_3dpi is the root after inoculation with the NodC mutant rhizobia for 3 days, and root_nodule is the root tissue containing nodules; Figure B shows the expression of LjPUB7 in roots, root hairs, nodule primordia and nodules of wild-type Lotus japonicus after inoculation with rhizobia. The original data is from (Mun et al 2016); Figure C shows the expression of LjPUB7 in different nodule symbiosis defective mutants after inoculation with rhizobia, where rootSZ is the RootSusceptible Zone, that is, the area of the root tissue that is more sensitive to rhizobia. The original data is from ( etal 2009).

[0051] Results showed that LjPUB7 was mainly expressed in roots at the early stage (1 day) after rhizobial inoculation, with a small amount of expression also detected in roots at the later stage (7 days and 21 days), and almost no expression in nodules ( Figure 2 A). This result indicates that LjPUB7 may mainly function at the early stage of nodule symbiosis. The same result was obtained after analyzing another independent RNAseq dataset. LjPUB7 transcription was not detected in nodules at 10 days and 21 days after inoculation, with a small amount of expression in roots at 3 days after inoculation and nodules at 7 days after inoculation, while the strongest expression was observed in root hair cells 24 h and 72 h after inoculation, which was more than 1000 times that of the control ( Figure 2 B). Root hair cells are the sites where the earliest signal exchange occurs between rhizobia and host plants. The recognition between the two triggers extensive transcriptional rearrangement of a large number of genes. The specifically induced expression of LjPUB7 in root hair cells suggests that this gene mainly functions at the early stage (24 h - 72 h) of the interaction between rhizobia and the host.

[0052] There is a tissue called the Root Susceptible Zone (root_RS) in the roots of leguminous plants, which is the most sensitive section of the plant roots to rhizobia, and early symbiotic signals also mostly occur here. RNAseq data analysis results showed that LjPUB7 had strong induced expression in the root_RS section of wild-type Lotus japonicus ( Figure 2 C). After comparing the expression data in different symbiotic defective mutants, it was found that the expression of this gene was not induced in the root_RS section of nfr1, nfr5, nup133, and cyclops mutants after rhizobial inoculation, but induced expression was still detected in nin and har1 mutant plants ( Figure 2 C). Genes such as LjNFR1, LjNFR5, LjNUP133, and LjCYCLOPS are all located upstream of the nodulation factor signaling pathway and are mainly responsible for the early signal transduction process. While LjNIN and LjHAR1 are located downstream and are mainly involved in the formation of infection threads, the development of nodule organs, and the autoregulation process. The induced expression of LjPUB7 in nin and har1 but not in nfr1 and other mutants indicates that the transcriptional level of this gene is indeed regulated by early symbiotic signals and may function downstream of genes such as NFR1, NFR5, NUP133, and CYCLOPS and upstream of genes such as NIN and HAR1.

[0053] Example 3:

[0054] Verification of LjPUB7 gene expression by qRT-PCR

[0055] 1. Rhizobial inoculation

[0056] The wild-type MG20 seeds of Lotus japonicus were treated with concentrated sulfuric acid and sodium hypochlorite and then dark-treated at 4°C overnight. After germination, they were planted in sterilized culture soil of perlite and vermiculite (perlite:vermiculite mass ratio is 1:2) until they grew into seedlings. After the rhizobium (Mesorhizobium loti MAFF303099) was activated on a TY solid plate, rhizobium was picked and cultured with shaking in TY liquid medium at 28°C until the OD 600 value was about 1.0. The cells were collected by centrifugation at 5000 rpm / min for 5 min, and then the cells were resuspended in nitrogen-free nutrient solution B&D medium to OD 600 = 0.04. Finally, the resuspended solution was inoculated onto the roots of Lotus japonicus seedlings, and the seedlings without inoculated rhizobium were used as the control group.

[0057] The wild-type MG20 seeds of Lotus japonicus were disclosed in

Feng Y, Wu P, Liu C, Peng L, Wang T, Wang C, Tan Q, Li B, Ou Y, Zhu h, Yuan S, Huang R, Stacey G, Zhang Z, Cao Y. Suppression of LjBAK1 mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus. Molecular Plant, 2021, 14: 1935-1950

[0058] The TY liquid medium was prepared as follows: 3 g Yeast Extract, 5 g Tryptone, 0.69 g CaCl 2 , ddH 2 O was made up to 1 L, the pH was adjusted to 7.0, and it was sterilized at 121°C for 20 min. Reagent sources: Tryptone (Sigma, T2559), Yeast Extract (Sigma, Y0875).

[0059] The nitrogen-free nutrient solution B&D medium was prepared as follows: 1.0 mM CaCl 2 ·2H 2 O, 0.5 mM KH 2 PO 4 , 10 μM Fe-Citrate, 0.25 mM MgSO 4 ·7H 2 O, 0.25 mM K 2 SO 4 , 1.0 μM MnSO 4 ·H 2 O, 2.0 μM H3 BO 3 ,0.5 μM ZnSO 4 ·7H 2 O,0.2 μM CuSO 4 ·5H 2 O,0.1 μM CoSO 4 ·7H 2 O,0.1 μM Na 2 MoO 4 ·2H 2 O。

[0060] 2. Detection of LjPUB7 Gene Expression in Lotus japonicus Roots

[0061] (1) Sampling: Dig out and wash the roots of Lotus japonicus that have not been inoculated and those inoculated with rhizobia at different times (1 day, 3 days, 5 days, 7 days).

[0062] (2) Detection of LjPUB7 Gene Expression by Fluorescent Quantitative PCR: Use an RNA extraction kit (Aidlab, RN3302) to extract total RNA from the washed roots in step (1). After RNA elution, use NanoDrop 2000 (Thermo) to detect the concentration and purity of the RNA. Perform reverse transcription using Primescript RT Reagent kit DRR047A (Takara, Japan), and operate qRT-PCR according to the one-step SYBR PrimeScript RT-PCR kit II (Takara) kit. Refer to the operation manual of the corresponding kit for the specific method of the experiment. Use the Roche Light Cycler apparatus fluorescent quantitative PCR instrument from Roche. The fluorescent quantitative PCR program is: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 15 s, 60 °C for 1 min, for 40 cycles. Among them, 2 pairs of primers, LjPUB7-qRT-F1, LjPUB7-qRT-R1 and LjPUB7-qRT-F2, LjPUB7-qRT-R2 are used to detect the expression of the LjPUB7 gene, and LjNIN-qRT-F, LjNIN-qRT-R are used to detect the expression of the LjNIN gene.

[0063] The fluorescent quantitative PCR primers are as follows (F at the end of the primer is the forward primer, R is the reverse primer):

[0064] LjPUB7-qRT-F1: 5’-TCCAAAGACAAGCCGCGTAT-3’, SEQ ID NO.4;

[0065] LjPUB7-qRT-R1: 5’-CTGTGACTGCATGCTCCTGA-3’, SEQ ID NO.5;

[0066] LjPUB7-qRT-F2: 5’-TTGCTGTGCTTCAAGGTTGC-3’, SEQ ID NO.6;

[0067] LjPUB7-qRT-R2: 5’-AACACTCCTTGGATTCGCCA-3’, SEQ ID NO.7;

[0068] LjNIN-qRT-F: 5’-AATGCTCTTGATCAGGCTG-3’, SEQ ID NO.8;

[0069] LjNIN-qRT-R: 5’-AGGAGCCCAAGTGAGTGCTA-3’, SEQ ID NO.9;

[0070] (3) Analysis of LjPUB7 gene expression

[0071] The expression level of genes was normalized using the Lotus japonicus housekeeping gene Ubiqutin as an internal reference gene, and the relative quantification ΔΔCt method was used for quantification. Figure 3 To verify the expression of the LjPUB7 gene by qRT-PCR; after wild-type Lotus japonicus was inoculated with rhizobia at different times (1 day, 3 days, 5 days, 7 days) and not inoculated, the spatio-temporal expression of LjPUB7 and the nodule symbiosis-related marker gene LjNIN in the roots was detected by qRT-PCR.

[0072] The results showed that LjPUB7 was hardly expressed before inoculation, but was significantly up-regulated after inoculation with rhizobia. The expression level of LjPUB7 was relatively high at 1-3 days after inoculation, and then showed a downward trend ( Figure 3 A, B), indicating that LjPUB7 was expressed in the early stage of nodule symbiosis, which was consistent with the results of RNAseq. At the same time, the expression of the symbiosis-related marker gene NIN was detected. NIN was continuously strongly up-regulated at 1-7 days after inoculation with rhizobia ( Figure 3 C), indicating that there was no problem with the inoculation process of the present invention.

[0073] Example 4:

[0074] Cloning of the LjPUB7pro promoter and construction of recombinant vectors

[0075] 1. Primer design: Primers were designed to amplify the promoter region 3 kb upstream of the start codon ATG of the LjPUB7 gene, that is, to obtain the LjPUB7pro fragment.

[0076] LjPUB7pro-F: 5'-GGGTTAGGAGATAAGAGCAAAGAGGGAAAT-3', SEQ ID NO.2;

[0077] LjPUB7pro-R: 5'-TGTGGTTGTTTGTTCTTACCTTATGAGG-3', SEQ ID NO.3.

[0078] 2. Vector construction: The DX2181G1-mCherry vector (containing the GUS reporter gene) was double digested with restriction enzymes HandIII / PstI to obtain a linearized vector, and then ligated with the LjPUB7pro fragment by Gibson reaction to fuse LjPUB7pro with the reporter gene GUS, resulting in a recombinant vector.

[0079] The Gibson (Yeasen, 10911ES20) reaction system was 5 μL: 2.5 μL of 2×Hieff Enzyme Premix, approximately 100 ng of gene fragment, approximately 300 ng of vector, and the remaining ddH 2 O; The Gibson reaction conditions were 50 °C for 1 h.

[0080] 3. The recombinant vector was introduced into Agrobacterium rhizogenes LBA1334, obtaining a recombinant strain of Lotus japonicus hairy root Agrobacterium LBA1334 containing the LjPUB7 promoter sequence.

[0081] Example 5:

[0082] Histochemical staining analysis of the expression characteristics of the LjPUB7 promoter in roots

[0083] 1. Lotus japonicus hairy root transformation

[0084] (1) Seed disinfection and germination: Seeds were added to a 50 mL sterile Erlenmeyer flask, and then concentrated H 2 SO 4 was poured in to cover the seeds and shaken evenly. The treatment time was 8 - 12 min. Then, concentrated H 2 SO 4Put them into a glass beaker filled with water, and add sterile water to the remaining seeds to quickly rinse the seeds. Repeat the rinsing 3 times until the color of the sterile water does not change. Then add 2% sodium hypochlorite solution (available chlorine concentration), cover the seeds, and gently shake. The treatment time is 8 - 12 min. After the treatment, discard the waste liquid, and add sterile water to rinse the seeds 3 times again until the color of the sterile water does not change. Finally, leave a little water to immerse the seeds, put them in a 4°C refrigerator, and place them for 1 - 2 days for vernalization. After vernalization, transfer the seeds onto an MS medium plate, and culture them in the dark at 23°C for 24 - 36 h, then culture them under light for 2 - 3 days until seedlings grow out.

[0085] The MS medium is prepared as follows: 4.43 g of MS salts (Sigma, M55191L), adjust the pH to 6.0, and make up to 1 L with ddH 2 O. Add 1.5 wt.% Agar (Yeasan, 70101ES76) for the solid medium, and sterilize at 115°C for 20 min.

[0086] (2) Strain preparation: Activate the recombinant Agrobacterium rhizogenes LBA1334 strain in Example 4 on a plate containing plasmid resistance (Kana, 50 μg / mL), and culture at 28°C for 1 - 2 days until single colonies grow. Inoculate the single colonies into 5 mL of LB liquid medium containing Kana antibiotic (50 μg / ml), and culture at 28°C, 200 rpm / min for 24 h to obtain a culture solution; then take the culture solution and inoculate it into 50 mL of LB liquid medium containing Kana antibiotic (50 μg / ml) at an inoculation amount of 2%, and continue to shake and culture at 28°C until OD 600 is 0.6 - 0.8. Then centrifuge at 4000 rpm / min for 5 min to collect the bacteria, and resuspend the bacteria with sterile water containing a final concentration of 20 - 40 μg / mL AS (Acetosyringone) to make the OD of the resuspended bacterial solution 600 about 0.8.

[0087] The LB liquid medium is prepared as follows: 10 g / L NaCl + 10 g / L Tryptone + 5 g / L Yeast Extract, (1.2% Agar), adjust to pH 7.0. Reagent sources: Tryptone (Sigma, T2559), Yeast Extract (Sigma, Y0875), Agar (Yeasan, 70101ES76).

[0088] Kana is prepared as follows: Weigh 1.00 g of Kanamycin sulfate (Yeasan, 60206ES10), dissolve it in 10 mL of ultrapure water, filter and sterilize, then dispense it into sterile 1.5 mL centrifuge tubes and store at -20°C.

[0089] (3) Infection and co-culture: Cut the germinated seedlings in step (1) on the plate with scissors at the hypocotyl of the seedlings, and cut slightly below the swollen part between the green stem and the white root. Transfer the cut seedlings to the resuspended bacterial solution in step (2) and infect for 30 min. Transfer the seedlings to an MS sucrose-free medium plate with sterile filter paper and seal the plate with a sealing film. Incubate in a light incubator in the upright position, first in the dark for 2 d, and then in the upright position with light for 2 - 3 d. Transfer the seedlings to a rooting medium containing HRE and 1% sucrose with 300 μg / mL Timentin, place two rows on each plate, 10 - 12 seedlings per row, seal the plate with a sealing film, and place it in a light incubator at 23 °C in a vertically inclined position for 12 - 20 d to obtain rooted seedlings.

[0090] The HRE rooting medium is prepared as follows: 50 mL of 20×SHA salt, 50 mL of 20×UM C vitamin, 10.0 g of sucrose, 3 mL of 1 mol / L MES, with a final concentration of 0.8 wt.% Agar (Yeasan, 70101ES76), made up to 1 L with ultrapure water, adjusted to pH 5.8 with KOH, and sterilized at 115 °C for 20 min.

[0091] The 20×SH A salt is prepared as follows: 8.0 g of MgSO 4 ·7H 2 O, 6.0 g of NH 4 ·H 2 PO 4 , 4.0 g of CaCl 2 ·2H 2 O, 0.2 g of MnSO 4 ·4H 2 O, 0.02 g of ZnSO 4 ·7H 2 O, 0.004 g of CuSO 4 ·5H 2 O, 0.002 g of NaMoO 4 ·2H 2 O, 0.002 g of CoCl·6H 2 O, 0.02 g of KI, 0.1 g of H 3 BO 3 , 50.0 g of KNO 3 , 0.3 g of FeSO 4 ·7H 2 O, 0.4 g of Na 2 EDTA, made up to 1 L with ultrapure water, and stored at -20 °C for later use.

[0092] 20×UM C vitamins was prepared as follows: 2.0 g Myoinositol, 0.1 g Nicotinic acid, 0.2 g Vitamin B6, 0.2 g Thiamine HCl, 0.04 g Glycine, made up to 1 L with ultrapure water, and stored at -20 °C for later use.

[0093] 1 mol / L MES was prepared as follows: 4.88 g MES, made up to 25 mL with ultrapure water, adjusted to pH 5.8, filtered and sterilized, and stored at -20 °C for later use.

[0094] 100 mmol / L Acetosyringone (AS) was prepared as follows: 196.2 mg Acetosyringone, made up to 10 mL with dimethyl sulfoxide, filtered and sterilized, and stored at -20 °C.

[0095] 300 mg / mL Timentin: 3 g Timentin was dissolved in ddH 2 O and made up to 10 mL, filtered and sterilized, then aliquoted into sterile 1.5 mL centrifuge tubes and stored at -20 °C.

[0096] 2. Root hair identification: Under a Nikon SMZ18 stereomicroscope with fluorescence, positive seedlings were selected from the rooted seedlings through the mCherry screening marker. The identified positive seedlings were acclimatized in a light incubator for 1 d, then transferred to sterilized perlite:vermiculite (ratio 1:2) culture soil, and cultured in a greenhouse at 23 °C with a 16 h / 8 h photoperiod. After 7 d of culture, rhizobia were inoculated.

[0097] 3. Inoculation of rhizobia: Rhizobium MesorhizobiumlotiMAFF303099 was cultured in TY liquid medium at 28 °C with shaking until the OD 600 value reached 1. The cells were collected by centrifugation at 5000 rpm / min for 5 min, and resuspended in nitrogen-free nutrient solution B&D medium to an OD 600 of 0.04, and inoculated onto the roots of positive Lotus japonicus seedlings. Seedlings without inoculated rhizobia were used as the control group.

[0098] The formulations of the above TY liquid medium and nitrogen-free nutrient solution B&D medium were the same as those in Example 3.

[0099] 4. GUS staining: Take the roots of the 3 plants without inoculation (A) and inoculation with rhizobia at different times and perform GUS staining and take photos. Specific operation: Clean the underground part of the plant and dry it, then add GUS staining solution to completely immerse the sample, evacuate for 5 minutes, and place it in a 37℃ incubator for staining for 8-12 hours. After staining, rinse with water, observe under a microscope and take photos. The results showed that when the rhizobia were not inoculated, the roots of the plant could not be stained blue ( Figure 4 A), and only after inoculation with rhizobia will the roots turn blue ( Figure 4 B, C, D), indicating that the promoter of the LjPUB7 ​​gene is only strongly active in the inoculated Lotus japonicus. At the same time, the promoter activity of the LjPUB7 ​​gene can be detected in the root vascular bundle and epidermal cells of the root meristem. And the staining is darker 1-3 days after inoculation with rhizobia, and lighter 5-7 days after inoculation ( Figure 4 E, F), indicating that the promoter of LjPUB7 ​​gene has higher activity mainly in the early stage of rhizobium inoculation.

[0100] The GUS staining solution was prepared as follows: 100 mM sodium phosphate buffer (pH 7.0), 0.1% Triton X-100, 0.1% N-laurylsarcosine, 10 mM Na 2 EDTA, 1 mM potassium ferrocyanide (K 3 Fe(CN) 6 ), 1 mM potassium ferrocyanide (K 4 Fe(CN) 6 ), 0.5mg / mL X-GluC.

[0101] In summary, the rhizobium-inducible promoter LjPUB7pro provided by the present invention is specifically expressed in roots, and under the induction conditions of rhizobium signals, can be used as an important regulatory element in transcriptional regulation to significantly enhance the expression of reporter genes. This promoter with rhizobium-inducible characteristics has good application value in plant genetic engineering, can be applied to the practical application of genetic breeding for symbiotic nitrogen fixation between plants and rhizobia, and is of great significance for studying the mechanism of symbiotic nitrogen fixation.

[0102] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A rhizobium inducible promoter LjPUB7pro, characterized in that The nucleotide sequence of the promoter LjPUB7pro is as shown in SEQ ID NO.1, or is a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion or addition of at least one nucleotide and having the same function.

2. A primer pair for amplifying the rhizobium inducible promoter LjPUB7pro according to claim 1, characterized in that: The nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO.2; the nucleotide sequence of the downstream primer of the primer pair is shown as SEQ ID NO.

3.

3. Use of the promoter LjPUB7pro according to claim 1 or an element containing the promoter LjPUB7pro according to claim 1 in specific induced expression in plant roots.

4. The use according to claim 3, characterized in that: The promoter LjPUB7pro or the element containing the promoter LjPUB7pro drives the exogenous gene to express specifically in plant roots.

5. The use according to claim 3, characterized in that: The elements include expression cassettes, expression vectors, recombinant microorganisms, transgenic plants or cells.

6. The use according to claim 5, characterized in that: The expression cassette includes a promoter region, a transcription initiation region, a target gene region, a transcription termination region and an optional translation termination region; the expression vector is a vector containing a promoter LjPUB7proDNA molecule or an expression cassette obtained by constructing a plant expression vector, wherein the plant expression vector includes DX2181G; The recombinant microorganism is a microorganism containing a DNA molecule of the promoter LjPUB7pro, an expression cassette or an expression vector as described in claim 5, wherein the microorganism is yeast, bacteria or fungi, and the bacteria can be Agrobacterium or Escherichia coli.

7. The use according to claim 5, characterized in that: The transgenic plant is a recipient plant transformed with the DNA molecule of the promoter LjPUB7pro according to claim 1 and its progeny, including seeds, callus tissues, complete plants or cells.

8. The use according to claim 3, characterized in that: The plants include leguminous plants; wherein the leguminous plants include lotus roots, alfalfa, soybeans, peanuts, kidney beans, cowpeas and chickpeas.

9. A method for driving and / or improving the specific expression of a target gene in roots using an inducible promoter LjPUB7pro, characterized in that: The steps include: The promoter LjPUB7pro described in claim 1 or an element containing the promoter LjPUB7pro described in claim 1 and a target gene are introduced into the plant genome to obtain a transgenic plant, thereby achieving driving and / or enhancing the expression of the target gene.

10. The method according to claim 9, characterized in that The specific operation is: constructing a recombinant expression vector with the promoter LjPUB7pro or an element containing the promoter LjPUB7pro and a target gene, then transferring the recombinant expression vector into Agrobacterium, and using the Agrobacterium-mediated transformation method to infect plant explants with Agrobacterium to obtain transgenic plants; Drive and / or enhance the expression of target genes under rhizobium-induced conditions.

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