CN110054671B - Method for regulating and controlling growth and aging of plant root nodules and application of method - Google Patents

Abstract

The invention discloses a method for regulating and controlling the growth and the aging of plant root nodules and application thereof. The invention also discloses an application of the GmCYP15 protein in any one of the following (1) to (8): (1) regulating and controlling symbiotic nitrogen fixation or symbiotic nodulation of plants; (2) regulating and controlling the growth of plant root nodules; (3) regulating and controlling the number of plant nodules; (4) regulating and controlling the plant root nodule aging process; (5) regulating and controlling the nitrogen fixation time of the plant root nodule; (6) regulating the expression level of a plant nodulin gene; (7) regulating and controlling the plant height; (8) regulating and controlling the fresh weight of the overground part of the plant. The invention provides gene resources for obtaining a new variety of the soybean edited by the high nitrogen-fixing gene, has important significance in agriculture and ecology, and has certain application prospect in nitrogen-fixing mechanism research and agricultural production of leguminous crops.

Description

Method for regulating and controlling growth and aging of plant root nodules and application of method

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for regulating and controlling the growth and senescence of plant nodules and application thereof.

Background

Symbiotic nitrogen fixation between leguminous plants and rhizobia is the most economic and environmentally friendly biological nitrogen fixation method. Therefore, the nitrogen fixation effect of leguminous plants is exerted, the application amount of nitrogen fertilizers is reduced, and the nitrogen fixation fertilizer has very important significance in the aspects of agriculture and ecology. The formation of symbiont nodules is a very complex process that is the result of microbial and plant recognition, and signal molecular interactions. The symbiotic nitrogen fixation efficiency of leguminous plants reaches a peak after 4-6 weeks of rhizobium inoculation, then thalli-like bodies are extremely differentiated, the nitrogen fixation capacity is gradually weakened, and finally rhizobium organs and cells are aged, the thalli-like bodies are released or died down, so that the symbiotic relationship of the rhizobium and the plants is disintegrated, and the nitrogen fixation effect is finished. The nitrogen fixation efficiency is improved, and the number of nodules is increased, and many genes related to nodule formation have been analyzed in recent years. Secondly, the activity of the azotase is improved, and the activity of the azotase is fully exerted mainly by improving the growing environmental conditions of leguminous plants. Thirdly, the root nodule aging is delayed, the nitrogen fixation time of the root nodule is prolonged, and the nitrogen fixation amount is increased.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the growth and the aging of the plant root nodule.

In order to solve the technical problems, the invention firstly provides a new application of the GmCYP15 protein or a biological material related to the GmCYP15 protein.

The invention provides application of GmCYP15 protein or biological materials related to the GmCYP15 protein in any one of the following (1) to (8):

(1) regulating and controlling the nitrogen fixation capacity or symbiotic nodulation capacity of the plant;

(2) regulating and controlling the growth of plant root nodules;

(3) regulating and controlling the number of plant nodules;

(4) regulating and controlling the plant root nodule aging process;

(5) regulating and controlling the nitrogen fixation time of the plant root nodule;

(6) regulating the expression level of a plant nodulin gene;

(7) regulating and controlling the plant height;

(8) regulating and controlling the fresh weight of the overground part of the plant;

the GmCYP15 protein is a protein of the following a) or b) or c):

a) the amino acid sequence is a protein shown in a sequence 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) and (b) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label 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 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, 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 5 amino acid residues.

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

The gene encoding the protein of c) 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 performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

The biomaterial is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding a GmCYP15 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

In the above application, the nucleic acid molecule of A1) is a gene as shown in 1) or 2) or 3) below:

1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a sequence 1;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes GmCYP15 protein;

3) hybridizes with the nucleotide sequence defined in 1) or 2) under strict conditions and codes a cDNA molecule or a genome DNA molecule of the GmCYP15 protein.

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.

The nucleotide sequence encoding the GmCYP15 protein of the invention can be readily 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 encoding the GmCYP15 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the GmCYP15 protein and have the same function.

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.

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

In the above applications, the expression cassette containing a nucleic acid molecule encoding a GmCYP15 protein (GmCYP15 gene expression cassette) described in a2) refers to a DNA capable of expressing the GmCYP15 in a host cell, and the DNA may include not only a promoter for initiating transcription of the GmCYP15 gene but also a terminator for terminating transcription of the GmCYP15 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter of cauliflower mosaic virus 35S: the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, oleosin, and soybeanThe promoter of beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627). In one embodiment of the invention, the promoter for promoting the expression of the GmCYP15 gene is a polyubiquitinated promoter.

The existing expression vector can be used for constructing a recombinant vector containing the GmCYP15 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may specifically be pU 1301.

In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. The agrobacterium may specifically be agrobacterium rhizogenes a. rhizogenes LBA 1334.

In the above applications, the transgenic plant cell line does not comprise propagation material.

In the application, the regulation and control of plant nitrogen fixation capacity, root nodule development, root nodule number, root nodule senescence process, root nodule nitrogen fixation time, expression level of nodulin genes, plant height and fresh weight of overground parts are specifically embodied as follows: when the expression amount and/or activity of the GmCYP15 protein in the plant is increased, the plant nitrogen fixation capacity is reduced, the number of nodules is reduced, the senescence of the nodules is accelerated, the nitrogen fixation time of the nodules is shortened, the expression level of nodulin genes is reduced, the plant height is reduced, and the fresh weight of the aerial parts is reduced; when the expression level and/or activity of the GmCYP15 protein in the plant is reduced, the plant nitrogen fixation capacity is improved, the number of nodules is increased, the senescence of the nodules is delayed, the nitrogen fixation time of the nodules is prolonged, the expression level of nodulin genes is improved, the plant height is improved, and the fresh weight of aerial parts is increased.

The nodulin gene is NIN gene and/or Lb gene and/or Enod40 gene.

The expression level is in particular an RNA expression level.

In one embodiment of the present invention, the regulation of plant nitrogen fixation capacity, root nodule number, root nodule senescence process, root nodule nitrogen fixation time, nodulation gene expression level, plant height and fresh weight of aerial parts is specifically reducing plant nitrogen fixation capacity, reducing plant root nodule number, accelerating plant root nodule senescence, shortening plant root nodule nitrogen fixation time, reducing nodulation gene expression level, reducing plant height, reducing fresh weight of aerial parts of plants.

The invention also provides application of the GmCYP15 protein or biological materials related to the GmCYP15 protein in culturing transgenic plants with improved nitrogen fixation capacity and/or increased nodule number and/or retarded nodule senescence and/or prolonged nodule nitrogen fixation time and/or improved plant height and/or increased fresh weight of aerial parts.

The invention also provides application of the GmCYP15 protein or biological materials related to the GmCYP15 protein in culturing transgenic plants with reduced nitrogen fixation capacity and/or reduced nodule number and/or accelerated nodule senescence and/or shortened nodule nitrogen fixation time and/or reduced plant height and/or reduced fresh weight of aerial parts.

The application of the GmCYP15 protein or the biological material related to the GmCYP15 protein in the cultivation of high nitrogen-fixing plant varieties or plant breeding also belongs to the protection scope of the invention.

In order to solve the above technical problems, the present invention finally provides a method for cultivating a transgenic plant.

The method for cultivating the transgenic plant comprises the steps of improving the expression quantity and/or activity of GmCYP15 protein in a target plant to obtain the transgenic plant; the transgenic plant has a lower nitrogen fixation capacity than the plant of interest and/or the transgenic plant has fewer root nodules than the plant of interest and/or the transgenic plant has faster senescence rate than the plant of interest and/or the transgenic plant has a shorter nitrogen fixation time than the plant of interest and/or the transgenic plant has a lower plant height than the plant of interest and/or the transgenic plant has less fresh weight of the aerial part than the plant of interest.

In the above method, the improvement of the expression level and/or activity of the GmCYP15 protein in the target plant is achieved by introducing a gene encoding the GmCYP15 protein into the target plant.

The nucleotide sequence of the coding gene of the GmCYP15 protein is a DNA molecule shown in a sequence 1.

The encoding gene of the GmCYP15 protein can be introduced into target plant cells by using Ti plasmids, plant virus carriers, direct DNA transformation, microinjection, electroporation and other conventional biotechnological methods.

In a specific embodiment of the invention, the gene encoding the GmCYP15 protein is introduced into a target plant through a recombinant vector pU1301-GmCYP 15. The pU1301-GmCYP15 is a vector obtained by replacing a DNA fragment between KpnI and BamHI enzyme cutting sites of a pU1301 vector with a GmCYP15 gene fragment shown in a sequence 1 and keeping other sequences of the pU1301 vector unchanged. The recombinant vector pU1301-GmCYP15 expresses GmCYP15 protein shown in sequence 2.

In the above application or method, the plant may be a monocotyledon or a dicotyledon. Further, the dicot may be a leguminous plant. Further on. The leguminous plant can be Soybean (Soybean), Lotus japonicus (Lotus japonicus), Astragalus sinicus (Astragalus sinicus), alfalfa (Medicago truncatula), and peanut (Arachis Hypogaea Linn). In one embodiment of the present invention, the Lotus corniculatus can be Lotus corniculatus MG 20.

The invention clones the GmCYP15 gene from soybean for the first time, and detects the positioning condition of the gene in the nodule inoculated for about 60 days by using a probe synthesized by using a digoxin marker based on a specific sequence thereof as a reference, and finds that the gene is mainly positioned on the cell membrane, the nuclear membrane and the symbiont membrane of the nodule cell. The result shows that the gene participates in the rhizobium symbiosis and the growth and the senescence of the rhizobium. The invention also constructs a plant expression vector which is expressed by the GmCYP15 gene driven by the polyubiquitination promoter, the plant expression vector is transferred into the crowtoe of the model leguminous plant through hairy root transformation, after a positive plant is obtained through GUS identification, the positive plant is planted in a sand and vermiculite 1:1 mixed pot culture, and rhizobium is inoculated. Functional analysis on transgenic crowtoe shows that after the GmCYP15 gene is over-expressed, plants are obviously weakened, the nodulation number is obviously reduced, and the nodulation aging is obviously accelerated. The result shows that the gene can participate and negatively regulate the Lotus corniculatus nodulation and accelerate the aging of the root nodule. In practical application, the GmCYP15 gene in a plant can be knocked out by means of gene editing, so that the number of plant nodules is effectively increased, the nitrogen fixation time of the nodules is prolonged, the nitrogen fixation capacity and soil fertility of the plant are further enhanced, and the use of chemical fertilizers and the like is effectively reduced. The invention provides gene resources for obtaining a new variety of the soybean edited by the high nitrogen-fixing gene, has important significance in agriculture and ecology, and has certain application prospect in nitrogen-fixing mechanism research and agricultural production of leguminous crops.

Drawings

FIG. 1 shows the results of the double digestion of the GmCYP15 gene.

FIG. 2 shows the in situ hybridization mapping of the GmCYP15 gene in nodules inoculated for 61 days. Red arrows indicate the symbiotic membrane, green arrows indicate the cell membrane, black arrows indicate the nuclear membrane.

Fig. 3 is a schematic diagram comparing the growth status of the GmCYP15 transgenic plants and the control plants 50 days after inoculation.

FIG. 4 is a statistical analysis of plant height, overground fresh weight and number of nodules of the GmCYP15 transgenic plants and the control plants at 50 days of inoculation. A is the plant height; b is the fresh weight of the aerial parts; c is the number of nodules.

FIG. 5 is a schematic representation of root nodule senescence observed 50 days after inoculation of GmCYP15 transgenic plants and control plants using paraffin sections. A is paraffin section of nodule 50 days after inoculation as control; b is paraffin section of the root nodule of the GmCYP15 transgenic plant 50 days after inoculation.

FIG. 6 shows the expression of related genes in hair roots of GmCYP15 transgenic plants and control plants detected by semi-quantitative PCR and qPCR. A is the expression condition of the GmCYP15 gene in the hair roots of the GmCYP15 transgenic plant and a control plant analyzed by semi-quantitative PCR; and B, the expression conditions of 3 nodulin genes in hair roots of the GmCYP15 transgenic plants and control plants are analyzed by qPCR.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The media and reagent formulations referred to in the following examples are as follows:

MS culture medium: 4.3g of MS basal salt mix (Sigma), 0.103g of MS vitamin powder, 0.7-0.8% (mass to volume) of agar powder and ddH₂And O is metered to 1000mL and is adjusted to pH 5.8.

YMA medium: 10.0g mannitol (or sucrose), 0.4g Yeast Extract, 0.5g K₂HPO₄，0.2g MgSO₄·7H₂O，0.1g CaCl₂·6H₂O, 0.1g NaCl, 4mL Rh microelement liquid, ddH₂And (4) metering the volume of O to 1000mL, adjusting the pH value to 6.8-7.0, and sterilizing at 115 ℃ for 20 min.

Rh trace element liquid: 5.0g H₃BO₃，5.0g Na₂MoO₄，ddH₂And O is metered to 1000 mL.

Fahraeus nitrogen-free nutrient solution: 0.10g of CaCl₂·2H₂O，0.12g MgSO₄·7H₂O，0.10g KH₂PO₄，0.15g Na₂HPO₄·12H₂O, 1mL of Gibson's microelement liquid, 5mg of ferric citrate, ddH₂And O is metered to 1000 mL.

Gibson trace element liquid: 2.86g H₃BO₃，0.22g ZnSO₄·7H₂O，2.03g MnSO₄·4H₂O，0.13g Na₂MoO₄·2H₂O，0.08g CuSO₄·5H₂O，ddH₂And O is metered to 1000 mL.

GUS dye solution: 100mM sodium phosphate buffer (pH 7.0), 0.1% Triton X-100, 0.1% N-laurylsarcosine,10mM Na₂EDTA, 1mM potassium ferricyanide (K)₃Fe(CN)₆) 1mM potassium ferrocyanide (K)₄Fe(CN)₆) And 0.5mg/mL X-GluC.

HRE medium: 20X SH-A salt, 20X UM-C vitamin, 10g sucrose, 3mL 1M MES, 0.7-0.8% (mass/volume ratio) agar powder, ddH₂And O is metered to 1000mL and is adjusted to pH 5.8. Sterilizing at 110 deg.C for 30 min.

20X SH-A salt: KNO₃ 50g，MgSO₄·7H₂O 8g，NH₄H₂PO₄ 6g，CaCl₂·2H₂O 4g，MnSO₄·4H₂O 0.2g，H₃BO₃ 0.1g，ZnSO₄·7H₂O 0.02g，KI 0.02g，CuSO₄·5H₂O 0.004g，NaMoO₄·2H₂O 0.002g，CoCl₂·6H₂O 0.002g，FeSO₄·7H₂0.3g of O and 0.4g of NaEDTA. Dissolving FeSO in 100mL of sterile water respectively₄·7H₂Dissolving O and NaEDTA and other salts in 700mL of sterile water, mixing to a constant volume of 1000mL, and subpackaging into 50mL tubes for preservation at-20 ℃.

20X UM-C vitamin: inositol 2.0g, nicotinic acid 0.1g, pyridoxine hydrochloride (vitamin B6)0.2g, thiamine hydrochloride 0.2g, glycine 0.04g, ddH₂And O is metered to 1000mL, and each tube is subpackaged with 50mL and stored at-20 ℃.

MES(2-[N-Morpholino]ethane-sulfonic acid)1M stock solution: weigh 29.28g MES (Sigma) in ddH₂And (4) metering the volume of O to 150mL, adjusting the volume to pH 5.8, subpackaging into 6mL tubes, and storing at-20 ℃.

The pU1301 vectors in the following examples are described in the following documents: yuan S, Zhu H et al.A. ubiquitin ligand of plasmid acceptor digested in non-double organic genetics.plant physiol.2012,160(1):106-17, the vector was digested with restriction enzymes BamHI and KpnI to form a linearized sticky end at 3', which was publicly available from the applicant, and the biomaterial was used only for repeating the experiments related to the present invention, but not for other uses.

Rhizogenes LBA1334 in the following examples is described in: offfran, I A; melchers, L S, et al, compliance of Agrobacterium tumefaciens organisms by the T (R) -region of the Ri plasmid of Agrobacterium rhizogenes, PNAS,1986,83(18):6935-9, publicly available from the Applicant, used only for the repetition of the experiments relating to the present invention and not for other uses.

Rhizobium loti MAFF303099 in the examples below is described in: myra L.Tangengco, Makoto Hayashi, et al crinkle, a Novel systematic variant of fat soil effects the Infection Thread Growth and organisms the Root Hair, Trichome, and Seed Development in Lotus japonica plant physiology, 2003,131(3):1054-63, publicly available from the Applicant, which biomaterial was used only for repeating the relevant experiments of the present invention, and was not used for other purposes.

Example 1 cloning of GmCYP15 Gene and mapping analysis thereof

Cloning of GmCYP15 Gene

The soybean cDNA is taken as a template and amplified by a PCR method to obtain a GmCYP15 gene with the size of 1089bp, the nucleotide sequence of the GmCYP15 gene is shown as a sequence 1 in a sequence table, and the amino acid sequence of the GmCYP15 protein coded by the GmCYP15 gene is shown as a sequence 2 in the sequence table. The method comprises the following specific steps:

1. extraction of RNA

Total RNA was extracted from soybean-inoculated roots or tumors using Trizol reagent (available from Invitrogen) according to the instructions of Trizol reagent.

2. Obtaining of cDNA

The RNA extracted in step 1 was reverse transcribed into the first strand cDNA using reverse transcriptase (purchased from TAKARA). The reaction conditions are as follows: 15min at 37 ℃ (reverse transcription reaction), 5sec at 85 ℃ (reverse transcriptase inactivation reaction).

3. PCR amplification of fragments of interest

Taking the cDNA obtained in the step 2 as a template, adopting an upstream primer: 5'-GGTACCATGGCAATGAAGAAGTTCTTG-3' and the downstream primer: 5'-GGATCCTCAAAGTTCATCTTTAGGAGATG-3' PCR amplification was performed to obtain PCR products.

PCR reaction procedure: 5min at 95 ℃; circulating for 30 times at 94 deg.C for 45s, 55 deg.C for 1s, and 72 deg.C for 1.5 min; 5min at 72 ℃ and 5min at 12 ℃.

4. Recovery and purification of target fragment

The target DNA fragment is recovered by agarose gel electrophoresis, the recovery method refers to a DNA agarose gel recovery kit of Beijing original Hao biotechnology Limited, and the detailed operation steps are shown in the specification.

5. Acquisition of GmCYP15 Gene

1) And carrying out double enzyme digestion on the recovered PCR product and the pU1301 vector by using KpnI and BamHI to obtain the PCR product and the pU1301 vector after enzyme digestion.

2) And connecting the PCR product after enzyme digestion with the pU1301 vector after enzyme digestion to obtain a recombinant vector pU1301-GmCYP 15. The linking system is as follows: 0.5 mu L of digested pU1301 vector, 4 mu L of digested PCR product and 5.5 mu L of Solution I. The connection conditions were as follows: the reaction was carried out at 16 ℃ overnight.

3) And carrying out double enzyme digestion identification and sequencing on the recombinant vector. The results of the double restriction enzyme identification are shown in FIG. 1. The sequencing result shows that: the nucleotide sequence of the GmCYP15 gene fragment is shown as a sequence 1 in a sequence table. The amino acid sequence of the GmCYP15 protein coded by the GmCYP15 gene is shown as a sequence 2 in a sequence table.

The recombinant vector pU1301-GmCYP15 is a vector obtained by replacing a DNA fragment between KpnI and BamHI enzyme cutting sites of the pU1301 vector with a GmCYP15 gene fragment shown in sequence 1 and keeping other sequences of the pU1301 vector unchanged. The recombinant vector pU1301-GmCYP15 expresses GmCYP15 protein shown in sequence 2.

II, in-situ hybridization positioning of GmCYP15 in nodule cells

The UTR sequence of the GmCYP15 gene was found from the soybean genomic database [ http:// www.phytozome.net/soybean ], and a digoxin-labeled gene probe was synthesized as follows: 5-CTCAAAGCCGTTACAGCATCCATTCCACAA-3'. Then the in-situ hybridization location of the GmCYP15 gene in the nodule cells is detected according to a method of BCIP/NBT chromogenic in-situ hybridization (CISH) of a plant paraffin section. The method comprises the following specific steps:

1. tissue fixation: the nodules inoculated for 61 days are taken out and washed, and then immediately placed into a fixing solution (prepared by DEPC water) for fixing for 2-12 h. And (5) exhausting air by using a vacuum pump.

2. And (3) dehydrating: after the nodule tissue is fixed, the nodule tissue is dehydrated by gradient alcohol, and then is soaked in wax and embedded. And (5) pumping air by a vacuum pump in the dehydration process.

3. Slicing: slicing the paraffin by a slicer, taking out the slices by a spreading machine, and baking the slices for 2 hours by an oven at 62 ℃.

4. Paraffin section dewaxing to water: putting the slices into xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5min-DEPC water washing in sequence.

5. Digestion: the gene is circled, and proteinase K (20ug/ml) is added dropwise to digest for 30min at 37 ℃. The membrane was washed with PBS 3 times with pure water and then 5 min.

6. And (3) hybridization: add the prehybridization solution dropwise and incubate for 1h at 37 ℃. The prehybridization solution was decanted, and the hybridization solution containing the GmCYP15 gene digoxin probe was added dropwise at a concentration of 10ng/ul and hybridized overnight in a thermostat at 37 ℃.

7. Post-hybridization wash and block: the hybridization solution was washed off, 2 XSSC, 10min at 37 ℃,2 XSSC, 2X 5min at 37 ℃ and 10min at 0.5 XSSC at room temperature. If there are more non-specific hybrids, formamide washing can be increased. Blocking serum BSA was then added dropwise and incubated at room temperature for 30 min.

8. Adding mouse anti-digoxin labeled alkaline phosphatase (anti-DIG-AP): the blocking solution is poured off, and anti-DIG-AP is added dropwise. Incubate at 37 ℃ for 40min, wash 4 times in TBS × 5 min.

9. BCIP/NBT coloration: and (4) dropwise adding a BCIP/NBT developing solution, and observing positive by a microscope. And (5) washing with pure water.

10. Mounting and observing: and (5) sealing the glycerol gelatin. Image acquisition analysis was then performed under a microscope.

The results are shown in FIG. 3. The results show that: the GmCYP15 gene is mainly localized in a membrane system such as a cell membrane, a nuclear membrane and a symbiont membrane in a nodule cell, and is presumed to be involved in a biological process related to metabolism of a substance in the disintegration of a symbiont.

Example 2 obtaining of transgenic Lotus corniculatus GmCYP15 and functional analysis of GmCYP15

Obtaining of transgenic crowtoe with GmCYP15

The recombinant vector pU1301-GmCYP15 constructed in example 1 was transformed into Agrobacterium rhizogenes LBA1334 for hairy root transformation of Lotus corniculatus. The transformation method of the hairy roots of the Lotus corniculatus is mainly referred to the Lotus corniculatus (Lotus japonica) Handbook. The method comprises the following specific steps:

1. plant material preparation

Lotus japonicus MG20 seeds were sanded with sandpaper and then treated in liquid nitrogen for 1 minute. Then, seeds are sterilized by 75 percent (mass volume ratio) of ethanol and 5 percent (effective chlorine concentration) of NaClO in sequence, and a little sterile water is left for vernalization for 1 day under dark conditions after the sterilization is finished. Then transferred to MS solid medium without sucrose for 2 days at 23 ℃ in the dark. Then placing the mixture in a light incubator (16h light and 8h dark) to continue culturing for 2 days at 23 ℃ for later use.

2. Infection with the Strain

Plasmids containing the GmCYP15 gene (recombinant vectors pU1301-GmCYP15 constructed in example 1) were introduced into Agrobacterium A. rhizogenes LBA1334 (spectinomycin resistance) by transformation. Then, a single colony was picked from the plate cultured for 2 days and inoculated into 5ml of LB medium (containing plasmid resistance), followed by shaking culture at 28-30 ℃ for 16-24 hours. Then, the small amount of the bacteria inoculated on the previous day is subjected to amplification culture, 1: inoculating at a ratio of 100, and culturing at 28-30 deg.C for about 8 hr under shaking. The mass-cultured bacterial solution was centrifuged at 6000 rpm for 10 minutes, and the cells were collected and resuspended in sterile water so that the OD600 became about 0.8. Cutting off hypocotyl base of wild type Lotus japonicus MG20 seedling, soaking cotyledonary part with the re-suspended bacteria solution for 30min, taking out explant, drying with filter paper, and co-culturing in MS culture medium without sucrose.

3. Positive transgenic root identification and culture

Co-culturing in dark for 3-5 days. The explants were then transferred to HRE medium containing 300. mu.g/mL cefotaxime for an additional 10 days during which time hair roots grew out of the hypocotyl incisions. Then, root segments with the length of about 0.5cm at the root tips are cut and put into GUS dye solution, dark culture is carried out at 37 ℃ overnight, and blue-appearing roots are positive trans-GmCYP 15 crow's hair roots. Non-transformed roots were cut off, and the seedlings were hardened in a petri dish (without lid) containing water for 1 day, and then transplanted in a flowerpot and cultured in a light incubator (16h light, 8h dark) at 23 ℃. The base material mixed by vermiculite (vermiculite) and sand (sand) according to the proportion of 1:1 is added in the flowerpot in advance. The nitrogen-free nutrient solution is poured once every 3 days.

The vector pU1301 was transformed into Agrobacterium rhizogenes LBA1334 for hairy root transformation of Lotus corniculatus to obtain the empty vector Lotus corniculatus.

Functional analysis of GmCYP15 gene overexpression in crowtoe

1. Inoculation of Rhizobium

Activating Rhizobium loti MAFF303099 on a YMA plate, inoculating into a liquid TY culture medium, and performing shake culture at 28 ℃ for 24-36 h; transferring the cultured rhizobia into a sterile centrifuge tube, centrifuging at 4 ℃ at 7000r/min for 5min, and collecting thalli; washing with sterile Fahraeus nitrogen-free nutrient solution and centrifuging for 2 times; adding nitrogen-free nutrient solution to re-suspend the thalli, and inoculating the thalli to the roots of the seedlings with the positive transgenic GmCYP15 crowtoe. Meanwhile, empty vector Lotus corniculatus and wild type Lotus corniculatus MG20 are used as controls.

2. Functional analysis of GmCYP15 gene overexpression in crowtoe

After inoculating rhizobium for 50 days, harvesting positive trans-GmCYP 15 crow's root (GmCYP15-OX) and trans-empty vector crow's root (CK), observing the phenotype of the whole plant (including overground part and underground root system), and photographing and recording. The plant height, the fresh weight of the overground part and the number of the root nodules of each plant are recorded, and then the whole is subjected to statistical comparative analysis. And randomly selecting 15 larger nodules for FAA fixation, and observing paraffin sections. Randomly taking root nodule materials of the positive trans-GmCYP 15 crowtoe and control root nodule materials to extract RNA, and detecting the expression abundance of the GmCYP15 in the positive trans-GmCYP 15 crowtoe hairy root materials and the control hairy root materials by a semi-quantitative method; the expression conditions of 3 nodulin genes in the positive transgenic GmCYP15 crowtoe hairy root material and the control hairy root material are detected by a qPCR method. 36 plants were obtained from transgenic GmCYP15 crowtoe (GmCYP15-OX), 35 plants were obtained from transgenic empty vector Crowtoe (CK), and the results were averaged.

The semi-quantitative PCR detection method of the GmCYP15 gene comprises the following steps: and extracting RNA from the mixed transgenic hairy root material, and performing reverse transcription to obtain cDNA. Then, PCR amplification was carried out using this cDNA as a template. The primer sequences used were as follows: an upstream primer (5'-GGTACCATGGCAATGAAGAAGTTCTTG-3') and a downstream primer (5'-GGATCCTCAAAGTTCATCTTTAGGAGATG-3'); ubiquitin (l. japonicum): an upstream primer (5'-TTCACCTTGTGCTCCGTCTTC-3') and a downstream primer (5'-AACAACCAGCACACACAGACAATC-3').

The qPCR detection method for three nodulin genes is as follows: and extracting RNA from the mixed transgenic hairy root material, and performing reverse transcription to obtain cDNA. Then, qPCR was performed using the cDNA as a template. The primer sequences used were as follows: ubiquitin (Lotus corniculatus): an upstream primer (5'-TTCACCTTGTGCTCCGTCTTC-3') and a downstream primer (5'-AACAACCAGCACACACAGACAATC-3'); NIN (Lotus corniculatus): an upstream primer (5'-AACTCACTGGAAACAGGTGCTTTC-3') and a downstream primer (5'-CTATTGCGGAATGTATTAGCTAGA-3'); lb (Lotus corniculatus): an upstream primer (5'-CTCCAAGCCCATGCTGAAAA-3') and a downstream primer (5'-TGGCATCTGCAAGTGTCACTTC-3'); enod40 (Lotus root): an upstream primer (5'-CAAAACTCGTTATGTTGCGG-3') and a downstream primer (5'-CACCTCAAAGGA AGAAGA ACA-3').

The specific steps for preparing the paraffin sections of the root nodules are as follows: paraffin section dewaxing to water: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing. Repairing: after the section is slightly dried, a circle is drawn around the tissue by a organizing pen (liquid is prevented from flowing away), protease K working solution is dripped into the circle to cover the tissue, and the tissue is incubated for 30min at 37 ℃. Slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. ③ breaking the membrane: after the section is slightly dried, the membrane-breaking working solution is dripped into the ring to cover the tissue, the incubation is carried out for 20min at normal temperature, and the slide is placed in PBS (PH7.4) and is shaken and washed on a decoloration shaking bed for 3 times, 5min each time. Adding reagent 1, 2: taking a proper amount of reagent 1(TdT) and reagent 2(dUTP) in a tunel reagent box according to the number of the slices and the size of the tissues, mixing the mixture according to a ratio of 2:29, adding the mixture into a ring to cover the tissues, placing the slices in a wet box, incubating the slices in a constant temperature box at 37 ℃ for 2 hours, and adding a small amount of water in the wet box to keep the humidity. Blocking endogenous peroxidase: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. The sections were placed in 3% hydrogen peroxide solution in methanol, incubated at room temperature in the dark for 15min, and the slides were washed in PBS (pH7.4) on a destaining shaker for 3 times, 5min each. Sixthly, adding a reagent 3: after the sections were spin-dried slightly, each section was covered with an appropriate amount of reagent 3 (coverer-POD), and the sections were placed flat in a wet box and incubated in an incubator at 37 ℃ for 30 min. Slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. Seventh, DAB color development: after the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive nucleus is brownish yellow, and the section is washed by tap water to stop color development. Eighthly, counterstaining cell nuclei: harris hematoxylin is counterstained for about 3min, washed with tap water, 1% hydrochloric acid alcohol is differentiated for several seconds, washed with tap water, returned to blue by ammonia water and washed with running water. Ninthly, dehydrating and sealing: placing the slices in 75% alcohol for 6 min-85% alcohol for 6 min-absolute ethanol I for 6 min-absolute ethanol II for 6 min-xylene I for 5min, dehydrating, taking out the slices from xylene, air drying, and sealing with neutral gum.

The results show that: after the GmCYP15 gene is over-expressed, compared with the control of an empty vector, the growth state of the trans-GmCYP 15 crow root is obviously deteriorated (figure 3), the plant is shortened, the fresh weight of the overground part is reduced, the number of nodules is obviously reduced (figure 4), the height of the trans-GmCYP 15 crow root plant is 7.5444cm, the fresh weight of the overground part is 0.4014g, and the number of nodules is 6.0833; the height of the control plant of the empty vector crowtoe is 11.3114cm, the fresh weight of the overground part is 0.6986g, and the number of root nodules is 11.5143. Compared with the empty vector control, the splitting phenomenon of the symbiont membrane of the crowtoe transformed by the GmCYP15 is relatively obvious, and the root nodule senescence is accelerated (figure 5). Compared with the empty vector control, the expression level of the GmCYP15 gene in the trans-GmCYP 15 crowtoe hairy root is obviously increased (figure 6A); the expression level of three nodulin genes in the transgenic crowtoe hairy roots of GmCYP15 is obviously reduced (FIG. 6B). The growth state, the plant height, the fresh weight of overground parts, the number of root nodules, the senescence of the root nodules and the expression quantity of related genes of the wild type Lotus corniculatus have no significant difference with the wild type Lotus corniculatus with an empty vector.

The above results show that: the GmCYP15 gene negatively regulates the number of the root nodules of the plant, can accelerate the senescence of the root nodules, is a negative regulation factor for symbiosis of leguminous plants, and can knock out the gene by means of gene editing so as to greatly increase the number of the root nodules of the plant, prolong the nitrogen fixation time of the root nodules and further improve the nitrogen fixation capacity of the plant, thereby having certain application prospect in the nitrogen fixation mechanism research and agricultural production of leguminous crops.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

<120> method for regulating and controlling plant root nodule development and senescence and application thereof

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<170>PatentIn version 3.5

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cagttcatca agcagaaggg aggcataaca acagaaagct attaccctta cacagctcaa 660

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Met Ala Met Lys Lys Phe Leu Trp Val Val Leu Ser Leu Ser Leu Val

1 5 10 15

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

20 25 30

Glu Glu Ser Leu Trp Asp Leu Tyr Glu Arg Trp Arg Ser His His Thr

35 40 45

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

50 55 60

Ala Asn Val Met His Val His Asn Thr Asn Lys Met Asp Lys Pro Tyr

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Lys Leu Lys Leu Asn Lys Phe Ala Asp Met Thr Asn His Glu Phe Arg

85 90 95

Ser Thr Tyr Ala Gly Ser Lys Val Asn His His Arg Met Phe Arg Asp

100 105 110

Met Pro Arg Gly Asn Gly Thr Phe Met Tyr Glu Lys Val Gly Ser Val

115 120 125

Pro Ala Ser Val Asp Trp Arg Lys Lys Gly Ala Val Thr Asp Val Lys

130 135 140

Asp Gln Gly His Cys Gly Ser Cys Trp Ala Phe Ser Thr Val Val Ala

145 150 155 160

Val Glu Gly Ile Asn Gln Ile Lys Thr Asn Lys Leu Val Ser Leu Ser

165 170 175

Glu Gln Glu Leu Val Asp Cys Asp Thr Glu Glu Asn Ala Gly Cys Asn

180 185 190

Gly Gly Leu Met Glu Ser Ala Phe Gln Phe Ile Lys Gln Lys Gly Gly

195 200 205

Ile Thr Thr Glu Ser Tyr Tyr Pro Tyr Thr Ala Gln Asp Gly Thr Cys

210 215 220

Asp Ala Ser Lys Ala Asn Asp Leu Ala Val Ser Ile Asp Gly His Glu

225 230 235 240

Asn Val Pro Gly Asn Asp Glu Asn Ala Leu Leu Lys Ala Val Ala Asn

245 250 255

Gln Pro Val Ser Val Ala Ile Asp Ala Gly Gly Ser Asp Phe Gln Phe

260 265 270

Tyr Ser Glu Gly Val Phe Thr Gly Asp Cys Ser Thr Glu Leu Asn His

275 280 285

Gly Val Ala Ile Val Gly Tyr Gly Ala Thr Val Asp Gly Thr Ser Tyr

290 295 300

Trp Ile Val Arg Asn Ser Trp Gly Pro Glu Trp Gly Glu Gln Gly Tyr

305 310 315 320

Ile Arg Met Gln Arg Asn Ile Ser Lys Lys Glu Gly Leu Cys Gly Ile

325 330 335

Ala Met Leu Ala Ser Tyr Pro Ile Lys Asn Ser Ser Asn Asn Pro Thr

340 345 350

Gly Pro Ser Ser Ser Pro Lys Asp Glu Leu

355 360

Claims (9)

1. Use of a GmCYP15 protein in any one of the following (1) to (8):

   (1) regulating and controlling nitrogen fixation capacity of plants or symbiotic nitrogen fixation or symbiotic nodulation;

   (2) regulating and controlling the growth of plant root nodules;

   (3) regulating and controlling the number of plant nodules;

   (4) regulating and controlling the plant root nodule aging process;

   (5) regulating and controlling the nitrogen fixation time of the plant root nodule;

   (6) regulating the expression level of a plant nodulin gene;

   (7) regulating and controlling the plant height;

   (8) regulating and controlling the fresh weight of the overground part of the plant;

   the GmCYP15 protein is a protein with an amino acid sequence shown in a sequence 2;

   the regulation is negative regulation.
2. 2. Use of a biomaterial related to GmCYP15 protein in any one of the following (1) to (8):

   (1) regulating and controlling nitrogen fixation capacity of plants or symbiotic nitrogen fixation or symbiotic nodulation;

   (2) regulating and controlling the growth of plant root nodules;

   (3) regulating and controlling the number of plant nodules;

   (4) regulating and controlling the plant root nodule aging process;

   (5) regulating and controlling the nitrogen fixation time of the plant root nodule;

   (6) regulating the expression level of a plant nodulin gene;

   (7) regulating and controlling the plant height;

   (8) regulating and controlling the fresh weight of the overground part of the plant;

   the GmCYP15 protein is a protein with an amino acid sequence shown in a sequence 2;

   the regulation is negative regulation;

   the biomaterial is any one of the following A1) to A8):

   A1) a nucleic acid molecule encoding a GmCYP15 protein;

   A2) an expression cassette comprising the nucleic acid molecule of a 1);

   A3) a recombinant vector comprising the nucleic acid molecule of a 1);

   A4) a recombinant vector comprising the expression cassette of a 2);

   A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

   A6) a recombinant microorganism comprising the expression cassette of a 2);

   A7) a recombinant microorganism comprising a3) said recombinant vector;

   A8) a recombinant microorganism comprising the recombinant vector of a 4).
3. 3. Use according to claim 2, characterized in that: A1) the nucleic acid molecule is a cDNA molecule shown in a sequence 1.
4. 4. Use according to claim 1 or 2, characterized in that: the nodulin gene is NIN gene and/or Lb gene and/or Enod40 gene.
5. 5. Use of the GmCYP15 protein according to claim 1 or of the biological material according to claim 2 or 3 for the cultivation of transgenic plants with reduced nitrogen fixation capacity and/or reduced number of nodules and/or accelerated senescence and/or shortened nitrogen fixation time and/or reduced plant height and/or reduced fresh weight of aerial parts.
6. 6. A method for producing a transgenic plant, comprising the step of increasing the expression level and/or activity of GmCYP15 protein according to claim 1 in a plant of interest to obtain a transgenic plant; the transgenic plant has a lower nitrogen fixation capacity than the plant of interest and/or the transgenic plant has fewer root nodules than the plant of interest and/or the transgenic plant has faster senescence rate than the plant of interest and/or the transgenic plant has a shorter nitrogen fixation time than the plant of interest and/or the transgenic plant has a lower plant height than the plant of interest and/or the transgenic plant has less fresh weight of the aerial part than the plant of interest.
7. 7. The method of claim 6, wherein: the improvement of the expression level and/or activity of the GmCYP15 protein according to claim 1 in a plant of interest is achieved by introducing a gene encoding the GmCYP15 protein according to claim 1 into a plant of interest.
8. 8. The method of claim 7, wherein: the nucleotide sequence of the coding gene of the GmCYP15 protein is a DNA molecule shown in a sequence 1.
9. 9. Use according to claim 1 or 2 or 5 or a method according to any of claims 6 to 8, wherein: the plant is a monocotyledon or a dicotyledon.

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