CN108220321B

CN108220321B - Method for monitoring rhizobia colonization process in non-leguminous plants

Info

Publication number: CN108220321B
Application number: CN201611127119.0A
Authority: CN
Inventors: 沈世华; 荆玉祥; 彭献军; 吴青青
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2016-12-09
Filing date: 2016-12-09
Publication date: 2020-07-24
Anticipated expiration: 2036-12-09
Also published as: CN108220321A

Abstract

The invention discloses a method for monitoring the colonization process of rhizobia in non-leguminous plants. The method disclosed by the invention comprises the following steps: introducing the EGFP coding gene into rhizobia to obtain recombinant rhizobia; inoculating the non-leguminous plant with the recombinant rhizobia, and monitoring the colonization process of the recombinant rhizobia in the non-leguminous plant; EGFP is a protein with the amino acid sequence of sequence 2; introduction of the gene encoding EGFP into rhizobia to introduce an expression vector containing the gene encoding EGFP into rhizobia. Experiments prove that after the recombinant rhizobia containing the EGFP coding gene is used for inoculating the non-leguminous plants, the detected rhizobia in the plant bodies are respectively obviously higher than that of the non-leguminous plants inoculated by the recombinant rhizobia containing the GFP coding gene, and the experiments show that the EGFP is used for marking the rhizobia, so that the detection accuracy can be improved, and the colonization process of the rhizobia in rice and wheat can be monitored more accurately.

Description

Method for monitoring rhizobia colonization process in non-leguminous plants

Technical Field

The invention relates to a method for monitoring the colonization process of rhizobia in non-leguminous plants in the field of biotechnology.

Background

The rhizobia not only can form symbiotic nitrogen-fixing root nodules on the roots of leguminous plants, but also can form endogenous combined action with the roots of important cereal crops under natural conditions. Although endophytes are widely present in plants, the manner in which rhizobia colonize plant roots is unclear. The manner in which the endogenous rhizobia enter non-legumes is different from the manner and distribution in which rhizobia enter the roots of most legumes. The rhizobia invades leguminous plants, mostly by means of root hairs forming invasion lines into local root cortex cells, and divides to form nodules. The rhizobia of only a few leguminous plants, such as peanuts and sesbania, form nodules by entering cortical cells of the root through lateral root fissures. Apart from the sesbania rhizobia entering sesbania roots to form nodules and the presence of endogenous rhizobia in the xylem, no other rhizobia has been found to exist as endogenous bacteria after entering the roots of legumes.

The current marker genes comprise a luminescent enzyme gene (L genes, luxAB), a β -glucosidase gene (β -Glucuronidase gene, gusA), a Green Fluorescent Protein gene (gfp) and a Red Fluorescent Protein gene (rfp), wherein the gfp gene marker has the advantages of rapidness, convenience and accuracy.

Sinorhizobium meliloti 1021 can promote growth of non-leguminous plants rice and wheat, however, the migration and colonization processes of Sinorhizobium meliloti 1021 as an endophyte in rice and wheat are not known, and the molecular mechanism of interaction with wheat is not clear at present. The method has the advantages that the Sinorhizobium meliloti is marked by gfp, the colonization process of the Sinorhizobium meliloti in the wheat body is monitored, and a foundation is laid for the deep research of the growth promoting mechanism of the Sinorhizobium meliloti on non-leguminous plants.

Disclosure of Invention

The invention aims to solve the technical problem of how to monitor the colonization process of rhizobia in plants.

In order to solve the technical problems, the invention firstly provides a method for monitoring the colonization process of rhizobia in plants

The method for monitoring the colonization process of rhizobia in the plant, provided by the invention, comprises the following steps: introducing the EGFP coding gene into rhizobia to obtain recombinant rhizobia; inoculating a plant with the recombinant rhizobia, and monitoring the process of colonization of the plant by the recombinant rhizobia.

In the above method, the EGFP may have the following sequence a1), a2), or A3):

A1) the amino acid sequence is the protein of sequence 2;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and codes EGFP;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein in A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 2 in the sequence table can be connected with the tags shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10(Usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The above A2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The EGFP in A2) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding EGFP in A2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

In the above method, the introduction of the gene encoding EGFP into rhizobia may be introducing an expression vector containing the gene encoding EGFP into the rhizobia.

In the above method, the EGFP-encoding gene in the expression vector may be promoted by lacZ gene promoter.

In one embodiment of the present invention, the expression vector is a vector expressing EGFP represented by the sequence 2, which is obtained by replacing the DNA fragment between the BamHI and XbaI recognition sequences of the plasmid pMP2444 with EGFP gene represented by the sequence 1.

In the above method, the EGFP-encoding gene may be any one of the following DNA molecules a11) -a 13):

A11) the coding sequence is cDNA molecule or genome DNA of sequence 1 in the sequence table;

A12) cDNA molecule or genomic DNA hybridizing under stringent conditions to the DNA molecule defined in a11) and encoding EGFP;

A13) a cDNA molecule or genomic DNA having 75% or more identity to a DNA molecule defined in a11) or a12) and encoding EGFP.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 encodes EGFP protein shown in the sequence 2.

The nucleotide sequence encoding EGFP of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the EGFP isolated according to the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode the EGFP and have the function of EGFP.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above method, the stringent conditions are hybridization and washing of the membrane at 68 ℃ for 2 times 5min in a solution of 2 × SSC, 0.1% SDS, and hybridization and washing of the membrane at 68 ℃ for 2 times 15min in a solution of 0.5 × SSC, 0.1% SDS, or hybridization and washing of the membrane at 65 ℃ in a solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above method, the plant may be any one of the following a1) -a 4):

a1) a monocot or dicot;

a2) non-leguminous plants or leguminous plants;

a3) a gramineous plant;

a4) wheat or rice.

In the above method, the rhizobia may be b1) or b2) below:

b1) rhizobia of the genus sinorhizobium;

b2) sinorhizobium meliloti (e.g., Sinorhizobium meliloti 1021).

In the above method, the monitoring of the colonization of the plant by the recombinant rhizobia may be performed by fluorescence microscopy (e.g., confocal laser microscopy) or by isolating the recombinant rhizobia from the plant tissue.

In the method, when the colonization process of the recombinant rhizobia in the plant is monitored by a fluorescence microscope, a 488nm wavelength filter can be used for capturing green fluorescence emitted by the recombinant rhizobia in the plant tissue.

In the method, the recombinant rhizobia in the plant tissue is separated, and the plant tissue to be detected can be crushed and then cultured on a culture medium to obtain the recombinant rhizobia in the plant tissue to be detected.

In order to solve the technical problems, the invention also provides application of the method in culturing nitrogen-fixing plants.

In the above application, the plant may be any one of the following a1) -a 4):

a1) a monocot or dicot;

a2) non-leguminous plants or leguminous plants;

a3) a gramineous plant;

a4) wheat or rice.

In the above application, the rhizobia may be b1) or b2) as follows:

b1) rhizobia of the genus sinorhizobium;

b2) sinorhizobium meliloti (e.g., Sinorhizobium meliloti 1021).

In order to solve the technical problem, the invention also provides any one of the following applications X1-X4:

the application of X1 and EGFP in monitoring the colonization process of rhizobia in plants;

the application of X2 and biological materials related to EGFP in monitoring the colonization process of rhizobia in plants;

the biomaterial is any one of the following B1) to B16):

B1) a nucleic acid molecule encoding an EGFP;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) transgenic plant tissue comprising the nucleic acid molecule of B1);

B12) transgenic plant tissue comprising the expression cassette of B2);

B13) a transgenic plant organ containing the nucleic acid molecule of B1);

B14) a transgenic plant organ containing the expression cassette of B2);

application of X3 and EGFP in cultivating nitrogen-fixing plants;

x4 and application of the biological material in culturing nitrogen-fixing plants.

In the above application, the nucleic acid molecule of B1) may specifically be the gene encoding EGFP in the above method.

In the above applications, the expression cassette containing a nucleic acid molecule encoding EGFP (EGFP gene expression cassette) described in B2) refers to a DNA capable of expressing EGFP in a host cell, and the DNA may include not only a promoter that initiates transcription of the EGFP gene but also a terminator that terminates transcription of the EGFP gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the EGFP gene expression cassette can be constructed by using the existing expression vector.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may specifically be pMP 2444.

B3) The recombinant vector can contain a DNA sequence shown in a sequence 1 and used for encoding EGFP; further B3) the recombinant vector may specifically be pMP-egfp. The pMP-EGFP is a recombinant vector of EGFP shown in an expression sequence 2 obtained by replacing a DNA fragment between BamHI and XbaI recognition sequences of a plasmid pMP2444 with an EGFP gene shown in a sequence 1.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. The bacteria may specifically be rhizobia. The rhizobia may be sinorhizobium. The Sinorhizobium meliloti can be Sinorhizobium meliloti, such as Sinorhizobium meliloti 1021.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

In the above application, the plant may be any one of the following a1) -a 4):

a1) a monocot or dicot;

a2) non-leguminous plants or leguminous plants;

a3) a gramineous plant;

a4) wheat or rice.

In the above application, the rhizobia may be b1) or b2) as follows:

b1) rhizobia of the genus sinorhizobium;

b2) sinorhizobium meliloti (e.g., Sinorhizobium meliloti 1021).

Experiments prove that after the recombinant rhizobia containing the EGFP coding gene is used for inoculating the non-leguminous plants, the detected rhizobia in the plant bodies are respectively obviously higher than that of the non-leguminous plants inoculated by the recombinant rhizobia containing the GFP coding gene, and the experiments show that the EGFP is used for marking the rhizobia, so that the detection accuracy can be improved, and the colonization process of the rhizobia in rice and wheat can be monitored more accurately. The method for monitoring the colonization process of sinorhizobium meliloti in a non-leguminous plant provided by the invention is to mark the sinorhizobium meliloti by egfp gene and observe the marked colonization process of the sinorhizobium meliloti in the plant by using a laser confocal microscope. The method for monitoring the colonization process of sinorhizobium in non-leguminous plants adopts plasmid pMP2444 of egfp gene, which is a broad-host plasmid and a lacZ promoter with constitutive expression. egfp is constitutively stably expressed and is not lost with passage in the absence of selective pressure. Therefore, the plant can be stably colonized in the plant body and can continuously grow. The method is used for detecting the colonization process of the rhizobia in the plant body, is favorable for deeply researching the growth promoting mechanism of the rhizobia to the plant, tracking the ecological and cytological positioning of the combined action of the rhizobia as an endophyte and the plant, is favorable for clarifying the biological significance of the endophyte to the plant, and lays the foundation of the form, the physiology and the molecule for determining the application of the rhizobia as a biological fertilizer with broad-spectrum significance.

Drawings

FIG. 1 is a confocal laser microscope observation of colonization of egfp gene-labeled Sinorhizobium meliloti 1021 in wheat. Wherein, the Sinorhizobium meliloti 1021 with A-B being egfp gene mark is colonized on the surface and the inside of the root hair of the wheat seedling, and some rhizobia enter the root through the root hair; C-D is the Sinorhizobium meliloti 1021 of egfp gene mark and colonizes in the side root fissure and root surface of the wheat seedling; the population number of the Sinorhizobium meliloti 1021 with the E-F as egfp gene marker and colonized at the root is gradually increased; g is a cross section of a leaf sheath, and the Sinorhizobium meliloti 1021 displaying egfp gene marker can be colonized in the wheat leaf sheath; h is the cross section of the leaf, shows that the egfp gene marked sinorhizobium meliloti 1021 can be colonized in the wheat leaf, and some egfp gene marked sinorhizobium meliloti 1021 are colonized in the mesophyll cell. White arrows indicate egfp gene-tagged Sinorhizobium meliloti 1021.

FIG. 2 shows the fluorescence detection and colony PCR detection results of 1021-pMP-egfp separated and cultured in wheat. Wherein A is green fluorescence emitted by 1021-pMP-egfp separated and cultured in wheat and observed and displayed by a fluorescence microscope; b is colony PCR result of nifA and egfp genes of 1021-pMP-egfp separated and cultured in wheat,

lanes

1 and 2 are respectively 1021-pMP-egfp and amplification of nifA gene fragment of 1021-pMP-egfp separated from wheat seedling; 3 and 4, respectively 1021-pMP-egfp and egfp gene fragment amplification of 1021-pMP-egfp separated from wheat seedling; m is DNA molecular weight Marker.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Sinorhizobium meliloti 1021(Chi et al, City University of New York, references of genomic analysis of rice seeds infected by biorhizobium meliloti 1021, Proteomics 2010,10, 1861-1874) in the examples described below is publicly available from the applicant, and this biomaterial is only used for the relevant experiments for repeating the present invention and is not used for other purposes.

Example 1 detection of the colonization Process of Rhizobium in wheat

Method for detecting colonization of rhizobia in wheat body by marking alfalfa sinorhizobium 1021 through egfp gene

1. Preparation of recombinant Rhizobium

The recombinant vector was obtained by replacing the DNA fragment between the BamHI and XbaI recognition sequences of plasmid pMP2444(Biovector plasmid strain cell gene Collection) with EGFP gene shown in sequence 1 (the sequence of EGFP protein encoded by EGFP gene is shown in sequence 2 in the sequence Listing), and was named pMP-EGFP.

The pMP-egfp was introduced into Sinorhizobium meliloti 1021 to obtain a recombinant Rhizobium meliloti, which was named as egfp gene-labeled Sinorhizobium meliloti 1021 (abbreviated as 1021-pMP-egfp).

2. Wheat inoculated with recombinant rhizobia

Culturing 1021-pMP-egfp of step 1 in culture medium 1 (culture medium 1 is prepared by adding tetracycline and streptomycin into L B liquid culture medium to obtain tetracycline concentration of 10 μ g/m L and streptomycin concentration of 50 μ g/m L) at 28 deg.C under constant temperature shaking at rotation speed of 180-₆₀₀When the concentration of the bacterial strain is 0.8, centrifuging the bacterial strain by a centrifugal machine at 4000 rpm, collecting the bacterial strain, washing the bacterial strain by PBS, and then resuspending the bacterial strain by PBS to obtain 1021-pMP-egfp bacterial liquid, wherein the concentration of the bacterial strain in the 1021-pMP-egfp bacterial liquid is OD₆₀₀0.6. Boiling 1021-pMP-egfp bacterial liquid in a microwave oven for 3 times to obtain 1021-pMP-egfp dead bacterial liquid.

Treating wheat seeds with 75% ethanol water solution for 10min, washing with sterile water for three times, and then using HgCl with the mass percentage concentration of 0.1%₂The aqueous solution was treated for 10 minutes and washed three times with sterile water. And (3) placing the sterilized wheat seeds into a sterilized culture dish containing sterile filter paper and a small amount of sterile water, and accelerating germination for two days in an incubator at 28 ℃. After germination of the seeds, selecting germinated seeds with consistent germination (i.e. root length is basically same as bud length), and transplanting into sterilized small bottles (9 cm high, 6cm diameter, 170cm containing)³Vermiculite and 1/4 Mucun B nutrient solution of 100m L, covering a transparent sealing film, and autoclaving at 120 ℃ for 20 minutes), 6 germinated seeds are put into each bottle, and cultured in a light incubator for 3 days (please show the time), wherein the culture conditions are 28 ℃ in the daytime, 14 hours, 25 ℃ at night and 10 hours, and wheat seedlings are obtained.

Sucking 1021-pMP-egfp bacterial liquid by a liquid transfer gun, injecting the liquid into the rhizosphere of a wheat seedling with 1m L per bottle to obtain wheat (1021-pMP-egfp processed wheat) processed by recombinant rhizobia, sucking 1021-pMP-egfp dead bacteria liquid by the liquid transfer gun, injecting the liquid into the rhizosphere of the wheat seedling with 1m L per bottle to obtain control wheat (1021-pMP-egfp dead bacteria processed wheat), marking the day of processing as the 1 st day of inoculation, and culturing the wheat processed by the recombinant rhizobia and the control wheat in a light culture box under the culture conditions of 28 ℃ in the daytime, 14h, 25 ℃ at night and 10 h.

During the culture process in the illumination incubator, 1/4 Mucun B nutrient solution is supplemented according to the evaporation condition to keep the normal growth of the seedlings.

Wherein, 1/4 Mucun B nutrient solution is obtained by diluting Mucun B nutrient solution mother liquor four times, the Mucun B nutrient solution mother liquor is composed of solvent and solute, the solvent is water, pH is 6.0, the solute and quality of 1L Mucun B nutrient solution mother liquor are as follows:

macroelements:

trace elements:

iron salt:

3. detection of colonization of recombinant rhizobia in wheat

3.1 detection Using confocal laser microscopy

On day 1, day 2, day 5 and day 8 of inoculation, roots, leaf sheaths and leaves of the wheat treated with recombinant rhizobia and the control wheat were taken, respectively, washed clean with sterile water, the plant tissue was sliced by hand, and then the colonization of the recombinant rhizobia in wheat was observed using a confocal laser microscope (ZEISS L SM 510META), filters of 568nm and 488nm wavelength were used to capture the red fluorescence emitted by the plant tissue and the green fluorescence emitted by the rhizobia, respectivelyhttp://imagej.nih.gov/ ij/) And carrying out subsequent processing on the picture. For each materialFeed and time points, 4 plants were selected for analysis, respectively.

The results show that: after the wheat seedlings are inoculated by the recombinant rhizobia, the recombinant rhizobia migrate to the rhizosphere range of the wheat seedlings, and a large number of rhizobia colonize the root hair and the lateral root cracks of the wheat seedlings on the 2 nd day after inoculation; repeated for many times, the rhizobia can enter the roots of the seedlings through the root hairs, and the rhizobia firstly colonizes at the top of the root hairs and then enters the interior of the root hairs (A in the figure 1 and indicated by white arrows), migrates in the root hairs and finally enters the intercellular spaces of the root cortex (B in the figure 1 and indicated by white arrows); in addition to entering the root through the root hair, rhizobia can enter the root through the lateral root fissure, and the rhizobia colonizes the surface of the seedling root at the 2 nd day of inoculation, and colonizes the lateral root fissure and enters the root (C and D in figure 1, white arrows); scanning the root tissues of the seedlings layer by a laser confocal microscope on the inoculation day 5, and showing that the quantity of rhizobia groups on the surface and in the roots is obviously increased, and a large number of rhizobia exist in the roots of the seedlings in an endophyte mode and are mainly distributed in intercellular spaces (E and F in figure 1); rhizobium as an endophyte migrated very fast in wheat seedlings, and on day 5 and 8 of inoculation, egfp-labeled Rhizobium colonized in leaf sheaths and leaves (G and H in FIG. 1) and some entered inside mesophyll cells (H in FIG. 1, white arrows). And the contrast wheat body obtained after the 1021-pMP-egfp dead bacteria liquid is inoculated has no green fluorescence, which indicates that the 1021-pMP-egfp dead bacteria can not enter the wheat body.

3.2PCR detection

On the 1 st day, the 2 nd day, the 5 th day and the 8 th day of inoculation, roots (seedling roots) and overground tissues (seedling overground parts) of the wheat and the control wheat treated by the recombinant rhizobia are respectively taken, the wheat tissue materials are washed clean by distilled water, the water-absorbing paper is wiped and weighed, the wheat tissue materials are disinfected in a disinfectant for 1 minute, the wheat tissue materials are washed by sterile water for 4 times after the disinfection and are placed on an antibiotic-free L B plate for 1 hour, the wheat tissue materials are taken down, then the antibiotic-free L B plate is cultured for 2 days at the temperature of 28 ℃, no bacterial colony is found, and the surface disinfection is thorough.

The sterilized roots and above-ground tissues of both the recombinant rhizobia treated wheat and the control wheat were then treated by grinding the roots and above-ground tissues of the recombinant rhizobia treated wheat and the control wheat, respectively, to homogenate using a sterilized mortar, gradient-diluting the homogenate with a 20% glycerol by volume in PBS, spreading onto L B plates to which antibiotics (tetracycline 10. mu.g/m L, streptomycin 50. mu.g/m L) had been added, and culturing at 28 ℃ until colonies appeared.

After picking single strains, transferring the single strains to a glass slide added with PBS, covering the glass slide, observing the single strains by using a computer-assisted fluorescence microscope, and collecting green fluorescence signals emitted by egfp gene-marked sinorhizobium meliloti 1021.

Single colonies were picked and the egfp and nifA genes of each Rhizobium colony were detected by colony PCR. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; stretching for 10min at 72 ℃. After the reaction, the PCR product was identified by electrophoresis on a 1% agarose gel.

The PCR reaction primers are as follows:

egfp: the length of the PCR product is 800bp

egfp-up：5’-CGATCCCGCGAAATTAATACGAC-3’

egfp-down：5’-TGTTAGCAGCCGGATCCTTTG-3’

Sm1021 nifA: the length of the PCR product is 300bp

Sm1021nifA-up：5’-CGCTGATGCGGTAGTAAAGGT-3’

Sm1021nifA-down：5’-AGCCTTCTCGAATCAGAG-3’

In order to determine the colonization of wheat seedlings and the migration of the wheat seedlings by the Egfp gene marked Rhizobium meliloti 1021 in the wheat body, the quantity of the rhizobium colonies which can be separately cultured in the wheat seedlings is determined on the 1 st, 2 nd, 5 th and 8 th days after the inoculation of the Rhizobium meliloti 1021, the quantity of the rhizobium colonies in the root and the overground part tissues of each wheat seedling shows a growing trend along with the increase of the number of days after the inoculation of the Rhizobium meliloti 1021, some rhizobium can invade the roots of the seedlings on the 1 st day after the wheat inoculation, and the average quantity can reach 1.2 × 10⁴The number of rhizobia in the aerial tissue was zero, indicating that this timeThe rhizobia has not migrated to the above ground tissue, and the average number of rhizobia in the root is increased to 3.6 × 10 at day 2 when the wheat is inoculated with the rhizobia⁴The rhizobia of each strain and some rhizobia migrate to the overground part tissues, and the average number of the overground part rhizobia is 1.5 × 10³The average number of rhizobia in the root and overground part tissues is respectively increased to 1.8 × 10 on the 5 th day of rhizobia inoculation of wheat⁶Su/strain and 9.6 × 10⁴The average number of rhizobia in the root and overground part tissues is respectively increased to 9.2 × 10 at the 8 th day of rhizobia inoculation of wheat⁶Suo/strain 6 × 10⁵One plant per strain.

The results of the examples show that egfp gene-tagged Sinorhizobium meliloti 1021 can be isolated from surface-sterilized wheat tissue by plate separation and re-cultured on solid media.

Colonies isolated and re-cultured were randomly picked and observed with a fluorescence microscope to detect green fluorescence (a in fig. 2). The nifA and egfp genes of Sinorhizobium meliloti 1021 were amplified by colony PCR to obtain the corresponding bands (B in FIG. 2). These results indicate that the rhizobia isolated from wheat tissue is a wheat-treated rhizobia marked with the egfp gene.

Secondly, detecting the colonization of rhizobia in wheat by using gfp gene marker alfalfa Sinorhizobium 1021

1. Preparation of recombinant Rhizobium

The DNA fragment between the BamHI and XbaI recognition sequences of plasmid pMP2444 was replaced with gfp gene shown in sequence 3 to give a recombinant vector, which was named pMP-gfp.

The pMP-gfp is introduced into Sinorhizobium meliloti 1021 to obtain recombinant rhizobia, and the recombinant rhizobia is named as the Sinorhizobium meliloti 1021 marked by the gfp gene (abbreviated as 1021-pMP-gfp).

2. Wheat inoculated with recombinant rhizobia

Replacing 1021-pMP-egfp with 1021-pMP-gfp according to the method in the step one (2) to obtain 1021-pMP-gfp treated wheat and 1021-pMP-gfp dead bacteria treated wheat.

3. Detection of colonization of recombinant rhizobia in wheat

Detecting the amount of rhizobia in the wheat at different time according to the PCR detection method of 3.2 in the step 3 in the step one, and the result shows that the average number of root bacteria is 0.3 × 10 on the 1 st day of 1021-pMP-gfp inoculation of the wheat²The number of rhizobia in the overground part tissue is 0 per rhizobia, the average number of the rhizobia is 1.3 × 10 on the 2 nd day of wheat inoculation 1021-pMP-gfp²The number of rhizobia in the overground part tissue is 0.5 × 10²The average number of root bacteria is 0.5 × 10 on the 5 th day of wheat inoculation 1021-pMP-gfp³The number of rhizobia in the overground part tissue is 1.6 × 10²The average number of root bacteria is 1.02 × 10 on the 8 th day of wheat inoculation 1021-pMP-gfp³The number of rhizobia in the overground part tissues is 6 × 10²One plant per strain.

The number of root rhizobia detected by the method is 400, 277, 3600 and 9020 times of the number of the root rhizobia detected by the gfp gene on the 1 st, 2 nd, 5 th and 8 th days after wheat is inoculated with 1021-pMP-egfp; the number of the above-ground rhizobia detected by the method of the present invention is 0, 30, 600 and 10000 times the number of the above-ground rhizobia detected by the gfp gene.

Example 2 detection of Rhizobium colonization in Rice by marking Sinorhizobium meliloti 1021 with egfp Gene

First, detecting rhizobium colonization in rice by using egfp gene marker alfalfa Sinorhizobium 1021

1. Preparation of recombinant Rhizobium

Same as example 1, step 1.

2. Recombinant rhizobium inoculated rice

The wheat seeds were replaced with dehulled rice seeds and the other steps were not changed according to the method of step 2 of example 1 to obtain 1021-pMP-egfp treated rice and 1021-pMP-egfp dead bacteria treated rice.

3. Detection of colonization of recombinant rhizobia in rice

Following the procedure in example 1The PCR detection method 3.2 in the step 3 detects the amount of rhizobia in the rice body at different time, and the result shows that the average number of root bacteria is 1.3 × 10 on the 1 st day of 1021-pMP-egfp rice inoculation⁴The number of rhizobia in the overground part tissues is 0 per plant, the average number of root bacteria is 1.8 × 10 on the 2 nd day of rice inoculation of 1021-pMP-egfp⁴The number of rhizobia in the overground part tissue is 1.25 × 10³The average number of root bacteria is 1.63 × 10 on the 5 th day of 1021-pMP-egfp rice inoculation⁶The number of rhizobia in the overground part tissues is 9.25 × 10⁴The average number of root bacteria is 9.42 × 10 on the 8 th day of 1021-pMP-egfp rice inoculation⁶The number of rhizobia in the overground part tissue is 7 × 10⁵One plant per strain.

Secondly, detecting the colonization of rhizobia in rice by using gfp gene marker alfalfa Sinorhizobium 1021

1. Preparation of recombinant Rhizobium

Same as example 1, step two, step 1.

2. Recombinant rhizobium inoculated rice

The method of step 2 of example 1 was followed to replace 1021-pMP-egfp with 1021-pMP-gfp as described above and to replace wheat seeds with dehulled rice seeds, to obtain 1021-pMP-gfp treated rice and 1021-pMP-gfp dead bacteria treated rice.

3. Detection of colonization of recombinant rhizobia in rice

The amount of rhizobia in rice plants at different times was measured by the PCR detection method according to 3.2 of step 3 of example 1, and the results showed that the average number of root bacteria was 5 × 10 on day 1 of 1021-pMP-gfp inoculation²The number of rhizobia in the overground part tissues is 0 per rhizobia, the average number of root bacteria is 2 × 10 on the 2 nd day of rice inoculation of 1021-pMP-gfp³The number of rhizobia in the overground part tissues is 2.5 × 10²The average number of root bacteria is 1 × 10 on the 5 th day of 1021-pMP-gfp inoculation of rice⁴The number of rhizobia in the overground part tissue is 4 × 10³Each plant; mean root inoculum of 1021-pMP-gfp at day 8 after inoculation of RiceThe quantity is 1.2 × 10⁴The number of rhizobia in the overground part tissues is 6 × 10³One plant per strain.

The number of root rhizobia detected by the method of the invention is 260, 90, 163 and 78.5 times of the number of root rhizobia detected by the method of the prior art on

days

1, 2, 5 and 8 of rice inoculation 1021-pMP-egfp; the number of rhizobia overground detected by the method of the present invention is 0, 5, 23 and 117 times the number of rhizobia overground detected by the prior art method.

Experiments prove that after wheat and rice are inoculated by the recombinant rhizobia containing the EGFP coding gene, the detected rhizobia in the plant body is respectively and obviously higher than that of wheat and rice inoculated by the recombinant rhizobia containing the GFP coding gene, and the experiments show that the EGFP is used for marking the rhizobia, so that the detection accuracy can be improved, and the colonization process of the rhizobia in the rice and the wheat can be more accurately monitored.

<110> institute of plant of Chinese academy of sciences

<120> method for monitoring rhizobia colonization process in non-leguminous plants

<160>3

<170>PatentIn version 3.5

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<211>793

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<213> Artificial sequence

<220>

<223>

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gtatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 60

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 120

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 180

ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 240

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 300

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 360

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 420

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 480

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 540

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 600

actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 660

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg 720

agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc 780

gtaccggtta gta 793

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<212>PRT

<213> Artificial sequence

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Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Glu

225 230 235 240

Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu

245 250 255

Asp Ser Thr Arg Thr Gly

260

<210>3

<211>721

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<213> Artificial sequence

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<223>

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aataatgtcg aagggcgagg agctgttcac cggcgtcgtc ccgatcctgg tcgagctgga 60

cggtgacgtc aacggccaca agttctccgt ctccggcgag ggtgagggcg acgccaccta 120

cggcaagctg accctgaagt tcatctgcac caccggtaag ctgccggtcc cgtggccgac 180

cctggtcacc accctgacct acggcgtcca gtgcttctcc cgctacccgg accacatgaa 240

gcgccacgac ttcttcaagt ccgccatgcc ggagggttac gtccaggagc gcaccatctc 300

cttcaaggac gacggtaact acaagacgcg tgccgaggtc aagttcgagg gcgacaccct 360

ggtcaaccgc atcgagctga agggcatcga cttcaaggag gacggtaaca tcctgggcca 420

caagctggag tacaactaca actcccacaa cgtctacatc accgcggaca agcagaagaa 480

cggcatcaag gccaacttca agacccgcca caacatcgag gacggtggcg tccagctagc 540

cgaccactac cagcagaaca ccccgatcgg cgacggcccg gtcctgctgc cggacaacca 600

ctacctgtcc acccagtccg ccctgtccaa ggacccgaac gagaagcgcg accacatggt 660

cctgctggag ttcgtcaccg ccgccggcat cacccacggc atggacgagc tgtacaagta 720

g 721

Claims

1. A method of monitoring the colonization of Sinorhizobium meliloti (Sinorhizobium meliloti)1021 in wheat, comprising: introducing an expression vector pMP2444 containing an EGFP coding gene into Sinorhizobium meliloti 1021 to obtain recombinant rhizobia; the coding gene of the EGFP is shown as a sequence 1 in a sequence table; the EGFP coding gene in the expression vector pMP2444 is started by a lacZ gene promoter; inoculating the wheat with the recombinant rhizobia, and monitoring the colonization process of the recombinant rhizobia in the wheat.