Red bayberry CRRSP1 gene and application thereof

Abstract

The invention discloses a waxberry CRRSP1 gene and application thereof, and belongs to the technical field of gene engineering. The invention provides a CRRSP1 gene specifically up-regulated in myrica rubra root nodules. The CDS sequence of the CRRSP1 gene is shown as SEQ ID NO. 1. The invention also provides an application of the red bayberry CRRSP1 gene in promoting symbiotic interaction of a nodulation plant and a symbiotic bacterium. The application comprises up-regulation expression of the red bayberry CRRSP1 gene in the nodulation plant by using a biological technical means. Symbiotic nodulation of the nodulated plant and symbiotic bacteria can be remarkably promoted by up-regulating expression of the gene in the nodulated plant, and the biomass of the plant is increased. The invention provides an important reference value for the research of symbiotic genes.

Description

Technical Field

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

[0002] Symbiosis in nature is mainly divided into two categories, the interaction between legumes and rhizobia and the interaction between actinomycete rhizobia and Frankia. Both types of symbiosis will form special symbiotic structures - nodules. The symbiotic bacteria use nitrogen in the atmosphere to convert it into ammonia that can be used by plants, thereby supplementing the nitrogen elements needed by plants.

[0003] The symbiotic relationship between legumes and rhizobia depends on the complex molecular signal exchange process between plants and rhizobia. The receptor-like kinases NFR1 (Nod Factor Receptor 1) and NFR5 on the cell membrane of legumes can recognize the nodulation factor secreted by rhizobia and initiate symbiotic signals.

[0004] Traditionally, it is believed that in order for rhizobia to form nodules with legumes, they must have nodulation genes, such as nodABC genes, and be able to synthesize nodulation factors. However, Giraud et al. found that two strains of photosynthetic bradyrhizobia, BTAi1 and ORS278, did not have conventional nodABC genes in their genomes and could not synthesize nodulation factors, but could nodulate and fix nitrogen on a tropical legume plant, the genus Aeschynomene (Giraud E, Moulin L, Vallenet D, et al. Legumes symbioses: absence of nod genes in photosynthetic bradyrhizobia [J]. Science, 2007, 316 (5829): 1307–1312.). This phenomenon has not been found in more rhizobia and legumes, and most rhizobia still rely on nodulation genes to establish symbiotic relationships with legumes (Chen Wenfeng. Progress and Prospects in Rhizobium Systematics Research [J]. Bulletin of Microbiology, 2016, 43(5): 1095-1100.).

[0005] Frankia is a genus of actinomycetes. Most Frankia can form symbiotic nodules and fix nitrogen with non-leguminous plants. The specificity of the symbiotic relationship between Frankia and its host is not as strict as that between rhizobia and leguminous plants. Studies have shown that the symbiosis between actinomycete rhizobium and Frankia does not depend on the Nodfactor pathway, and has certain similarities with the symbiotic nodulation of syngenetic and photosynthetic bradyrhizobia. Through the analysis of several transcriptomes, the genes that positively promote symbiotic nodulation in the interaction between leguminous plants and rhizobia and the interaction between actinomycete rhizobium and Frankia have a certain degree of overlap, and there are also some differences.

[0006] Bayberry, as a typical actinomycete, interacts with Frankia and other auxiliary strains to establish an effective symbiotic system to help plants grow healthily. In-depth research on the specific genes in bayberry that promote symbiotic nodulation will help discover new symbiotic genes. Summary of the invention

[0007] The purpose of the present invention is to conduct in-depth research on the specific genes promoting symbiotic nodulation in bayberry and apply them to nodulation plants to enhance their symbiotic interaction ability with symbiotic bacteria.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention screens out a gene specifically up-regulated in bayberry nodules by transcriptome analysis of bayberry nodules, and the gene is annotated as Cysteine-rich repeat secretory protein, which is named as CRRSP1 gene.

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

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

[0012] The molecular weight of the protein is 25.2kD. The prediction results of functional domain and protein structure analysis showed that there is a signal peptide region and two regions containing DUF26 motifs.

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

[0014] Therefore, the present invention provides the use of the bayberry CRRSP1 gene in promoting the symbiotic interaction between nodulation plants and symbiotic bacteria, and the use comprises: using biological technology means to up-regulate the expression of the bayberry CRRSP1 gene in the nodulation plants.

[0015] Furthermore, the symbiotic interaction includes: accumulation of plant biomass and increase in the number of nodules in the root system. The growth ability of nodulated plants is enhanced by promoting the symbiotic interaction with symbiotic bacteria.

[0016] Furthermore, the application includes: cloning the bayberry CRRSP1 gene into an overexpression vector to construct a recombinant plasmid, introducing the target gene in the recombinant plasmid into a recipient plant through Agrobacterium-mediated technology, and then co-culturing the transgenic plant with symbiotic bacteria.

[0017] In the present invention, suitable overexpression vectors and Agrobacterium are selected for transgenic operation according to the characteristics of the recipient plant. The method for up-regulating the expression of the waxberry CRRSP1 gene in the present invention is not limited to the use of transgenic technology.

[0018] Furthermore, the nodulation plants and symbiotic bacteria may be leguminous plants and rhizobia that are not dependent on the Nod factor pathway.

[0019] Furthermore, the legume plant is a plant of the genus Aeschynomene, and the rhizobium is Bradyrhizobium sp.

[0020] Furthermore, the plant of the genus Aeschynomene is Aeschynomene indica, and the bradyrhizobium is Bradyrhizobium sp. ORS285. The present invention shows that heterologous overexpression of the Myrica rubra CRRSP1 gene in Aeschynomene can significantly promote the symbiotic nodulation process of A. indica and Bradyrhizobium sp. ORS285.

[0021] Furthermore, the nodulation plants and symbiotic bacteria may also be actinomycorrhizal plants and Frankia.

[0022] Furthermore, the actinomycete rhizome plant is Myrica rubra.

[0023] The present invention also provides a method for promoting symbiotic nodulation of bradyrhizobium, comprising the following steps:

[0024] (1) cloning the bayberry 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) cutting off the hypocotyl of the fertilized seedlings, soaking the cut surface in an Agrobacterium infection solution, transferring it to a culture medium for cultivation, and screening to obtain transgenic plants;

[0026] (3) The transgenic seedlings were transplanted into a culture medium containing Bradyrhizobium and cultured. The heterologous expression of the Bayberry CRRSP1 gene promoted symbiotic nodulation with Bradyrhizobium.

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

[0028] Furthermore, in step (1), the Agrobacterium rhizogenes adopts the AR1193 strain.

[0029] When preparing the infection solution, the positive transformants were activated and expanded, and then resuspended in sterile water to a concentration of OD 600 =1.0 bacterial suspension, add AS with a final concentration of 20 μg / mL to prepare the infection solution.

[0030] Furthermore, in step (2), the hypocotyl of the newly grown seedling is cut off with a clean knife, and the seedling is picked up with clean tweezers, and the cut surface is quickly immersed in the Agrobacterium infection solution for infection for 20-30 seconds, and then transferred to the BNM (buffered nodal medium) culture medium, the cut surface is in contact with the culture medium, and the culture is carried out at 22° C. in the dark for 2-3 days, and then at 22° C. in the light for 2-3 days, and then transferred to the rooting culture medium for rooting culture. The rooting culture medium contains cephalosporin, which is used to remove excess Agrobacterium.

[0031] The present invention has the following beneficial effects:

[0032] (1) The present invention provides a CRRSP1 gene that is specifically upregulated in bayberry root nodules. Upregulating the expression of this gene in nodulated plants using biological techniques can significantly promote symbiotic nodulation with symbiotic bacteria and increase plant biomass. The present invention provides important reference value for the study of symbiotic genes.

[0033] (2) The present invention introduces the Myrica rubra CRRSP1 gene into the legume plant zygote through Agrobacterium-mediated technology to achieve heterologous overexpression. Compared with the zygote plants expressing no vector, the total number of nodules and the aboveground biomass of the zygote plants overexpressing the Myrica rubra CRRSP1 gene are significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the electrophoresis diagram of cDNA amplification of Bayberry CRRSP1 gene; wherein each lane is DNA marker; CRRSP1 gene.

[0035] Figure 2 This is a map of the Bayberry CRRSP1 gene expression vector.

[0036] Figure 3 The morphological pictures of transgenic plants expressing the control vector and overexpressing the Bayberry CRRSP1 gene without inoculation of rhizobia.

[0037] Figure 4 These are the morphological pictures of the zygote plants expressing the control vector and overexpressing the Bayberry CRRSP1 gene after inoculation with rhizobia.

[0038] Figure 5 These are images of nodules of zygotic plants expressing the control vector and overexpressing the Bayberry CRRSP1 gene after inoculation with rhizobia under white light and GFP fluorescence.

[0039] Figure 6 The number of nodules per plant was counted after rhizobia were inoculated on the control vector-expressing and overexpressing bayberry CRRSP1 gene zymophytes.

[0040] Figure 7 Statistics of the aboveground fresh weight of each plant (A) and the total fresh weight of each plant (B) after inoculation of rhizobia in the control vector-expressing and overexpressing bayberry CRRSP1 gene zygotes. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] Example 1: Amplification of the full length of the CRRSP1 gene in bayberry

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

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

[0046] Forward Primer:

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

[0048] Reverse Primer:

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

[0050] 2. Extract RNA from the nodule tissue of Bayberry using the CTAB method as follows:

[0051] (1) Weigh an appropriate amount of ground bayberry nodule sample (0.3 g from the root tissue of Bayberry cultivar) in a 2 mL clean centrifuge tube, add 1 mL of CTAB and mix quickly, then add 3 mL of CTAB, vortex for 20 seconds, and place in a 65 °C water bath for 5 minutes;

[0052] (2) Add 1 mL of chloroform:isoamyl alcohol (24:1) to each tube and vortex thoroughly; then centrifuge at 15°C, 10,000 rpm for 10 min;

[0053] (3) Take the supernatant and transfer it to a new centrifuge tube. Add 1 mL of chloroform:isoamyl alcohol (24:1) and vortex thoroughly. Centrifuge at 15°C, 10,000 rpm for 10 min.

[0054] (4) Pipette the supernatant into a new centrifuge tube, and try not to aspirate the middle layer;

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

[0056] (6) Refrigerate at 4°C overnight;

[0057] (7) The next day, prepare the centrifuge to precool to 4°C, heat SSTE to 65°C, and precool anhydrous ethanol to -20°C;

[0058] (8) Centrifuge at 10,000 rpm for 30 min at 4°C; prepare ice cubes;

[0059] (9) Discard the supernatant and gently aspirate and discard the remaining supernatant. Add 400 μL SSTE and mix well;

[0060] (10) Add 400 μL of chloroform:isoamyl alcohol (24:1), vortex to mix, and transfer to a 1.5 mL centrifuge tube;

[0061] (11) Centrifugation at 10,000 rpm for 10 min at 4°C;

[0062] (12) Remove the supernatant and transfer it to a new centrifuge tube. Add twice the volume of -20°C precooled anhydrous ethanol, mix by inverting, and place at -80°C for 4 h.

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

[0064] (14) Remove the supernatant, aspirate the residual liquid, and blow dry the precipitate in a fume hood;

[0065] (15) Add an appropriate amount of sterile water to each tube to dissolve the precipitate and detect the RNA quality by gel electrophoresis.

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

[0067] PCR reaction system: 10×KOD Plus Neo Buffer 5μL, 2mM dNTP 5μL, 25mM MgSO 4 3μL, 1.5μL each of upstream and downstream primers, 1μL template cDNA, 1μL KOD Plus Neo, 1μL ddH 2 Add HO to make up to 50 μL.

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

[0069] The PCR products were tested by gel electrophoresis. Figure 1 shown.

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

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

[0072] The correctly sequenced plasmid was subjected to LR recombination reaction with the PUB-GW-GFP empty vector. The ligation product was transformed into the E. coli DH5α strain by heat shock. The positive clone was picked and added to the LB liquid medium containing Kan resistance. The culture was shaken at 37℃ and 220rpm for 10-12 hours. The plasmid was extracted. After restriction digestion and PCR identification, the PUB-CRRSP1 vector construction was completed. Figure 2 .

[0073] Example 3: Transformation of Myrica rubra with the CRRSP1 gene

[0074] 1. Seed disinfection and germination: Prepare fresh A. indica seeds (purchased from Longyou County Farmers' Market, Quzhou City, Zhejiang Province), disinfect with 95% concentrated sulfuric acid for 5-7 minutes, discard the waste liquid, place the centrifuge tube on ice, and rinse with ddH 2 After washing 3 times with 10% NaClO, disinfect for 5-7 min, discard the waste solution, and replace with ddHO. 2 O, wash 5-7 times, incubate at 4°C overnight, plant the seeds on 0.8% water agar plates the next day, and then culture under light for 1-2 days until the seeds germinate.

[0075] 2. Infection of Agrobacterium rhizogenes: The plasmid to be transformed constructed in Example 2 was transferred into Agrobacterium rhizogenes AR1193, and the positive transformants were identified by PCR. The strain was preserved and activated by streaking, and cultured at 28°C for 1-2 days. One day before infection, a single colony was inoculated in 5 mL LB liquid culture medium (if there are no special circumstances, LB+Kan+Rif should be used here, Kan is the resistance carried by the binary vector, and Rif is the resistance carried by AR1193), and cultured at 28°C, 200 rpm for 18 hours. 4,200 rpm, 10 min centrifugation was performed to collect the bacteria, resuspended in sterile water, and a final concentration of 20 μg / mL AS was added to the resuspended bacteria.

[0076] Cut the hypocotyl of the seedlings on the plate, cut below the green stem and white root, then transfer the cut seedlings to the resuspended bacterial solution and soak the slices for tens of seconds. Transfer the seedlings to the BNM slant culture medium, with the cut contacting the culture medium, seal the plate with sealing film, and culture in the dark at 22℃ for 2-3 days. Transfer the seedlings after 2-3 days of dark culture to the light, and culture them in the light at 22℃ for 2-3 days.

[0077] 3. Rooting culture and identification of positive seedlings: Move the seedlings to BNM rooting medium (containing 100 μg / mL cef, used to inhibit and kill Agrobacterium), keep the seedlings in the same direction (cotyledons on top and wounds on the bottom), seal with sealing film, place in a 22°C light incubator, tilt 70-90°, and culture for 8-10 days. Under a stereo fluorescence microscope, place the rooting plate under a black bottom plate, select positive seedlings through the fluorescent baffle, and remove non-positive roots without GFP tags.

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

[0079] The positive seedlings were transplanted into pots and cultured in a light culture room (16h light, 8h dark) at 25℃. A matrix of sterilized vermiculite:perlite (3:1) was added to the pots in advance. After 2-3 days, the rhizobium Bradyrhizobium sp.ORS285 strain grown to the logarithmic growth phase was inoculated, and an appropriate amount of equal volume was poured into each pot. Four weeks after inoculation, the physiological phenotypes of the zygote plants expressing no vector and the zygote plants overexpressing the Bayberry CRRSP1 gene were statistically analyzed.

[0080] like Figure 3 As shown, without inoculation of rhizobia, the morphological differences between the control vector-expressing and overexpressing bayberry CRRSP1 gene plants were not significant, and their growth potentials were similar.

[0081] like Figure 4-7 As shown, 4 weeks after inoculation with rhizobia, compared with the control vector expression, the zygote plants overexpressing the Bayberry CRRSP1 gene grew more robustly, accumulated more biomass in the aboveground part, and the number of nodules in the root system was significantly increased.

[0082] In summary, we discovered a specific symbiotic gene CRRSP1 in bayberry, 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 invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A Myrica rubra CRRSP1 gene, characterized in that: Its CDS sequence is shown in SEQ ID NO.

1.

2. The use of the waxberry CRRSP1 gene in promoting the symbiotic interaction between nodulation plants and symbiotic bacteria as claimed in claim 1, characterized in that: The application comprises: utilizing biological technology to up-regulate the expression of the Myrica rubra CRRSP1 gene in the nodulation plant.

3. The use according to claim 2, characterized in that The symbiotic interaction includes accumulation of plant biomass and increase in the number of nodules in the root system.

4. The use according to claim 2, characterized in that The application comprises: cloning the bayberry CRRSP gene into an overexpression vector to construct a recombinant plasmid, introducing the target gene in the recombinant plasmid into a recipient plant through Agrobacterium-mediated technology, and then co-culturing the transgenic plant with symbiotic bacteria.

5. The use according to any one of claims 2 to 4, characterized in that: The nodulation plant and the symbiotic bacteria are leguminous plants and rhizobia that are not dependent on the Nod factor pathway, or actinomycete rhizobium plants and Frankia.

6. The use according to claim 5, characterized in that The legume plant is a plant of the genus Aeschynomene, and the rhizobium is Bradyrhizobium sp.

7. The use according to claim 5, characterized in that The actinomycete root plant is bayberry.

8. A method for promoting symbiotic nodulation of bradyrhizobium, characterized in that: The following steps are involved: (1) Cloning the Myrica rubra CRRSP1 gene as described in claim 1 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; (2) cutting off the hypocotyl of the fertilized seedlings, soaking the cut surface in an Agrobacterium infection solution, transferring the cut surface to a culture medium for cultivation, and screening to obtain transgenic plants; (3) The transgenic seedlings were transplanted into a culture medium containing Bradyrhizobium and cultured. The heterologous expression of the Bayberry CRRSP1 gene promoted symbiotic nodulation with Bradyrhizobium.

9. The method according to claim 8, characterized in that In step (1), the overexpression vector uses the PUB-GW-GFP vector, and the promoter in the gene overexpression module uses the LjUbq promoter.

10. The method according to claim 8, characterized in that In step (2), the cut surface of the seedlings is infected in the Agrobacterium infection solution for 20-30 seconds and then transferred to BNM medium, with the cut surface in contact with the medium, cultured at 22° C. in the dark for 2-3 days, and then cultured at 22° C. in the light for 2-3 days, and then transferred to rooting medium for rooting culture.

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