Red bayberry crrsp1 gene and application thereof

Abstract

The application discloses a waxberry CRRSP1 gene and application thereof, and belongs to the technical field of genetic engineering. The application provides a CRRSP1 gene which is specifically up-regulated in the root nodule of a waxberry, and the CDS sequence of the CRRSP1 gene is shown as SEQ ID NO. 1. The application also provides application of the waxberry CRRSP1 gene in promoting symbiotic interaction between a nodule plant and symbiotic bacteria, and the application comprises: up-regulating expression of the waxberry CRRSP1 gene in the nodule plant by using a biological technical means. By up-regulating expression of the gene in the nodule plant, symbiotic nodulation of the nodule plant and the symbiotic bacteria can be significantly promoted, and the plant biomass is increased. The application provides an important reference value for research on symbiotic genes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and in particular to a waxberry CRRSP1 gene and its application in promoting symbiotic interaction between nodulation plants and symbiotic bacteria. BACKGROUND

[0002] Symbiosis in nature mainly includes two categories: the interaction between legume plants and rhizobium, and the interaction between actinomycete plants and frankia. Both types of symbiosis form a special symbiotic structure, i.e., nodule, and the symbiotic bacteria convert atmospheric nitrogen into ammonia that can be used by plants to supplement the nitrogen elements required by plants.

[0003] The symbiotic relationship between legume plants and rhizobium depends on a complex molecular signal exchange process between plants and rhizobium. The receptor-like kinase NFR1 (Nod Factor Receptor 1) and NFR5 on the cell membrane of legume plants can recognize the nodule factor secreted by rhizobium, and initiate the symbiotic signal.

[0004] According to the traditional concept, rhizobium must have nodule genes, such as nodABC genes, and be able to synthesize nodule factors to form nodules with legumes. However, Giraud et al. found that two photosynthetic slow-growing rhizobium BTAi1 and ORS278 do not have conventional nodABC genes in their genomes and cannot synthesize nodule factors, but they can form nodules and fix nitrogen on a tropical legume Aeschynomene (Giraud E, Moulin L, Vallenet D, et al. Legume symbioses: absence of nod genes in photosynthetic bradyrhizobia [J]. Science, 2007, 316(5829): 1307-1312.). This phenomenon has not been found in more rhizobium and legume plants, and most rhizobium still depends on nodule genes to establish symbiotic relationship with legume plants (Chen Wenfeng. Research progress and prospect of rhizobium systematics [J]. Microbiology Bulletin, 2016, 43(5): 1095-1100.).

[0005] Frankia is a genus of actinobacteria, most of which can form symbiotic nodules with non-leguminous plants, and the symbiotic relationship between Frankia and the host is specific, which is not as strict as the symbiotic relationship between rhizobium and leguminous plants. Studies have shown that the symbiosis of actinomycete plants and Frankia is not dependent on the Nod factor pathway, and has some similarities with the symbiotic nodulation of Aeschynomene and photosynthetic slow-growing rhizobium. Through several transcriptome analyses, there is a certain degree of overlap between the genes that positively promote symbiotic nodulation in the interaction between leguminous plants and rhizobium and the interaction between actinomycete plants and Frankia, and there are also some differences.

[0006] As a typical actinomycete plant, Myrica rubra can establish an effective symbiotic system with Frankia and other auxiliary strains to help the plant grow healthily. In-depth study of the specific symbiotic nodulation-promoting genes in Myrica rubra can help discover new symbiotic genes. SUMMARY

[0007] The purpose of the present application is to improve the symbiotic interaction ability of nodulation plants with symbiotic bacteria by in-depth study of the specific symbiotic nodulation-promoting genes in Myrica rubra and applying them to the nodulation plants.

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

[0009] The present application screens a gene that is specifically up-regulated in Myrica rubra nodules through transcriptome analysis of Myrica rubra nodules, and the gene is annotated as Cysteine-rich repeat secretory protein, which is named CRRSP1 gene.

[0010] Cloning and analysis of CRRSP1 gene: CRRSP1 gene is cloned from Myrica rubra nodule tissue, and the CDS sequence of the gene is shown as SEQ ID NO. 1.

[0011] The CDS region of CRRSP1 gene is 693 bp in length, and the amino acid sequence encoded by the CDS region contains 230 amino acid residues, and the amino acid sequence is shown as SEQ ID NO. 2.

[0012] The protein has a molecular weight of 25.2 kD. The functional domain and protein structure analysis prediction result shows that there is a signal peptide region and two regions containing DUF26 motif.

[0013] The present application studies and analyzes the function of Myrica rubra CRRSP1 gene, and finds that the gene has the effect of promoting symbiotic interaction between nodulation plants and symbiotic bacteria. By overexpressing Myrica rubra CRRSP1 gene in nodulation plants, the number of nodules in the root system of the plants can be significantly increased, and the biomass accumulation of the plants can be increased.

[0014] Therefore, the application provides the application of the Myrica rubra CRRSP1 gene in promoting symbiotic interaction of nodule plant and symbiotic bacteria, and the application comprises: up-regulating expression of the Myrica rubra CRRSP1 gene in the nodule plant by using a biological technical means.

[0015] Further, the symbiotic interaction comprises: plant biomass accumulation, and increase of nodule number in the root system.

[0016] Further, the application comprises: cloning the Myrica rubra CRRSP1 gene into an overexpression vector to construct a recombinant plasmid, introducing the target gene in the recombinant plasmid into a receptor plant by using an agrobacterium-mediated technology, and then co-culturing the transgenic plant with the symbiotic bacteria.

[0017] In the application, a suitable overexpression vector and agrobacterium are selected according to the characteristics of the receptor plant to perform the transgenic operation.

[0018] Further, the nodule plant and the symbiotic bacteria can be legume plants and rhizobium independent of a Nod factor channel.

[0019] Further, the legume plant is an Aeschynomene plant, and the rhizobium is Bradyrhizobium sp.

[0020] Further, the Aeschynomene plant is Aeschynomene indica, and the Bradyrhizobium sp. is Bradyrhizobium sp. ORS285.

[0021] Further, the nodule plant and the symbiotic bacteria can also be actinomycete root plants and Frankia.

[0022] Further, the actinomycete root plant is Myrica rubra.

[0023] The application further provides a method for promoting symbiotic nodulation of Aeschynomene and Bradyrhizobium sp.

[0024] (1) cloning the Myrica rubra CRRSP1 gene into an overexpression vector to construct a recombinant plasmid; then transforming the recombinant plasmid into Agrobacterium rhizogenes to obtain positive transformants, and then preparing an infection solution;

[0025] (2) cut the lower hypocotyl of the Aeschynomene indica seedling, immerse the cut surface in the Agrobacterium infection liquid, and then transfer to the culture medium for culture to screen and obtain the transgenic plant;

[0026] (3) transplant the transgenic plant seedling to the culture medium containing Bradyrhizobium slow-growing to culture, and the heterologous expression of the Myrica CRRSP1 gene promotes the Aeschynomene indica and Bradyrhizobium slow-growing to form symbiotic nodulation.

[0027] Further, in step (1), the overexpression vector is PUB-GW-GFP vector, and the promoter in the gene overexpression module is LjUbq promoter.

[0028] Further, in step (1), the Agrobacterium rhizogenes is AR1193 strain.

[0029] When the infection liquid is prepared, the positive transformant is activated and expanded, resuspended in sterile water to prepare a bacterial suspension with an OD 600 = 1.0 concentration, and 20 μg / mL AS is added to prepare the infection liquid.

[0030] Further, in step (2), a clean knife is used to cut the lower hypocotyl of the newly born Aeschynomene indica seedling, a clean forceps is used to clamp the Aeschynomene indica seedling, the cut surface is quickly immersed in the Agrobacterium infection liquid for 20-30 s, and then transferred to the BNM (buffered nod medium) culture medium, the cut surface contacts the culture medium, and then cultured at 22 ℃ in the dark for 2-3 days, cultured at 22 ℃ under light for 2-3 days, and then transferred to the rooting culture medium for rooting culture. The rooting culture medium contains cefotaxime, which is used to remove excess Agrobacterium.

[0031] The present application has the beneficial effects:

[0032] (1) The present application provides a CRRSP1 gene specifically up-regulated in Myrica nitraria root nodules, and the up-regulation of the gene in the nodulation plant by using biological technical means can significantly promote the symbiotic nodulation with symbiotic bacteria and increase the plant biomass. The present application provides an important reference value for the research of symbiotic genes.

[0033] (2) The present application introduces the Myrica nitraria CRRSP1 gene into the legume Aeschynomene indica by Agrobacterium-mediated technology to realize heterologous overexpression. Compared with the Aeschynomene indica plant expressing empty vector, the total number of root nodules of the Aeschynomene indica plant overexpressing the Myrica nitraria CRRSP1 gene is significantly increased, and the aboveground biomass is significantly increased. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a cDNA amplification electrophoretogram of the Myrica nitraria CRRSP1 gene, wherein each lane is DNA marker; and CRRSP1 gene.

[0035] Figure 2 Figure for the expression vector of CRRSP1 gene in Myrica rubra.

[0036] Figure 3 Figure for the morphology of transgenic plants expressing and overexpressing CRRSP1 gene in Myrica rubra without inoculation of rhizobia.

[0037] Figure 4 Figure for the morphology of transgenic plants expressing and overexpressing CRRSP1 gene in Myrica rubra after inoculation of rhizobia.

[0038] Figure 5 Figure for the nodule of transgenic plants expressing and overexpressing CRRSP1 gene in Myrica rubra after inoculation of rhizobia under white light and under GFP fluorescence.

[0039] Figure 6 Figure for the nodule number of each plant of transgenic plants expressing and overexpressing CRRSP1 gene in Myrica rubra after inoculation of rhizobia.

[0040] Figure 7 Figure for the fresh weight of aboveground part of each plant (A) and total fresh weight of each plant (B) of transgenic plants expressing and overexpressing CRRSP1 gene in Myrica rubra after inoculation of rhizobia. DETAILED DESCRIPTION

[0041] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application, and are not used to limit the application scope. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0042] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0043] Example 1: Amplification of the full-length of CRRSP1 gene in Myrica rubra

[0044] 1. The sequence of CRRSP1 gene in Myrica rubra was obtained by searching the root nodule RNA-seq data of Myrica rubra (the search number of CRRSP1 gene was gene_scaffold_9_g34767_t1), the nucleotide sequence of the protein coding region was shown as SEQ ID NO. 1, and the amino acid sequence of the protein coding region was shown as SEQ ID NO. 2. The functional domain and protein structure analysis prediction results showed that there was a signal peptide region and two regions containing DUF26 motif, which was named CRRSP1.

[0045] According to the Gateway manual, the forward primer and the reverse primer were designed as follows:

[0046] Forward primer:

[0047] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGTCTCTCTT GTTGCTAACCT-3';

[0048] Reverse primer:

[0049] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCGAAGGTTTCGTA TGCCGTG-3'.

[0050] 2, using CTAB method to extract the nodule tissue RNA of Myrica rubra, the method is as follows:

[0051] (1) with 2ml clean centrifuge tube, take the right amount of ground Myrica rubra nodule sample (from the root system tissue of Myrica rubra, 0.3g), add 1ml CTAB, mix quickly, then add 3ml CTAB, vortex for 20s, and put into 65℃ water bath for 5min;

[0052] (2) add 1ml chloroform: isopropyl alcohol (24:1) to each tube, vortex fully; then centrifuge at 10000rpm for 10min at 15℃;

[0053] (3) take the supernatant to a new centrifuge tube, add 1ml chloroform: isopropyl alcohol (24:1), vortex fully; then centrifuge at 10000rpm for 10min at 15℃;

[0054] (4) take the supernatant to a new centrifuge tube, try not to suck the middle layer;

[0055] (5) add 1 / 4 of 10M LiCl;

[0056] (6) 4℃ refrigerator overnight;

[0057] (7) the next day, prepare the centrifuge to be cooled at 4℃, SSTE 65℃ heating, anhydrous ethanol-20℃ precooling;

[0058] (8) centrifuge at 10000rpm for 30min at 4℃; prepare ice;

[0059] (9) discard the supernatant, and gently suck out the residual supernatant and discard. Add 400ul SSTE, mix well;

[0060] (10) add 400ul chloroform: isopropyl alcohol (24:1), vortex mix, transfer to 1.5ml centrifuge tube;

[0061] (11) centrifuge at 10000rpm for 10min at 4℃;

[0062] (12) The supernatant was sucked into a new centrifuge tube, and two volumes of anhydrous ethanol pre-cooled at -20°C were added. The mixture was inverted up and down to mix, and was placed at -80°C for 4 hours;

[0063] (13) The sample was taken out from -80°C, and was centrifuged at 4°C and 10,000 rpm for 20 minutes;

[0064] (14) The supernatant was removed, and the residual liquid was sucked out. The precipitate was blown dry in a fume hood;

[0065] (15) An appropriate amount of sterilized water was added to each tube to dissolve the precipitate. The quality of the RNA was detected by gel electrophoresis.

[0066] 3. cDNA was obtained by reverse transcription, and PCR amplification was performed using KOD high-fidelity enzyme of TOYOBO Company.

[0067] The PCR reaction system was as follows: 10x KOD Plus Neo Buffer 5 μL, 2 mM dNTP 5 μL, 25 mM MgSO4 3 μL, 1.5 μL of each of the upper and lower primers, 1 μL of template cDNA, 1 μL of KOD Plus Neo, and ddH2O was added to 50 μL.

[0068] The PCR cycle conditions were as follows: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 68°C for 1 minute, cycle 2-4 for 29 cycles, extension at 68°C for 10 minutes, and low-temperature storage at 10°C for 30 minutes.

[0069] The PCR product was detected by gel electrophoresis, and the detection results are shown in Figure 1 .

[0070] Example 2: Construction of overexpression vector of CRRSP1 gene of Myrica rubra

[0071] The PCR product of Example 1 was subjected to BP recombination reaction with the vector pDONR-Zeo. The ligation product was transformed into Escherichia coli DH5α strain by heat shock. Positive clones were picked and added into LB liquid medium containing Zeocin resistance. The culture was incubated at 37°C for 10-12 hours on a 220 rpm shaker. The plasmid was extracted, and was subjected to enzyme digestion and PCR identification. After correct identification, the plasmid was sent to TSINGKE Biological Technology Company for sequence sequencing.

[0072] The correct sequencing plasmid was subjected to LR recombination reaction with PUB-GW-GFP empty vector, and the ligation product was transformed into E. coli DH5a strain by heat shock. Positive clones were selected and added into LB liquid medium containing Kan resistance, and cultured at 37°C for 10-12 hours at 220 rpm. The plasmid was extracted, and after enzyme digestion and PCR identification, the construction of PUB-CRRSP1 vector was completed, as shown in Figure 2 .

[0073] Example 3: Transformation of CRRSP1 gene of Myrica rubra into mung bean root

[0074] 1. Seed disinfection and germination: Fresh mung bean (A. indica) seeds (purchased from Longyou County Agricultural Market in Quzhou City, Zhejiang Province) were prepared, and 95% concentrated sulfuric acid was used for disinfection for 5-7 minutes. The centrifuge tube was placed on ice, and ddH2O was used for washing 3 times. Then, 10% NaClO was used for disinfection for 5-7 minutes, and the waste liquid was discarded. ddH2O was used for washing 5-7 times, and the seeds were stored at 4°C overnight. The next day, the seeds were inoculated on 0.8% water agar plates, and then cultured under light for 1-2 days until the seeds germinated.

[0075] 2. Agrobacterium rhizogenes infection: The plasmid constructed in Example 2 was transformed into Agrobacterium rhizogenes AR1193, and PCR identification showed that it was a positive transformant. The strain was preserved and streaked for activation, and cultured at 28°C for 1-2 days. The day before infection, a single colony was inoculated into 5 mL of LB liquid medium (if there is no special situation, LB+Kan+Rif should be used here, Kan is the resistance carried by the binary vector, and Rif is the resistance carried by AR1193). The culture was incubated at 28°C, 200 rpm for 18 hours. The bacterial cells were collected by centrifugation at 4,200 rpm for 10 minutes, resuspended in sterile water, and 20 μg / mL AS was added to the resuspended bacterial solution.

[0076] The lower hypocotyls of mung bean seedlings were cut on the plate, and the cuttings were cut at the lower position of the green stem and white root. Then, the cut seedlings were transferred to the resuspended bacterial solution and soaked for several seconds. The seedlings were transferred to BNM slant medium, the cut surface was in contact with the medium, and the plate was sealed with parafilm. The plate was incubated at 22°C in the dark for 2-3 days. The seedlings after dark incubation for 2-3 days were transferred to light, and incubated at 22°C under light for 2-3 days.

[0077] 3. Rooting culture and identification of positive seedlings: The seedlings were transferred to BNM rooting medium (containing 100 μg / mL cef for inhibiting and killing Agrobacterium), and the seedlings were aligned (cotyledons on top, wounds on bottom). After sealing with parafilm, the plate was placed in a 22°C light incubator at an angle of 70-90°, and incubated for 8-10 days. Under the stereoscopic fluorescence microscope, the rooting plate was placed under a black bottom plate, and the positive seedlings were selected by passing through the fluorescence barrier. The non-positive roots without GFP label were removed.

[0078] Example 4: Inoculation of Bradyrhizobium sp. ORS285 strain and phenotype statistics

[0079] The identified positive seedlings were transplanted into flowerpots and cultured in a light culture room (16 h light, 8 h dark) at 25°C. Sterile vermiculite: perlite (3:1) mixed substrate was added in advance in the flowerpots. After 2-3 days, Bradyrhizobium sp. ORS285 strain growing to the logarithmic growth phase was inoculated, and an appropriate amount of equal volume was poured for each pot. After 4 weeks of inoculation, the physiological phenotype of the A. crassicarpa plants expressing empty vector and the A. crassicarpa plants overexpressing the Myrica rubra CRRSP1 gene was counted.

[0080] As shown in Figure 3 , there was little difference in morphology between the A. crassicarpa plants expressing empty vector and the A. crassicarpa plants overexpressing the Myrica rubra CRRSP1 gene without inoculation of Bradyrhizobium, and the growth was similar.

[0081] As shown in Figures 4-7 , after 4 weeks of inoculation of Bradyrhizobium, compared with the control vector expression, the A. crassicarpa plants overexpressing the Myrica rubra CRRSP1 gene grew more robustly, the aboveground biomass accumulation was more, and the number of root nodules in the root system was significantly increased.

[0082] In summary, we found a specific symbiotic gene CRRSP1 in Myrica rubra, which significantly promoted the symbiotic nodulation process of A. indica and Bradyrhizobium sp. ORS285 after heterologous expression.

[0083] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solution obtained by equivalent substitution or equivalent transformation falls within the protection scope of the present application.

Claims

1. A raspberry CRRSP1 gene, characterized in that, The CDS sequence thereof is shown as SEQ ID NO.

1.

2. The raspberry of claim 1 CRRSP1 The use of the gene in promoting the symbiotic interaction of nodulated plants with symbiotic bacteria, characterized in that, The applications include: using biological techniques to upregulate the expression of Myrica rubra in the nodulating plants. CRRSP1 Genes; the nodulating plant is a syn-budding plant ( Aeschynomene indica The symbiotic bacteria are slow-growing rhizobia ( Bradyrhizobium sp.); The symbiotic interaction is: plant biomass accumulation and an increase in the number of root nodules in the root system.

3. Use according to claim 2, wherein the compound is ###0002### The application comprises cloning the Myrica rubra CRRSP1 gene into an overexpression vector to construct a recombinant plasmid, introducing the target gene in the recombinant plasmid into a receptor plant through an agrobacterium-mediated technique, and then co-culturing the transgenic plant with symbiotic bacteria.

4. A method of promoting symbiotic nodulation of Aeschynomene with Bradyrhizobium sp., characterized by, The method comprises the following steps: (1) The waxberry as claimed in claim 1 CRRSP1 The gene is cloned into an overexpression vector to construct a recombinant plasmid; the recombinant plasmid is transformed into Agrobacterium rhizogenes to obtain positive transformants, and then the positive transformants are prepared into an infection solution; (2) The lower embryo axis of the Aeschynomene indica seedling is cut, the cut surface is soaked in the agrobacterium infection solution, and then the cut surface is transferred to the culture medium for culture, and a transgenic plant is obtained through screening; (3) Transgenic plant seedlings are transplanted into culture medium containing Bradyrhizobium, and the seedlings are cultured in the medium. The Bradyrhizobium is capable of promoting the seedlings to form symbiotic nodules with Bradyrhizobium. CRRSP1 The Bradyrhizobium is capable of promoting the seedlings to form symbiotic nodules with Bradyrhizobium after the heterologous expression of the gene.

5. The method of claim 4, wherein, In step (1), the overexpression vector is PUB-GW-GFP vector, and the promoter in the gene overexpression module is LjUbq promoter.

6. The method of claim 4, wherein, In step (2), the cut surface of the Aeschynomene indica seedling is infected in the agrobacterium infection solution for 20-30 s, and then transferred to the BNM culture medium, the cut surface contacts the culture medium, and then cultured at 22 DEG C in the dark for 2-3 days, and then cultured at 22 DEG C in the light for 2-3 days, and then transferred to the rooting culture medium for rooting culture.

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