CN110627886B

CN110627886B - Method for cultivating transgenic leguminous plant with increased root nodule size and application of transgenic leguminous plant

Info

Publication number: CN110627886B
Application number: CN201910903078.7A
Authority: CN
Inventors: 袁松丽; 李�荣; 张晓娟; 张婵娟; 陈李淼; 郝青南; 陈海峰; 陈水莲; 郭葳; 单志慧; 杨中路; 邱德珍; 曹东; 杨红丽; 黄毅; 周新安
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2021-06-01
Anticipated expiration: 2039-09-24
Also published as: CN110627886A

Abstract

The invention discloses a method for cultivating transgenic leguminous plants with increased root nodule sizes and application thereof. The method comprises the following steps: improving the expression quantity and/or activity of protein GmCYS2 in the original leguminous plant to obtain a transgenic leguminous plant; the transgenic legume plants have an increased nodule size compared to the starting legume. Experiments prove that the GmCYS2 gene is introduced into a soybean variety Tianlong I to obtain transgenic soybean; the root nodule size of transgenic soybeans is increased compared to the soybean variety Tianlong No. one. Therefore, the protein GmCYS2 can regulate and control the nitrogen fixation efficiency of the soybeans. The invention has important application value.

Description

Method for cultivating transgenic leguminous plant with increased root nodule size and application of transgenic leguminous plant

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for cultivating transgenic leguminous plants with increased root nodule sizes and application of the transgenic leguminous plants.

Background

Symbiotic nitrogen fixation between leguminous plants and rhizobia is an important natural nitrogen source in an agricultural ecological system, and is the most economical and environment-friendly nitrogen fixation mode. The soybean seed has protein content of 40% and oil content of 20%, and is the most important raw material for oil crop and livestock feed in the world, and is also an excellent crop for crop rotation and intercropping in agricultural production. However, soybeans require a large amount of nitrogen source for planting, and the nitrogen in soybeans, without excessive application of nitrogen fertilizer, is estimated to be 58-68% from symbiotic nitrogen fixation. After harvesting, 30-60% of the nitrogen in the underground roots and nodules of the plants can be used for replenishing the surrounding soil. Therefore, the biological nitrogen fixation function of the soybean is fully exerted, and the method has great significance for weight reduction and efficiency improvement and development of environment-friendly sustainable ecological agriculture.

The formation of root nodules and nitrogen fixation in leguminous plants is a very complex process that is the result of the interaction between microorganisms and plants, and signal molecules. Generally, methods for increasing nitrogen fixation efficiency of soybeans include three methods: firstly, the number of nodules is increased; secondly, improving the nitrogen fixation capacity of the nodule, which mainly comprises the increase of the size of the nodule and the improvement of the activity of the nitrogen fixation enzyme; thirdly, the root nodule aging is delayed, the nitrogen fixation time of the root nodule is prolonged, and the nitrogen fixation amount is increased. Therefore, the method has important significance in the aspect of symbiotic nitrogen fixation of the soybeans by increasing the size of the root nodules and improving the nitrogen fixation capacity of the root nodules per unit to improve the nitrogen fixation amount of the soybeans and guarantee the nitrogen supply in the growth period of the soybeans.

Disclosure of Invention

The invention aims to increase the size of soybean root nodules so as to improve the nitrogen fixation efficiency of soybeans.

The invention firstly protects the application of the protein GmCYS2, which can be at least one of the following D1) -D5):

D1) regulating the size of the root nodule of the leguminous plant;

D2) regulating and controlling the plant height of the plant;

D3) regulating and controlling plant biomass;

D4) cultivating transgenic leguminous plants with altered nodule size;

D5) and (3) cultivating the transgenic plant with the changed plant height and/or the changed biomass.

In the above application, the protein GmCYS2 can be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) and (b) the protein which 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 the sequence table, is related to the size of the root nodule of the leguminous plant and/or the plant height and/or the plant biomass.

Wherein, the sequence 2 in the sequence table is composed of 130 amino acid residues.

In order to facilitate the purification of the protein in a1), 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
FLAG
		8	DYKDDDDK
Strep-tag II				8	WSHPQFEK
	c-myc	10	EQKLISEEDL

The protein according to a3), 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 protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The invention also protects the application of a nucleic acid molecule for coding the protein GmCYS2, which can be at least one of the following D1) -D5):

D1) regulating the size of the root nodule of the leguminous plant;

D2) regulating and controlling the plant height of the plant;

D3) regulating and controlling plant biomass;

D4) cultivating transgenic leguminous plants with altered nodule size;

In the above application, the nucleic acid molecule encoding the protein GmCYS2 can be a DNA molecule shown in b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule which has 75 percent or more identity with the nucleotide sequence defined by b1) or (b2) and codes the protein GmCYS 2;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under strict conditions and codes for the protein GmCYS 2.

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 in the sequence table consists of 393 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence of the protein GmCYS2 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 and have 75% or more identity with the nucleotide sequence of the protein GmCYS2 isolated in the invention are derived from the nucleotide sequence of the invention and are identical with the sequence of the invention as long as the nucleotide sequence encodes the protein GmCYS 2.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of protein GmCYS2 consisting of the amino acid sequence shown in sequence No. 2 of the coding sequence Listing 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 any of the above applications, the controlling the size of the root nodule of the legume may be increasing the size of the root nodule of the legume.

In any of the above applications, the regulating the plant height may be increasing the plant height.

In any of the above applications, the regulating plant biomass may be increasing plant biomass.

Any of the transgenic leguminous plants described above having altered root nodule size may in particular be transgenic leguminous plants having increased root nodule size.

Any of the transgenic plants with altered plant height and/or altered biomass can be specifically a transgenic plant with increased plant height and/or increased biomass.

The invention also provides a method for cultivating the transgenic leguminous plant, which comprises the following steps: improving the expression quantity and/or activity of the protein GmCYS2 in the starting leguminous plant to obtain a transgenic leguminous plant; the transgenic legume plants have an increased nodule size compared to the starting legume.

In the method, the expression quantity and/or activity of the protein GmCYS2 in the starting leguminous plant can be improved by a method of multi-copy, promoter, regulatory factor or transgene, so that the effect of improving the expression quantity and/or activity of the protein GmCYS2 in the starting leguminous plant is achieved.

In the above method, the "improvement of the expression level and/or activity of the protein GmCYS2 in the starting leguminous plant" may be achieved by introducing a nucleic acid molecule encoding the protein GmCYS2 into the starting leguminous plant.

In the above method, the "introducing into the starting leguminous plant a nucleic acid molecule encoding the protein GmCYS 2" may be carried out by introducing into the starting leguminous plant a recombinant vector; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule coding for protein GmCYS2 into an expression vector.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: improving the expression quantity and/or activity of the protein GmCYS2 in the starting plant to obtain a transgenic plant; the transgenic plants have an increased plant height and/or increased biomass compared to the starting plants.

In the method, the expression quantity and/or activity of the protein GmCYS2 in the starting plant can be improved by a method of multi-copy, promoter, regulatory factor or transgene, so that the effect of improving the expression quantity and/or activity of the protein GmCYS2 in the starting plant is achieved.

In the above method, the "improvement of the expression level and/or activity of the protein GmCYS2 in the starting plant" may be achieved by introducing a nucleic acid molecule encoding the protein GmCYS2 into the starting plant.

In the above method, the "introducing into the starting plant a nucleic acid molecule encoding the protein GmCYS 2" may be carried out by introducing into the starting plant a recombinant vector; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule coding for protein GmCYS2 into an expression vector.

Any one of the recombinant vectors can be specifically a recombinant plasmid pB2GW7-GmCYS 2.

The construction method of the recombinant plasmid pB2GW7-GmCYS2 can be as follows:

(1) the cDNA of soybean variety Tianlong No. is used as a template, a primer pair consisting of 5'-ATGGCGGCGTTGATAAGGTC-3' and 5'-TCACTGCGTGGAAGGAGCGA-3' is adopted for PCR amplification, and a PCR amplification product with the size of 393bp is recovered.

(2) The entry vector pGWC was digested with the restriction enzyme AhdI and the vector backbone of about 2500bp was recovered.

(3) And (2) connecting the PCR amplification product recovered in the step (1) with a vector framework to obtain a recombinant plasmid pGWC-GmCYS 2.

(4) And carrying out LR recombination reaction on the recombinant plasmid pGWC-GmCYS2 and the pB2GW7 vector to obtain a recombinant plasmid pB2GW7-GmCYS 2.

The rotating baseThe plant or transgenic leguminous plant can be GmCYS2OX-1-T₃、GmCYS2OX-2-T₃、GmCYS2OX-3-T₃、GmCYS2OX-1-T₀、GmCYS2OX-2-T₀And GmCYS2OX-3-T₀. At the moment, the starting plant is soybean, in particular to a soybean variety Tianlong No. I.

The invention also protects K1) or K2).

K1) A method of breeding leguminous plants, comprising the steps of: increasing the expression level and/or activity of the protein GmCYS2 in the leguminous plant, so that the size of the root nodule of the leguminous plant is increased.

K2) A method of plant breeding comprising the steps of: the expression quantity and/or activity of the protein GmCYS2 in the plant is increased, so that the plant height and/or biomass of the plant are increased.

Any of the above-mentioned biomass may be, in particular, fresh weight of aerial parts.

Any of the above leguminous plants may be c4) or c 5); c4) soybean; c5) soybean variety tianlong number one.

Any of the plants described above may be any of c1) -c 5); c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) soybean; c5) soybean variety tianlong number one.

Any one of the above nodule sizes may specifically be a nodule volume size. The size of the nodule volume or the size of the nodule can be evaluated using the nodule diameter. The larger the diameter of the nodule, the larger the volume of the nodule, i.e., the larger the nodule.

Experiments prove that the GmCYS2 gene is introduced into a soybean variety Tianlong I to obtain transgenic soybean; compared with the soybean variety Tianlong No. one, the transgenic soybean has increased root nodule size, plant height and biomass. Therefore, the protein GmCYS2 can regulate and control the nitrogen fixation efficiency of the soybeans. The invention has important application value.

Drawings

FIG. 1 is a structural schematic diagram of recombinant plasmid pB2GW7-GmCYS 2.

FIG. 2 shows the identification result of Bar immunoassay test paper.

FIG. 3 is T₀GmCYS2 in soybean with pseudo-transferred GmCYS2 geneThe result of detecting whether the gene is integrated.

FIG. 4 is T₀And (3) simulating the detection result of whether the Bar gene is integrated in soybean transformed with the GmCYS2 gene.

FIG. 5 is T₃And (3) statistics results of the growth state, the plant height and the fresh weight of the overground part of the soybean subjected to the soybean homozygous transgenic GmCYS2 gene inoculation for 55 days.

To be significantly different, P<0.05；

Is very significantly different, P<0.01。

FIG. 6 is T₃And (4) carrying out statistics on the root nodules and the diameter of the root nodules of the soybean inoculated with the soybean rhizobia by the generation homozygous transgenic soybean with the GmCYS2 gene for 55 days.

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 soybean variety tianlong No. is described in the following documents: the hybrid seed is prepared by sexual hybridization and pedigree breeding of the institute of oil crop research of Chinese agricultural academy of sciences, wherein the female parent of the Tianlong I of the soybean variety is the middle bean 32, and the male parent of the Tianlong I of the soybean variety is the middle bean 29 in 2015 year 05. Hereinafter, the soybean variety "Tianlong No.", is abbreviated as "Tianlong No.".

Entry vectors pGWC are described in the following documents: chen QJ, Zhou HM, Chen J et al, using a modified TA cleaning method to critical entities Biochemistry, 2006, 358: 120-.

The pB2GW7 vector is described in the following documents: karimi M, Inz é D, decapicker a. gateway vectors for Agrobacterium-mediated Plant transformation. trends in Plant Science, 2002, 7 (5): 193-195.

Agrobacterium tumefaciens EHA105 is described in: duan X, Zheng L et al Co-cutting on dry filter paper marking the information of the microorganisms-mediated transformations of mail immMATure organizations Physiol Mol Biol plants.2019, 25 (2): 549-560.

Rhizobium japonicum 113-2 is described in the following documents: song L Yeast, Rong Li, et al, RNA-Seq analysis of minor definition at specific definition stages of nutrient (Glycine max) unsaturated with Bradyrhizobium japonicum strain113-2.Scientific Reports, 2017, 7:42248. hereinafter, Rhizobium japonicum 113-2 is abbreviated as Rhizobium japonicum.

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

MS culture medium: to the right amount of ddH₂Adding 4.3g MS basal salt mix (Sigma), 0.103g MS vitamin powder (Sigma) and 7-8g agar powder into O, mixing, and adding ddH₂And (4) metering the volume of O to 1000mL, adjusting the pH value to 5.8, and sterilizing at 121 ℃ for 20 min.

YMA medium: to the right amount of ddH₂Adding 10.0g mannitol (or sucrose), 0.4g Yeast Extract, and 0.5g K into O₂HPO₄、0.2g MgSO₄·7H₂O、0.1g CaCl₂·6H₂O, 0.1g NaCl and 4mL Rh microelement, mixing well and using 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: to the right amount of ddH₂Adding 5.0g H into O₃BO₃And 5.0g of Na₂MoO₄Mixing and then using ddH₂And O is metered to 1000 mL.

YMA plate: to the right amount of ddH₂Adding 10.0g mannitol (or sucrose), 0.4g Yeast Extract, and 0.5g K into O₂HPO₄、0.2g MgSO₄·7H₂O、0.1g CaCl₂·6H₂O, 0.1g NaCl, 4mL Rh microelement liquid and 7-8g agar powder, mixing uniformly, and then using ddH₂O is metered to 1000mL, the pH value is adjusted to 6.8-7.0, and sterilization is carried out for 20min at 115 ℃; after cooling to about 55 ℃, the mixture was poured into sterile petri dishes (the diameter of each dish was 9cm, and 20mL of each dish was poured), and cooled naturally to obtain a YMA plate.

Nitrogen-free nutrient solution: to the right amount of ddH₂0.10g of CaCl was added to O₂·2H₂O、0.12g MgSO₄·7H₂O、0.10g KH₂PO₄、0.15g Na₂HPO₄·12H₂O, 1mL of Gibson microelement liquid and 5mg of ferric citrate, mixing uniformly, and adding ddH₂And O is metered to 1000 mL.

Gibson trace element liquid: to the right amount of ddH₂O with 2.86g H₃BO₃、0.22g ZnSO₄·7H₂O、2.03g MnSO₄·4H₂O、0.13g Na₂MoO₄·2H₂O and 0.08g CuSO₄·5H₂O, mixing and then using ddH₂And O is metered to 1000 mL.

Example 1 cloning of the GmCYS2 Gene

1. Total RNA was extracted from the Tianlong-No. one roots using Trizol reagent (Invitrogen) to obtain total RNA of soybean.

2. After step 1 is completed, the total RNA of the soybean is taken and reverse transcription is carried out by reverse Transcriptase (TAKARA), and cDNA of the soybean is obtained.

The reaction conditions are as follows: 15min at 37 ℃ and 5sec at 85 ℃.

3. After the step 2 is completed, the cDNA of the soybean is taken as a template, a primer pair consisting of 5'-ATGGCGGCGTTGATAAGGTC-3' and 5'-TCACTGCGTGGAAGGAGCGA-3' is adopted for PCR amplification, and a PCR amplification product with the size of 393bp is recovered.

4. After step 3 is completed, the PCR amplification product is connected with a vector pMD18-T (TaKaRa) to obtain a recombinant plasmid pMD18-T-GmCYS 2.

The recombinant plasmid pMD18-T-GmCYS2 was sequenced. Sequencing results show that the recombinant plasmid pMD18-T-GmCYS2 contains a DNA molecule (named as GmCYS2 gene) shown as a sequence 1 in a sequence table.

Example 2, T₃Acquisition and phenotypic identification of generation homozygous transgenic soybean with GmCYS2 gene

Construction of recombinant plasmid pB2GW7-GmCYS2

1. The recombinant plasmid pMD18-T-GmCYS2 is used as a template, a primer pair consisting of 5'-ATGGCGGCGTTGATAAGGTC-3' and 5'-TCACTGCGTGGAAGGAGCGA-3' is adopted for PCR amplification, and a PCR amplification product with the size of 393bp is recovered.

2. The entry vector pGWC was digested with the restriction enzyme AhdI and the vector backbone 1 of about 2500bp was recovered.

3. And (3) connecting the PCR amplification product recovered in the step (1) with a vector framework 1 to obtain a recombinant plasmid pGWC-GmCYS 2.

4. And carrying out LR recombination reaction on the recombinant plasmid pGWC-GmCYS2 and the pB2GW7 vector to obtain a recombinant plasmid pB2GW7-GmCYS 2.

The structural schematic diagram of the recombinant plasmid pB2GW7-GmCYS2 is shown in figure 1.

The recombinant plasmid pB2GW7-GmCYS2 was sequenced. The sequencing result shows that the recombinant plasmid pB2GW7-GmCYS2 contains a DNA molecule shown in a sequence 1 in a sequence table.

The recombinant plasmid pB2GW7-GmCYS2 expresses protein GmCYS2 shown in a sequence 2 in a sequence table.

II, obtaining recombinant agrobacterium

The recombinant plasmid pB2GW7-GmCYS2 is introduced into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium tumefaciens which is named as EHA105/pB2GW7-GmCYS 2.

The pB2GW7 vector is introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which is named as EHA105/pB2GW 7.

III, T₀Obtaining of soybean with GmCYS2 gene

1. The method adopts an Agrobacterium-mediated soybean cotyledonary node genetic transformation method (Paz M, Martinez JC, Kalvig AB et al. improved cottonly node method using an alternative active expression from the seed for the transformation of EHA105/pB2GW7-GmCYS2 into Tianlong I (screening by using herbicide base) to obtain T₀Simulating a soybean plant with GmCYS2 gene,after maturation, the single plant is harvested.

2. Planting seeds of different strains in nutrient soil, and culturing at 25 ℃ until compound leaves are unfolded and flat; and then spraying Basta (spraying concentration is 100mg/L) to the compound leaves, if the ratio of the number of the soybean plants (resistant seedlings) with green compound leaves to the number of the soybean plants (non-resistant seedlings) with yellow compound leaves in a certain strain is 3: 1, the strain is a strain with one copy of the GmCYS2 gene inserted.

3. Bar immune test paper identification

(1) Planting the seeds identified as a strain with one copy of the GmCYS2 gene inserted into the strain in the step 2 in nutrient soil, and culturing at 25 ℃ until the compound leaves are unfolded and flat; then 0.1g of compound leaves are taken from each plant, and total protein is extracted to obtain protein extracting solution.

(2) And (3) restoring the transgenic test paper to room temperature, vertically inserting the protein extracting solution, taking out after 1min, and flatly reading the detection result.

The transgenic test paper is a product of the institute of oil crop of Chinese academy of agricultural sciences, and can be obtained by the public from the institute of oil crop of Chinese academy of agricultural sciences (namely the applicant).

(3) The detection line and the control line can appear within 1-2min generally, and the detection standard is as follows: only one mauve quality control line appears on the detection strip, and the result is negative; two purple-red strips appear on the detection strip, one is a purple-red detection line, and the other is a purple-red quality control line, which is positive.

If the ratio of the number of soybean plants in a line from which two purplish-red bands are obtained to the number of soybean plants from which one purplish-red band is obtained is 3: 1, the strain is a strain with one copy of the GmCYS2 gene inserted.

The plants in the method are replaced by the Tianlong I plants, and other steps are not changed and are used as negative control.

The plant in the method is replaced by a soybean (disclosed in Chinese invention patent document CN 106397562A) plant transformed with GmGATA44 gene, and other steps are not changed and are used as positive control.

The results of the partial detection are shown in FIG. 2.

Randomly selected 8 GmCYS2 gene insertionsOne copy strain (i.e., 8T's)₀A soybean plant transformed with GmCYS2 gene) for subsequent experiments.

A strain in which the 1 st GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-1.

A strain in which the 2 nd GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-2.

A strain in which the 3 rd GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-3.

A strain in which the 4 th GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-4.

A strain in which the 5 th GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-5.

A strain in which the 6 th GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-6.

A strain in which the 7 th GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-7.

A strain in which the 8 th GmCYS2 gene is inserted into one copy and the descendants thereof is named as GmCYS2 OX-8.

According to the method, EHA105/pB2GW7-GmCYS2 is replaced by EHA105/pB2GW7, and other steps are not changed to obtain the trans-empty vector soybean.

4. Molecular level detection

(1) By T₀Soybean with pseudo-transferred GmCYS2 gene (GmCYS2 OX-1-T)₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀Or GmCYS2OX-8-T₀) Genomic DNA of leaf was used as template, and OSH 177F: 5'-CATTTGGAGAGGACTCCGG-3' and GmCYS 2R: 5'-TCACTGCGTGGAAGGAGCGA-3', and then carrying out PCR amplification on the primer pair as follows: if the PCR amplification product contains a 493bp DNA band, the corresponding T₀The GmCYS2 gene in soybean transformed by the transfer of the GmCYS2 gene is successfully integrated into the genome of Tianlong I.

The reaction system was 20. mu.L, consisting of 1. mu.L of template, 0.5. mu.L of upstream primer (at a concentration of 10. mu. mol/L), 0.5. mu.L of downstream primer (at a concentration of 10. mu. mol/L), 2. mu.L of 10 XBuffer (TaKaRa), 0.5. mu.L of dNTP (at a concentration of 10mM), 0.2. mu.L of Taq DNA polymerase (at a concentration of 5U/. mu.L) and 15.3. mu.L of sterile water. The upstream primer is a primer containing "F" in the name of the primer. The downstream primer is the primer containing "R" in the name of the primer.

Reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 0.5min, annealing at 58 deg.C for 0.5min, extension at 72 deg.C for 0.5min, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

The template in the method is replaced by a recombinant plasmid pB2GW7-GmCYS2, and other steps are not changed and are used as a positive control.

The template in the method is replaced by the genomic DNA of the Tianlong first leaf or the genomic DNA of the empty vector soybean leaf, and other steps are not changed and are used as negative control.

The partial results are shown in FIG. 3 (lanes 1-8 are GmCYS2OX-1-T in turn)₀-GmCYS2OX-8-T₀Lane 9 is the recombinant plasmid pB2GW7-GmCYS2, lane 10 is the genomic DNA of Tianlong No. one leaf, M is DNA Marker). The detection result shows that the PCR amplification products of the empty vector soybean and the Tianlong I do not contain a DNA band with the size of 493bp, and the recombinant plasmids pB2GW7-GmCYS2 and GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀The PCR amplification products of (1) all contained a 493bp DNA band. As can be seen, GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀The medium GmCYS2 gene has been successfully integrated into the genome of Tianlong No. one.

(2) By T₀Soybean with pseudo-transferred GmCYS2 gene (GmCYS2 OX-1-T)₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀Or GmCYS2OX-8-T₀) Taking the genome DNA of the leaf as a template, and collectingWith a BarF: 5'-TCAGATTTCGTGACGGGCAG-3' and BarR: 5'-GTGCCACCGAGGCGGACAT-3', and then carrying out PCR amplification on the primer pair as follows: if the PCR amplification product contains DNA band with the size of 552bp, the corresponding T₀The Bar gene in soybean transformed with the GmCYS2 gene has been successfully integrated into the genome of Tianlong No. one.

The reaction system is the same as that in the step (2).

The reaction procedure is the same as that in step (2).

The partial results are shown in FIG. 4 (

lanes

1, 2, 3, 4, 5, 6, 8, 9 are GmCYS2OX-1-T in sequence)₀-GmCYS2OX-8-T₀Lane 10 is the recombinant plasmid pB2GW7-GmCYS2, lane 11 is the genomic DNA of tianlong leaf No. one, M is DNA Marker). The detection result shows that the PCR amplification products of the empty transfer vector soybean and the Tianlong I do not contain DNA bands with the size of 552bp, and the recombinant plasmids pB2GW7-GmCYS2 and GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀The PCR amplification products of (1) all contain DNA bands with the size of 552 bp. As can be seen, GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀The middle Bar gene has been successfully integrated into the genome of Tianlong No. one.

The above results show that GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀The gene GmCYS2 and the Bar gene have been successfully integrated into the genome of Tianlong No. one, and a copy of the gene GmCYS2 is inserted. GmCYS2OX-1-T₀、GmCYS2OX-2-T₀、GmCYS2OX-3-T₀、GmCYS2OX-4-T₀、GmCYS2OX-5-T₀、GmCYS2OX-6-T₀、GmCYS2OX-7-T₀And GmCYS2OX-8-T₀Are all T₀The GmCYS2 gene soybean is transferred.

Four, T₃Generation of Generation homozygous lines

GmCYS2OX-1-T₀Selfing for three generations continuously to obtain T₃Homozygous strain of generation GmCYS2OX-1, named GmCYS2OX-1-T₃. To T₃The homozygous lines of the GmCYS2OX-1 and the compound leaves of the descendants thereof are sprayed with Basta (the spraying concentration is 100mg/L), and the compound leaves are all green.

GmCYS2OX-2-T₀Selfing for three generations continuously to obtain T₃Homozygous strain of generation GmCYS2OX-2, named GmCYS2OX-2-T₃. To T₃The homozygous lines of the GmCYS2OX-1 and the compound leaves of the descendants thereof are sprayed with Basta (the spraying concentration is 100mg/L), and the compound leaves are all green.

GmCYS2OX-3-T₀Selfing for three generations continuously to obtain T₃Homozygous strain of generation GmCYS2OX-3, named GmCYS2OX-3-T₃. To T₃The homozygous lines of the GmCYS2OX-1 and the compound leaves of the descendants thereof are sprayed with Basta (the spraying concentration is 100mg/L), and the compound leaves are all green.

Subjecting empty vector-transferred soybean to three-generation selfing to obtain T₃The generation pure transgenic empty vector soybean.

Five, T₃Phenotypic identification of generation homozygous transgenic soybean with GmCYS2 gene

1. Activating Rhizobium japonicum on YMA plate, inoculating into YMA culture medium, and shake culturing at 28 deg.C for 2-3 days to obtain OD_600nm0.8 of rhizobium japonicum bacterial liquid.

2. Taking 40 seeds to be tested (Tianlong I seed, T)₃Seed of soybean with homozygous empty vector, GmCYS2OX-1-T₃Seed of GmCYS2OX-2-T₃Seed of (1) or GmCYS2OX-3-T₃Seed of (2), the paper roll germinates for 5-7 days.

3. After the step 2 is completed, planting 15 plants to be tested with good growth vigor and relatively consistent growth vigor in a sterilized substrate (vermiculite: perlite: 1), and simultaneously inoculating 1mL of soybean rhizobium bacterial liquid to each plant to be tested; culturing at 25 deg.C, and irrigating nitrogen-free nutrient solution during culturing period.

4. After the rhizobium japonicum liquid is inoculated for 55 days, cutting off the rhizobium japonicum liquid from cotyledonary nodes to photograph the overground part, simultaneously counting the plant height and the fresh weight of the overground part, and then taking an average value; and (4) grading the diameter of the root nodules and photographing the diameter of each root nodule aiming at the underground part, and meanwhile counting the diameter of each single root nodule.

The results of the above-ground part are shown in FIG. 5(A is the above-ground phenotype, B is the statistical result of plant height and fresh weight of the above-ground part, GmCYS2OX-1 is GmCYS2OX-1-T₃The GmCYS2OX-2 is GmCYS2OX-2-T₃The GmCYS2OX-3 is GmCYS2OX-3-T₃CK is tianlong number one) and table 2. The results show that T is comparable to Tianlong No. one₃Generation homozygous soybean transformed with GmCYS2 gene (such as GmCYS2 OX-1-T)₃、GmCYS2OX-2-T₃Or GmCYS2OX-3-T₃) The growth state of the strain is good, and the plant height and the fresh weight of the overground part are obviously increased; tianlong number one and T₃The growth state and plant height of the generation homozygous empty vector-transferred soybean and the fresh weight average of the overground part have no obvious difference.

TABLE 2

The results of the partial experiments in the underground part are shown in FIG. 6(A is a root nodule, B is a statistical result of the diameter of a single root nodule, GmCYS2OX-1 is GmCYS2OX-1-T₃The GmCYS2OX-2 is GmCYS2OX-2-T₃The GmCYS2OX-3 is GmCYS2OX-3-T₃And CK is Tianlong No. one). The results show that T is comparable to Tianlong No. one₃Generation homozygous soybean transformed with GmCYS2 gene (such as GmCYS2 OX-1-T)₃、GmCYS2OX-2-T₃Or GmCYS2OX-3-T₃) Root nodule ofThe diameter is obviously increased, and the proportion of the root nodules with the root nodule diameter larger than 3mm is obviously increased; tianlong number one and T₃The root nodule diameter of the soybean with the generation-homozygous empty vector has no obvious difference.

The results show that the GmCYS2 gene is over-expressed in Tianlong I, the diameter of the root nodule is obviously increased, and the plant height and the fresh weight of the overground part are obviously increased. Therefore, the protein GmCYS2 can regulate the diameter, the plant height and the biomass of the root nodule of the soybean.

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

<120> a method for breeding transgenic leguminous plants with increased root nodule size and applications thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 393

<212> DNA

<213> Glycine max（Linn.）Merr.

<400> 1

atggcggcgt tgataaggtc accggcggtg atactggcga tcctgacgat ctcggcgtgc 60

atcgcgtgta cggcgtcgta cgggggattg gtcgggggaa ggtcgaagat ccctgacgtg 120

aaggcgaaca agaaggtgca ggatctaggg cggttctcgg tggaggagca taaccggatg 180

ctgaggcagg cgcagaagga ggaggagcaa gtcacgttcg tggaagtggt ggaggcgcaa 240

caacaagtgg tgtctgggat caagtactac atgaagatat cggccacgca gggtggcgac 300

ggtggagatt ccagaatatt cgaatccgtt gtggtggtga agccgtggct tcgttccaag 360

cagcttctca atttcgctcc ttccacgcag tga 393

<210> 2

<211> 130

<212> PRT

<213> Glycine max（Linn.）Merr.

<400> 2

Met Ala Ala Leu Ile Arg Ser Pro Ala Val Ile Leu Ala Ile Leu Thr

1 5 10 15

Ile Ser Ala Cys Ile Ala Cys Thr Ala Ser Tyr Gly Gly Leu Val Gly

20 25 30

Gly Arg Ser Lys Ile Pro Asp Val Lys Ala Asn Lys Lys Val Gln Asp

35 40 45

Leu Gly Arg Phe Ser Val Glu Glu His Asn Arg Met Leu Arg Gln Ala

50 55 60

Gln Lys Glu Glu Glu Gln Val Thr Phe Val Glu Val Val Glu Ala Gln

65 70 75 80

Gln Gln Val Val Ser Gly Ile Lys Tyr Tyr Met Lys Ile Ser Ala Thr

85 90 95

Gln Gly Gly Asp Gly Gly Asp Ser Arg Ile Phe Glu Ser Val Val Val

100 105 110

Val Lys Pro Trp Leu Arg Ser Lys Gln Leu Leu Asn Phe Ala Pro Ser

115 120 125

Thr Gln

130

Claims

1. The application of the protein GmCYS2 is at least one of the following D1) -D5):

D1) increasing the root nodule size of the soybean;

D2) increasing the plant height of the soybean;

D3) increasing soybean biomass;

D4) cultivating transgenic soybean with increased nodule size;

D5) cultivating transgenic soybean with increased plant height and/or increased biomass;

the protein GmCYS2 is a1) or a 2):

a2) and (b) fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table.

2. The use of a nucleic acid molecule encoding the protein GmCYS2 according to claim 1, which is at least one of the following D1) -D5):

D1) increasing the root nodule size of the soybean;

D2) increasing the plant height of the soybean;

D3) increasing soybean biomass;

D4) cultivating transgenic soybean with increased nodule size;

D5) and (3) cultivating the transgenic soybean with increased plant height and/or increased biomass.

3. Use according to claim 2, characterized in that: the nucleic acid molecule of the protein GmCYS2 is a DNA molecule shown as b1) or b 2):

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.

4. A method of breeding transgenic soybeans comprising the steps of: increasing the expression level and/or activity of the protein GmCYS2 in claim 1 in starting soybeans to obtain transgenic soybeans; the transgenic soybeans have increased nodule size compared to the starting soybeans.

5. The method of claim 4, wherein:

the expression quantity and/or activity of the protein GmCYS2 in the starting soybean is improved by introducing a nucleic acid molecule coding the protein GmCYS2 into the starting soybean.

6. The method of claim 5, wherein:

the introduction of the nucleic acid molecule coding for the protein GmCYS2 into the starting soybean is achieved by introducing a recombinant vector into the starting soybean;

the recombinant vector is a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein GmCYS2 into an expression vector.

7. A method of breeding transgenic soybeans comprising the steps of: increasing the expression level and/or activity of the protein GmCYS2 in claim 1 in starting soybeans to obtain transgenic soybeans; the transgenic soybean has increased plant height and/or increased biomass compared to the starting soybean.

8. The method of claim 7, wherein:

9. The method of claim 8, wherein:

10. A soybean breeding method comprising the steps of: increasing the expression level and/or activity of the protein GmCYS2 of claim 1 in soybean, whereby the root nodule size of soybean is increased.

11. A soybean breeding method comprising the steps of: increasing the expression level and/or activity of the protein GmCYS2 in the soybean according to claim 1, thereby increasing the plant height and/or biomass of the soybean.