CN115896131A - Soybean salt tolerance gene and method for regulating and controlling salt tolerance - Google Patents
Soybean salt tolerance gene and method for regulating and controlling salt tolerance Download PDFInfo
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- CN115896131A CN115896131A CN202211269790.4A CN202211269790A CN115896131A CN 115896131 A CN115896131 A CN 115896131A CN 202211269790 A CN202211269790 A CN 202211269790A CN 115896131 A CN115896131 A CN 115896131A
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Abstract
The invention belongs to the field of genetic engineering, and provides a soybean salt tolerance gene GmBaSS5, a recombinant vector containing the GmBaSS5 gene, a method for improving soybean salt tolerance and application thereof. The invention utilizes plant genetic engineering technology to simultaneously introduce a GmBaSS5 gene, a GmPLGG1 gene, a CrGDH-Ma gene and a ZmMS-Ma gene into soybeans by an agrobacterium-mediated method, and the salt tolerance, photosynthetic efficiency, biomass and yield of the obtained transgenic soybean plants are obviously improved.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a soybean salt tolerance gene GmBASS5, a recombinant vector containing the GmBASS5 and a method for improving soybean salt tolerance.
Background
Soybeans are a worldwide important source of edible oil and high-quality plant protein, and the cultivated soybeans are continuously influenced by stress factors such as drought, salt and alkali, cold damage, freezing damage, waterlogging and the like in the whole growth and development process, so that large-scale production reduction of crops can be caused in severe cases. Among them, soil salinization is an abiotic stress that has a large influence on the growth of soil. Although soybean is a moderately salt-tolerant crop, salt damage inhibits the germination and vegetative growth of soybean seeds, prevents the formation of root nodules in soybeans, and significantly reduces the yield of soybeans at salinity values exceeding 5 dS/m. Therefore, the cultivation of soybean salt-tolerant varieties by means of genetic engineering becomes one of effective strategies for salt damage.
At present, the method for cultivating the soybean salt-tolerant variety mainly comprises the steps of inhibiting the expression of a gene for negatively regulating and controlling the soybean salt tolerance or over-expressing the soybean salt tolerance gene. For example, patent CN111620933B discloses application of protein GmNAC2 in regulation of salt tolerance of plants, and proves that reducing expression of protein GmNAC2 and its coding gene can significantly improve salt tolerance and yield of soybean. Patent CN108504664B discloses application of soybean cation excreting protein GmCDF1 coding gene, wherein the GmCDF1 coding gene can negatively regulate and control the salt tolerance of soybean, inhibit the expression of the gene and can obviously improve the salt tolerance of the soybean. The salt tolerance of soybean can be weakened by over-expressing the gene. Patent CN108570472B discloses application of soybean transcription factor GmZF351 in plant stress tolerance regulation, wherein coding genes of the transcription factor GmZF351 are transferred into receptor soybeans, and the drought tolerance and salt tolerance of the obtained transgenic soybean strain are obviously improved. Patent CN108642066B discloses an application of protein from wild soybean and a coding gene thereof in improving salt tolerance of plants, wherein the salt tolerance of the plants is obviously increased after GsMYB15 gene is introduced into the wild soybean and expressed.
Glycolate Dehydrogenase (GDH) can convert glycolate into glyoxylate. Malate Synthase (MS) catalyzes the conversion of acetyl-CoA and glyoxylate into malate and CoA. Patent CN110184293B over-expresses GDH gene and MS gene in soybean chloroplast, can improve soybean photosynthetic efficiency, and then improves soybean biomass and yield.
Sodium Bile Acid Symporter (BASS) and plastidial glycolate/glycerate transporter 1 (plgg1) are key proteins in photophoresis for transporting glycolate from chloroplasts to peroxisomes. Both BASS and PLGG1 genes have the function of transporting glycolic acid, but PLGG1 also has the function of transporting glycolic acid and glyceric acid. Previous researches show that the biomass of tobacco can be remarkably improved by over-expressing GDH and MS genes in tobacco chloroplasts and simultaneously inhibiting the expression of PLGG1 genes. Patent CN110564760B improves the photosynthetic efficiency of soybean and rice, improves the biomass or yield thereof and increases drought resistance by inhibiting the expression of BASS6 gene in rice and soybean and combining with the overexpression of GDH gene and MS gene in chloroplast.
The prior art only relates to that the combination of over-expression of GDH and MS gene and inhibition of PLGG1 gene can improve soybean yield and biomass, but no literature report on the improvement of soybean salt tolerance by regulating PLGG1 gene and BASS gene exists at present.
Disclosure of Invention
The invention aims to provide a soybean salt-tolerant gene GmBaSS5, a recombinant vector GMGmTG containing the GmBaSS5 and a method for improving the salt tolerance of soybeans. The invention overexpresses glycollic acid dehydrogenase gene CrGDH-Ma and malic acid synthase Z gene mMS-Ma which are subjected to codon optimization and amino acid substitution according to the codon preference in the soybean in soybean chloroplasts so as to improve the activity of the two enzymes in the soybean; meanwhile, the gene editing technology is utilized to micro-regulate the expression of GmPLGG1 (glycollic acid transport protein) and GmBaSS5 (bile acid/sodium transport protein) genes in the soybean aiming at a key region of a promoter, and the salt tolerance and the yield of the soybean are improved under the condition of not influencing the normal growth of plants.
In conclusion, the invention can improve the salt tolerance, biomass and yield of soybean by inhibiting the expression of the BaSS5 gene and the PLGG1 gene and simultaneously over-expressing GDH and MS genes.
The technical scheme provided by the invention is as follows:
in a first aspect of the invention, a soybean salt-tolerant gene GmBASS5 is provided.
The GmBASS5 gene is derived from soybean, and the nucleotide sequence of the coding region of the GmBASS5 is shown in SEQ ID NO:3, the nucleotide sequence of the promoter editing region of the GmBASS5 is shown as SEQ ID NO:4, respectively.
In a second aspect of the invention, a recombinant vector is provided.
The recombinant vector comprises the soybean salt-tolerant gene GmBASS5 of the first aspect of the invention.
Further, the recombinant vector also comprises a GmPLGG1 gene, a ZmMS-Ma gene and a CrGDH-Ma gene;
the GmPLGG1 gene codes a glycollic acid transporter and is derived from soybean, and the nucleotide sequence of a coding region of the glycollic acid transporter is shown as SEQ ID NO:1, the sequence of the promoter editing region is shown as SEQ ID NO:2 is shown in the specification;
the ZmMS-Ma gene coding malate synthase is derived from corn and is obtained by codon optimization and amino acid substitution according to the preference of soybean codons, and the nucleotide sequence of a coding region is shown as SEQ ID NO:5, the amino acid sequence is SEQ ID NO:6 is shown in the specification;
the gene coding the CrGDH-Ma gene is a glycollic acid dehydrogenase gene which is derived from Chlamydomonas reinhardtii and is obtained by codon optimization and amino acid substitution according to the preference of soybean codons, and the nucleotide sequence of the coding region is shown as SEQ ID NO:7, the amino acid sequence is shown as SEQ ID NO: shown in fig. 8.
Further, the recombinant vector also comprises a target sequence of a GmPLGG1 promoter editing region, a p35S promoter, a pUbi promoter, an OsU3 promoter, a chloroplast localization signal peptide coding sequence and an Nos terminator;
the target sequence of the GmBASS5 promoter editing region is shown as SEQ ID NO:9 is shown in the figure;
the target sequence of the GmPLGG1 promoter editing region is shown as SEQ ID NO:10 is shown in the figure;
and (3) constructing a gene promoter region editing expression frame by using the predicted promoter editing region target sequences of the GmBASS5 and the GmPLGG1 by using a CRISPR-Cas9 gene editing technology, and finely adjusting the gene expression of the GmBASS5 and the GmPLGG 1.
The nucleotide sequence of the p35S promoter is shown as SEQ ID NO:11 is shown in the figure;
the nucleotide sequence of the pUbi promoter is shown as SEQ ID NO:12 is shown in the specification;
the nucleotide sequence of the OsU3 promoter is shown as SEQ ID NO:13 is shown in the figure;
the p35S promoter, pUbi promoter and OsU3 promoter are used for mediating the over-expression of ZmMS-Ma gene and CrGDH-Ma gene, and the promoters are derived from eukaryote or prokaryote, can also be obtained by artificial synthesis, and can be constitutive promoters or specific promoters. For example, the pUbi promoter is derived from maize and the OsU3 promoter is derived from rice.
The chloroplast localization signal peptide coding sequence is shown as SEQ ID NO:14 fused at the N end of the ZmMS-Ma and CrGDH-Ma genes; the chloroplast localization signal peptide coding sequence mediates the overexpression of the ZmMS-Ma gene and the CrGDH-Ma gene in chloroplasts, and is derived from the chloroplast localization signal peptide sequence of a small subunit of ribulose-1, 5-diphosphate carboxylase/oxygenase (Rubisco) of a plant.
The nucleotide coding sequence of the Nos terminator is shown as SEQ ID NO:15 is shown in the figure; the Nos terminator mediates the termination of ZmMS-Ma and CrGDH-Ma gene overexpression, can be derived from eukaryotes or prokaryotes, and can also be obtained by artificial synthesis.
In a third aspect of the present invention, a method for improving salt tolerance of soybeans is provided.
The method comprises the following steps:
(1) Constructing a recombinant vector according to the second aspect of the invention;
(2) Transforming the recombinant vector obtained in the step (1) into a soybean host.
Further, the construction method of the recombinant vector in the step (1) comprises the following steps:
(1) Artificially synthesizing the ZmMS-Ma gene, the CrGDH-Ma gene, the pUbi promoter, the chloroplast signal peptide coding sequence, the p35S promoter and the Nos terminator to construct a pUbi-ZmMS-Ma-Nos-p35S-CrGDH-Ma-Nos gene overexpression frame;
(2) Artificially synthesizing the GmBASS5 gene, the GmPLGG1 gene, the target sequence of the editing area of the GmPLGG1 promoter, the OsU3 promoter and the gRNA scaffold sequence, and constructing an OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (GmBASS 5) -scaffold gene editing expression frame;
(3) The pUbi-ZmMS-Nos-p35S-CrGDH-Nos gene overexpression cassette in the above step (1) was ligated into a T-DNA vector via BamHI and KpnI sites, and the OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (GmBASS 5) -scaffold gene editing cassette in the above step (2) was ligated into a T-DNA vector via SpeI and PacI to obtain a recombinant vector bar-OEZmMS-Ma-OECrGDH-Ma-Cas9-gRNA (GmPLGG 1) -gRNA (GmBASS 5) designated as GmGMTG.
Further, the transformation method in step (2) is agrobacterium transformation.
In a fourth aspect of the invention, an application is provided.
The application includes any one of:
(1) The application of the soybean salt-tolerant gene GmBASS5 of the first aspect of the invention, the recombinant vector of the second aspect of the invention and the method of the third aspect of the invention in improving the salt tolerance of soybean;
(2) The soybean salt-tolerant gene GmBASS5 of the first aspect of the invention, the recombinant vector of the second aspect of the invention and the method of the third aspect of the invention are applied to the improvement of soybean biomass or yield.
Advantageous effects
The invention constructs GmPLGG1 and GmBASS5 gene promoter regions to accurately regulate the expression levels, overexpresses glycollic dehydrogenase CrGDH-Ma and malic acid synthase ZmMS-Ma genes in soybean chloroplasts, constructs a recombinant vector GMGmTG which simultaneously expresses the GmPLGG1 gene, the GmBASS5 gene, the CrGDH-Ma gene and the ZmMS-Ma gene by utilizing a gene editing technology, reduces the transportation of glycolic acid and Na + dependent protein, and obviously improves the salt tolerance, the biomass and the yield of soybeans by reducing the energy consumption and the Na + transportation.
Drawings
FIG. 1 is a structural diagram of a recombinant vector GMGmTG;
FIG. 2 shows the proline content of soybean leaves under the salt stress conditions of 0mM, 60mM, 120mM, 180mM and 240mM, wherein the salt is prepared by mixing NaCl and Na2SO4 according to the molar ratio of 1;
FIG. 3 shows the soluble sugar content of soybean leaves under 0mM, 60mM, 120mM, 180mM, 240mM salt stress conditions, wherein the salt is NaCl and Na 2 SO 4 Mixing and preparing according to the molar ratio of 1;
FIG. 4 shows the biomass of transgenic soybean under normal soil conditions, in which NaCl was not added to the screened dryland, and under salt-stressed soil conditions, in which 4 g.kg was added in accordance with the salt content of the soil (NaCl addition/soil content) -1 NaCl of (2);
FIG. 5 shows the transgenic soybean yields under normal soil conditions, in which NaCl was not added to the screened air-dried soil, and under salt-stressed soil conditions, in which 4 g.kg was added in accordance with the salt content of the soil (NaCl addition/soil content) -1 The NaCl of (2);
fig. 6 is the glycolic acid content of different transgenic soybean leaves as a function of time and illumination intensity, with time points of 10;
FIG. 7 shows the expression levels of GmPLGG1 and GmBASS5 in different transgenic soybean plants.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present 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 backbone vector used in the examples described below was pcmcia 1305.
Agrobacterium LBA4404 Agrobacterium tumefaciens used in the examples below
The soybeans used in the examples described below were of the Jack variety.
Example 1: vector construction
1. Construction of overexpression cassettes for MS Gene and GDH Gene
The gene overexpression frame is formed by artificially synthesizing a promoter pUbi, a chloroplast signal peptide, an MS coding gene ZmMS-Ma, a promoter p35S, a GDH coding gene CrGDH-Ma and a Nos terminator, and the 5 'end and the 3' end of the gene overexpression frame are respectively provided with an AvrII site and a BamHI site.
Acquisition modes of the MS gene and GDH gene: obtaining coding region sequences and amino acid sequences of ZmMS genes and CrGDH genes through an NCBI website (https:// www.ncbi.nlm.nih.gov /), improving the activity of expressed proteins by analyzing and replacing partial amino acids in the gene amino acid sequences, performing codon optimization by utilizing common codons of corn according to the amino acid sequences, and artificially synthesizing ZmMS-Ma genes and CrGDH-Ma gene coding sequences after codon optimization and amino acid substitution.
The MS gene is a ZmMS-Ma gene subjected to codon optimization and amino acid substitution, and the amino acid sequence of the ZmMS-Ma gene is shown as SEQ ID NO. 6;
the GDH gene is a CrGDH-Ma gene subjected to codon optimization and amino acid substitution, and the amino acid sequence of the gene is shown as SEQ ID NO. 8.
2. Construction of promoter gene editing expression cassette of GmPLGG1 gene and GmBASS5 gene
The GmPLGG1 and GmBASS5 genes are obtained in a mode that: the full length of GmPLGG1 and GmBASS5 genes is obtained by NCBI homologous alignment according to tobacco PLGG1 and BASS6, and the sequence of a gene promoter region is obtained by searching the upstream sequence of the genes. sgRNAs were designed for the promoter regions of the GmPLGG1 and GmBASS5 genes, respectively, using the website (https:// ports. Branched. Organization. Org/gpp/public/analysis-tools/sgrnan-design).
And realizing accurate regulation and control of the expression of the GmPLGG1 gene and the GmBASS5 gene by using gene editing, and constructing a GmPLGG1 gene and GmBASS5 gene promoter gene editing expression cassette. Due to complete functional deletion caused by the gene knockout of PLGG1 and BASS5, the plants are weak in growth and even die, so that the expression level of the trace regulatory gene at a specific site in the promoter region of the editing gene is selected, and the transformation event that the yield is increased, the salt tolerance is realized and the plants can grow normally is achieved.
An OsU3 promoter, a PLGG1 promoter region target site, a BASS5 promoter region target site sequence and a gRNA scaffold sequence are artificially synthesized to form an OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (GmBASS 5) -scaffold gene editing expression frame, and AscI and PacI sites are respectively arranged at the 5 'end and the 3' end.
GmPLGG1 and GmBASS5 are derived from soybean, and the target sequences of the promoter regions are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.
3. Construction of recombinant vector GMGmTG
The gene overexpression frame of pUbi-ZmMS-Nos-p35S-CrGDH-Nos is connected into a T-DNA vector through BamHI and KpnI sites, and an OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (BASS 5) -scaffold gene editing expression frame is connected into the T-DNA vector through SpeI and PacI to construct a final vector, the soybean vector utilizes a Bar screening marker, and the recombinant vector is named as GMGmTG: the bar-OEZmMS-Ma-OECrGDH-Ma-Cas9-gRNA (GmPLGG 1) -gRNA (GmBASS 5), and the structure of the recombinant vector is shown in figure 1.
As a control, 2 recombinant vectors for inhibiting the expression cassettes of the GmPGGL1 and the GmBASS5 genes were constructed in the same manner as bar-OEZmMS-Ma-OECrGDH-Ma-Cas9-gRNA (GmPLGG 1) and bar-OEZmMS-Ma-OECrGDH-Ma-Cas9-gRNA (GmBASS 5) and named as GMGmTP and GMGmTB, respectively.
Finally, the T-DNA plasmid was transferred to Agrobacterium LBA4404 by electroporation, and positive clones were selected by YEP solid medium containing 15. Mu.g/mL tetracycline and 50. Mu.g/mL kanamycin and maintained for the subsequent plant transformation.
Example 2 Soybean transformation
1. Culture medium
The culture medium and the specific components used in this example were as follows:
(1) Soybean germination culture medium: b5 salt + B5 vitamins +20g/L sucrose +8g/L agar powder (pH 5.6);
(2) YP medium:
(3) Infection culture medium: MS salt 2.15g/L + B5 vitamin + sucrose 20g/L + glucose 10g/L + Acetosyringone (AS) 40mg/L + MES 4g/L + zeatin 2mg/L (pH5.3);
(4) Co-culture medium: MS salt 4.3g/L + B5 vitamin + sucrose 20g/L + glucose 10g/L + MES 4g/L + zeatin 2mg/L + agar 8g/L (pH5.6);
(5) And (3) recovering the culture medium: 3.1g/L of B5 salt, MS ferric salt, B5 vitamin, 1g/L of MES, 30g/L of sucrose, 2mg/L of Zeatin (ZT), 8g/L of agar, 150mg/L of cefamycin, 100mg/L of glutamic acid and 100mg/L of aspartic acid (pH 5.6);
(6) Screening a culture medium: b5 salt 3.1g/L + MS iron salt + B5 vitamin + MES 1g/L + sucrose 30g/L + 6-benzyl adenine (6-BAP) 1mg/L + agar 8g/L + cefuroxime 150mg/L + glutamic acid 100mg/L + aspartic acid 100mg/L + glufosinate 8mg/L (pH 5.6);
(7) Regeneration culture medium: 3.1g/L of B5 salt, MS iron salt, B5 vitamin, MES 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cefamycin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L and glufosinate 6mg/L (PH 5.6);
(8) B5 rooting culture medium: 1/2MS salt, B5 vitamin, MES 1g/L, sucrose 30g/L, agar 8g/L, cefamycin 150mg/L and IBA 1mg/L;
(9) Soaking soybean in a dye solution: n6 salt + B5 vitamin, sucrose 30g/L, proline 0.5g/L, glutamine 0.5g/L,2, 4-D2 mg/L, hydrolyzed casein 0.3g/L, AS 40 mg/L)
2. Experimental methods
(1) Soybean seed germination
Germinating mature soybean seeds in a soybean germination culture medium, inoculating the soybean seeds on the germination culture medium, and culturing according to the following conditions: the temperature is 24-26 ℃; the photoperiod is 16/8h. Taking the soybean aseptic seedling expanded at the fresh green cotyledonary node after germinating for 4-6 days, cutting off hypocotyl at 3-4mm position below the cotyledonary node, longitudinally cutting cotyledon, and removing terminal bud, side bud and seed root.
(2) Agrobacterium activation
The Agrobacterium slide containing the GMGmTG plasmid constructed in example 1 was taken. A single colony is selected and inoculated, and agrobacterium for transformation is prepared. The strain was removed from the-80 ℃ incubator, activated on YP medium (containing the corresponding antibiotic) and cultured at 28 ℃ for 2 days. Single colonies on the plates were picked and reactivated onto new YP plates and incubated overnight at 28 ℃ for experiments. Agrobacterium was collected from YP medium and resuspended in 25-40ml of soybean-infected solution (final concentration of 100umol/L containing AS) in an EP tube, and the concentration of the bacterial solution (OD 660 value of 0.1-0.5) was measured spectrophotometrically for further use.
(3) Co-cultivation
The wound was done with the back of a scalpel at the cotyledonary node, the wounded cotyledonary node tissue was contacted with agrobacterium suspension, and the cotyledonary node tissue was immersed in agrobacterium suspension (OD 660=0.5-0.8, infection medium) to initiate infection. After infection, the Agrobacterium was aspirated, and the cotyledonary node tissue was transferred to a co-culture medium for co-culture with Agrobacterium for a period of time (3 days).
(4) Obtaining regenerated plants
Following this co-cultivation phase, the cotyledonary node tissues were transferred to recovery medium and recovery culture was continued for 5-7 days. After the recovery culture is finished, the regenerated tissue block of the cotyledonary node is cultured on a screening culture medium, and the transformed cells grow on the screening culture medium.
The transformed resistant tissue pieces are then cultured on regeneration medium to regenerate the plants. Transferring the regenerated plantlet to a B5 rooting culture medium, culturing at 25 ℃ to a height of about 10cm, and transferring to a greenhouse for culturing until the plantlet is fruited. In the greenhouse, the culture was carried out at 26 ℃ for 16 hours and at 20 ℃ for 8 hours each day.
And finally washing the regenerated plant to remove agar, transplanting the agar into a greenhouse, and selecting a transgenic plant line which has high salt tolerance or high biomass and can improve the yield of the soybean to obtain a transgenic soybean plant containing the transformation vector.
Example 3 identification of transgenic Soybean
Salt stress treatment method in this example:
5 parts of transgenic and control group seeds (each concentration is set to 5 parallel groups) are respectively sown in plastic flowerpots containing cleaned fine sand with the diameter of 25cm, after emergence of seedlings, the seedlings are thoroughly irrigated for 1 time (about 400 ml) by Hoagland nutrient solution every day, and 2-3 plants are fixed seedlings in each pot. Two neutral salts, naCl and Na 2 SO 4 As salt stress treatment, 5 treatments were carried out at 0mM, 60mM, 120mM, 180mM and 240mM in a molar ratio of 1. When the plants grow the third compound leaves, carrying out saline solution treatment, irrigating 400ml every day, starting photosynthesis related data detection and leaf collection when the plants treated with the highest concentration show wilting signs (about 8 days), then continuing carrying out salt stress on the materials, observing the growth conditions of the materials, and finally selecting 1 transgenic material leaf sample with the best growth condition to carry out physiological and biochemical index experiments.
1. Changes in proline and soluble sugar content in GMGmTG transgenic soybean plants
Grouping experiments: GMGmTP transgenic soybean plant group (only transferred with PLGG1 gene) and GMGmTG transgenic soybean plant group (simultaneously transferred with PLGG1 gene and BASS5 gene).
Under the adverse circumstances, the plant can accumulate micromolecular osmotic substance to improve the resistance, and proline and soluble sugar are both micromolecular osmotic substance and are used for detecting the contents of proline and soluble sugar in the leaves of the soybean plant under the salt stress.
The method for measuring the proline content in the leaves comprises the following steps:
first, a standard curve is drawn, and the absorbance at 520nm at different proline concentrations is measured using zero concentration as a control to prepare a proline standard curve. Then, weighing the plant leaves subjected to salt stress treatment, wherein each treatment is carried out in three parts, and each part is 0.5g; cutting, placing into test tube, adding 3% sulfosalicylic acid 5ml, sealing, and placing in boiling water for water bath for 10min while shaking at intervals. After the solution is cooled, centrifuging at 3000r/min for 10min, taking 2ml of supernatant, adding 2ml of water, 2ml of glacial acetic acid and 4ml of 2.5% acidic ninhydrin, sealing the pipe orifice, and carrying out boiling water bath for 1h until the solution is red. Cooling, adding 5ml toluene into 4ml toluene, shaking for 30s, standing, collecting supernatant, and measuring light absorption value at 520 nm; and (4) calculating a result: proline content (. Mu. Mol. G-1) = (CV/a)/w/M, and the proline content were determined. Wherein, C: checking the microgram of proline according to a standard curve; v: total volume (ml) of the extractive solution; a: measuring the volume (ml) of the solution; w: fresh weight of sample (g); m: molar mass of proline.
The soluble sugar content in the leaves is determined as follows:
firstly, making a soluble sugar standard curve by referring to an anthrone colorimetric method, numbering 6 large test tubes from 0 to 5 respectively, quickly shaking and uniformly mixing the tubes, boiling the tubes in a boiling water bath for 10min, taking out the tubes for cooling, measuring optical density by blank zeroing at a wavelength of 620nm, and drawing the standard curve by taking the optical density as an ordinate and the content of glucose (mu g) as an abscissa. Then, the plant leaves after salt stress treatment are respectively taken, three parts of each treatment, 0.5g of each part, are put into a large test tube, 15mL of distilled water is added, the plant leaves are boiled in boiling water bath for 20min, taken out and cooled, filtered into a 100mL volumetric flask, residues are washed by distilled water for several times, and the volume is fixed to the scale. Taking 1.0mL of the extracting solution of the sample to be detected, adding 5mL of anthrone reagent, and performing color development and optical density determination by the same operation. Repeat 3 times. And (4) calculating a result: soluble sugar content (. Mu.g.g) -1 ) = CxVT × dilution factor/[ V1 xW × 106](ii) a Wherein, C: the glucose amount (μ g) was determined from the standard curve; VT: total sample extract volume (mL); v1: sample liquid volume (mL) at the time of color development; w: the sample weighed (g).
As shown in the results of FIG. 2 and FIG. 3, the proline content and the soluble sugar content of both groups of plants increased with the increase of the salt concentration; but the content of soluble sugar of the GMGmTG transgenic soybean plant is obviously higher than that of the GMGmTP transgenic soybean plant, in addition, under the concentrations of 180mM and 240mM, the proline content of the GMGmTG transgenic soybean plant respectively reaches 2.36 times and 2.11 times of the GMGmTP transgenic soybean plant, and the content of soluble sugar respectively reaches 1.80 times and 1.84 times of the GMGmTP transgenic soybean plant. The transgenic soybean plant with the GmBASS5 gene editing expression box has higher salt tolerance.
2. Changes in biomass and yield of GMGmTG transgenic soybean plants
In order to further identify the performance change of the GMGmTG transgenic soybean plants, the biomass and the yield of the transgenic plants are evaluated and compared, and normal soil conditions (no NaCl is added) and salt stress soil conditions (calculated according to the soil salt content (the soil salt content = NaCl addition amount/soil amount), and 4 g.kg is added -1 (69 mM) salt stress additive NaCl), experimental groupings were as follows: control group (non-transgenic plant), GMGmTP transgenic group (only transferred into PLGG1 gene), GMGmTB transgenic group (only transferred into BASS5 gene), and GMGmTG transgenic group (both PLGG1 gene and BASS5 gene).
The method for detecting the salt tolerance biomass and yield of the soybean adopts a barrel cultivation random block test design: the growth conditions of the plants are observed early and late every day during the growth period, the salt content of the soil of the plough layer in the barrel is monitored every day by using a soil salt content tester, so that the relative stability of the average salt content of the soil of the plough layer of the plants in the whole growth period is ensured, and the biomass and the yield are detected after the plants are mature.
Results as shown in fig. 4 and 5, the biomass and yield of gmgmgmtg transgenic soybean plants under normal soil conditions were significantly increased compared to GMGmTP, gmgmgmtb transgenic plants and non-transgenic controls. Compared with a non-transgenic control, the biomass of the transgenic soybean plant can be increased by 65.6 percent and the yield can be increased by 60.5 percent; under the condition of salt stress soil, the biomass and yield of the GMGmTG transgenic plant are still remarkably increased compared with the GMGmTP transgenic plant, the GMGmTB transgenic plant and a non-transgenic control. Compared with a non-transgenic control, the biomass of the GMGmTG transgenic soybean plant is increased by up to 60.5 percent, and the yield is increased by 58.7 percent.
The results show that the biomass and yield of the GMGmTG transgenic soybean plant are obviously improved under the soil conditions of normal and salt stress.
3. Expression condition of related gene in GMGmTG transgenic soybean plant
In order to further prove the performance change of the GMGmTG transgenic soybean, the substrate glycolic acid content of the CrGDH gene in the non-transgenic soybean, the GMGmTP, the GMGmTB and the GMGmTG leaf is measured. The method for measuring the content of the glycolic acid adopts the prior art (by virtue of Xiumei et al, 2005, high performance liquid chromatography for measuring the content of the glycolic acid and a plurality of keto acids in plant leaves),
the results are shown in FIG. 6. The glycolic acid content of the non-transgenic plants is not obviously changed along with the change of time and light intensity, the glycolic acid content of the three groups of transgenic plants is increased along with the increase of the light intensity, and the accumulated glycolic acid increase of the GMGmTG transgenic plants along with the increase of the light intensity is the largest. The results show that the salt tolerance and photosynthetic efficiency improvement of the GMGmTG provided by the invention are more glycolic acid accumulation caused by the inhibition of 2 glycolic acid transporters, and are converted into energy required by plant growth through a glycolic acid metabolic pathway.
In order to identify the correlation between the expression of PLGG1 and BASS5 in GMGmTG transgenic soybeans and the change of plant performance, the expression of GmPLGG1 gene and GmBASS5 gene in non-transgenic soybeans, GMGmTP, GMGmTB and GMGmTG leaves is analyzed.
The results are shown in FIG. 7. The expression of the GmPLGG1 gene is reduced in a GMGmTP transgenic plant, the expression of the GmBASS5 gene has no obvious difference, while the expression of the GmBASS5 gene is reduced in a GMGmTB transgenic plant, and the expression of the GmPLGG1 gene is increased by 3.78 times. The results show that the optimal salt-tolerant high-photosynthetic-efficiency plant cannot be obtained by independently inhibiting the PLGG1 or BASS5 genes, and the salt tolerance and the photosynthetic efficiency of the plant can be simultaneously improved by inhibiting 2 glycollic acid transporters.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A soybean salt-tolerant gene GmBASS5, wherein the nucleotide sequence of a coding region of the GmBASS5 is shown as SEQ ID NO:3, the nucleotide sequence of the promoter editing region of the GmBASS5 is shown as SEQ ID NO:4, respectively.
2. A recombinant vector comprising the soybean salt-tolerant gene GmBASS5 of claim 1.
3. The recombinant vector according to claim 2, further comprising a GmPLGG1 gene, a ZmMS-Ma gene, and a CrGDH-Ma gene;
the GmPLGG1 gene codes a glycollic acid transport protein, and the nucleotide sequence of a coding region of the glycollic acid transport protein is shown as SEQ ID NO:1, the sequence of the promoter editing region is shown as SEQ ID NO:2 is shown in the specification;
the ZmMS-Ma gene codes malic acid synthase, and the nucleotide sequence of the coding region is shown as SEQ ID NO:5 is shown in the specification;
the CrGDH-Ma gene codes a glycollic acid dehydrogenase gene, and the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: shown in fig. 7.
4. The recombinant vector according to claim 3, wherein the recombinant vector further comprises a GmPLGG1 promoter editing region target sequence, a p35S promoter, a pUbi promoter, an OsU3 promoter, a chloroplast localization signal peptide coding sequence, a Nos terminator;
the target sequence of the GmBASS5 promoter editing region is shown as SEQ ID NO:9 is shown in the figure;
the target sequence of the GmPLGG1 promoter editing region is shown as SEQ ID NO:10 is shown in the figure;
the nucleotide sequence of the p35S promoter is shown as SEQ ID NO:11 is shown in the figure;
the nucleotide sequence of the pUbi promoter is shown as SEQ ID NO:12 is shown in the specification;
the nucleotide sequence of the OsU3 promoter is shown as SEQ ID NO:13 is shown in the figure;
the chloroplast localization signal peptide coding sequence is shown as SEQ ID NO:14 is shown in the figure;
the nucleotide coding sequence of the Nos terminator is shown as SEQ ID NO: shown at 15.
5. A method for improving salt tolerance of soybeans is characterized by comprising the following steps:
(1) Constructing a recombinant vector as claimed in claim 4;
(2) Transforming the recombinant vector obtained in the step (1) into a soybean host.
6. The method according to claim 5, wherein the recombinant vector is constructed in the step (1) by a method comprising the steps of:
(1) Artificially synthesizing the ZmMS-Ma gene, the CrGDH-Ma gene, the pUbi promoter, the chloroplast signal peptide coding sequence, the p35S promoter and the Nos terminator to construct a pUbi-ZmMS-Ma-Nos-35S-CrGDH-Ma-Nos gene overexpression cassette;
(2) Artificially synthesizing the GmBASS5 gene, the GmPLGG1 gene, the target sequence of the editing region of the GmPLGG1 promoter, the OsU3 promoter and the gRNA scaffold sequence to construct an OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (GmBASS 5) -scaffold gene editing expression cassette;
(3) The pUbi-ZmMS-Nos-p35S-CrGDH-Nos gene overexpression cassette in the above step (1) was ligated into the T-DNA vector via BamHI and KpnI sites, and the OsU3-gRNA (GmPLGG 1) -scaffold-OsU3-gRNA (GmBASS 5) -scaffold gene editing expression cassette in the above step (2) was ligated into the T-DNA vector via SpeI and PacI to obtain the bar recombinant vector bar-OEZmMS-Ma-OECrGDH-Ma-Cas9-gRNA (GmPLGG 1) -gRNA (GmBASS 5).
7. The method according to claim 5, wherein the transformation in step (2) is Agrobacterium transformation.
8. An application, characterized in that the application comprises any of the following:
(1) The use of the soybean salt-tolerant gene GmBASS5 of claim 1, the recombinant vector of any one of claims 2 to 4, the method of any one of claims 5 to 7 for improving the salt tolerance of soybean;
(2) The use of the soybean salt-tolerant gene GmBASS5 of claim 1, the recombinant vector of any one of claims 2-4, and the method of any one of claims 5-7 for increasing soybean biomass or yield.
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