RICE SERINE HYDROXYMETHYLTRANSFERASE CODED GENE OsSHM4 MUTANTS AND APPLICATION THEREOF

Abstract

The present invention discloses rice serine hydroxymethyltransferase coded gene OsSHM4 mutants and application thereof. The rice serine hydroxymethyltransferase coded gene mutants are obtained in a manner that T at a 461st position of a CDS sequence region of a wild type rice OsSHM4 gene is mutated to C, so that coded amino acids thereof are mutated from leucine to proline. A CDS sequence of the wild type rice OsSHM4 gene is shown in SEQ ID NO. 1. After mutation of serine hydroxymethyltransferase coded genes OsSHM4, under a field experiment condition, S and Se content of rice shoots is remarkably improved without influencing biomass of rice. After wild type serine hydroxymethyltransferase coded genes OsSHM4 are genetically modified to complement the mutants, S and Se content of shoots of complemented strains is restored to that of wild type rice water planting.

Claims

1. A rice serine hydroxymethyltransferase coded mutant gene, wherein the rice serine hydroxymethyltransferase mutant gene has a C instead of a T at a 461st position of the coding sequence (CDS) region of a wild type rice OsSHM4 gene, so as to encode a proline instead of a leucine at the corresponding coded amino acid thereof.

2. The mutant gene of claim 1, wherein the CDS sequence of the wild type rice OsSHM4 gene is set forth as SEQ ID NO. 1.

3. A protein encoded by the rice serine hydroxymethyltransferase coded gene mutant according to claim 1.

4. The protein of claim 3 having an amino acid sequence set forth as SEQ ID NO.4.

5. An expression vector comprising the rice serine hydroxymethyltransferase coded mutant gene according to claim 1.

6. A host cell comprising the rice serine hydroxymethyltransferase coded mutant gene according to claim 1.

7. A method for enhancing an absorbing capacity of rice roots to sulfate and improving total sulfur concentration of rice shoots, for enhancing an absorbing capacity of roots to selenate and improving total selenium concentration of rice shoots, or for improving total S and Se concentrations in brown rice without influencing another agronomic trait, the method comprising growing rice which contains the rice serine hydroxymethyltransferase coded mutant gene according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows that after OsSHM4 gene mutation, concentrations of sulfur and selenium in shoots are remarkably improved.

[0024] A: through water planting in a ½ KimuraB nutrient solution for three weeks, total S concentration of the shoots of OsSHM4 mutant materials is more than twice that of wide type rice and total S concentration of roots of the OsSHM4 mutant materials has no difference with that of the wide type rice; and

[0025] B: through processing with 1 μM of sodium selenate for 72 hours after water planting in a ½ KimuraB nutrient solution for three weeks, total Se concentration of the shoots of the OsSHM4 mutant materials is about three times that of the wide type rice and total Se content of the roots of the OsSHM4 mutant materials has no difference with that of the wide type rice.

[0026] FIG. 2 shows that compared with wide type rice, an absorbing capacity of roots of OsSHM4 gene mutant materials to selenate is remarkably improved.

[0027] FIG. 3 shows that in a field experiment, compared with wide type rice, more S and Se elements are concentrated in brown rice of OsSHM4 mutant materials.

[0028] A: S concentrations in brown rice of the wide type rice and S concentrations in brown rice of the OsSHM4 mutant materials when planted in different rice fields; and

[0029] B: Se concentrations in brown rice of the wide type rice and Se concentrations in brown rice of the OsSHM4 mutant materials when planted in different rice fields.

[0030] FIG. 4 is a plasmid spectrum of a complementing vector pZH2B.

[0031] FIG. 5 shows that after wide type OsSHM4 genes complement mutants, total S and Se content of shoots is restored to the level of wide type rice.

[0032] A: through water planting in a ½ KimuraB nutrient solution for three weeks, total sulfur content of shoots of transgenic strains complemented by the wide type OsSHM4 genes is restored to the level of the wide type rice water planting, and complementation succeeds; and

[0033] B: through processing with 5 μM of sodium selenate for 72 hours after water planting in a ½ KimuraB nutrient solution for three weeks, total selenium content of the shoots of the transgenic strains complemented by the wide type OsSHM4 genes is restored to the level of the wide type rice water planting, and complementation succeeds.

[0034] FIG. 6 is a vector pOS-sgRNA spectrum and a vector pOS-cas9 spectrum.

DETAILED DESCRIPTION

[0035] The following embodiments are intended to illustrate the present invention and are not intended to limit the scope thereof. Modifications or substitutions to the methods, steps or conditions of the present invention are within the scope of the present invention without departing from the spirit or essence of the present invention.

[0036] Embodiment 1 Obtaining of Rice Serine Hydroxymethyltransferase Gene OsSHM4 Mutants

[0037] 1) A rice ethyl methanesulfonate (EMS) chemical mutagenesis library is constructed, specifically, after about 1 kg of rice seeds (Zhongjiazao 17) are soaked at 37° C. for 24 hours, chemical mutagenesis is carried out with EMS at a final concentration of 1% (V/V) for 8 hours, and then the rice is rinsed with tap water to remove residual EMS. Germination is accelerated for 48 hours under the condition of 37° C. Then the mutagenized rice seeds are sown into a rice field. M2 generations of seeds are harvested from each single plant after the rice is mature, dried and preserved. The construction of a rice mutant library is completed.

[0038] 2) Screening and gene cloning of rice mutants are carried out, specifically, when a rice mutant library is screened, one cadmium-resistant rice mutant is screened by 2 cadmium chloride and subjected to rice whole genome sequencing. A mutant gene is located as a rice serine hydroxymethyltransferase coded gene OsSHM4. A mutation site is mutation of T at a 461st position of a first exon of a CDS sequence region of the OsSHM4 gene to C, and single amino acid substitution (leucine-proline) is caused.

[0039] One serine hydroxymethyltransferase coded gene OsSHM4 mutant of Zhongjiazao 17 rice background is obtained in the present embodiment, and other positions of a genome do not mutate except for T-C mutation at the 461st position of the first exon of the CDS sequence region of the rice serine hydroxymethyltransferase coded gene OsSHM4.

[0040] Embodiment 2

[0041] Comparison on total S concentrations of shoots and roots of osshm4 mutant materials and wide type rice and total Se concentrations of the shoots and the roots of the osshm4 mutant materials and the wide type rice specifically includes the following implementation process.

[0042] 1) Wild type rice seeds and osshm4 mutant seeds are soaked at 37° C. for 48 hours, rinsed with tap water, sown on a suspended plastic small black mesh, and cultured with a 0.5 mM CaCl.sub.2 solution for 1 week, and the solution is changed every three days.

[0043] 2) Wild type rice seedlings and osshm4 mutant seedlings are transferred to 4 L plastic black barrels for culture and cultured with ½ KimuraB (pH 4.5-5.0) for two weeks. Four wild type rice seedlings and four osshm4 mutant seedlings are planted in the 4 L black barrels respectively, a total of three barrels serve as three biological replicates, and then the seedlings are treated with a sodium selenate nutrient solution with a final concentration of 1 mM for 72 hours.

[0044] 3) Shoot and root samples are collected, washed three times with deionized water to remove ions and impurities absorbed on the surface, and placed in a 65° C. oven to be dried for 3 days for digestion.

[0045] 4) All the shoot or root samples are weighed to be put into a graphite digestion tube. 5 mL of mixed acid solution (85%: 15%, V/V, nitric acid: perchloric acid, guaranteed reagent) is added. Then the graphite digestion tube is placed in a graphite digestion furnace for sample digestion, and temperature control conditions are as follows: 60° C. for 1 hour, 90° C. for 1 hour, 120° C. for 1 hour and 150° C. for 1 hour. Then the residual acid solution in the tube is completely drained off under the 190° C. condition. The volume is made up to 25 mL with a 2% nitric acid solution. The solution is mixed well and then transferred to a 50 mL plastic centrifuge tube.

[0046] 5) The S and Se content in the sample solution is determined by an inductively coupled plasma mass spectrometry (ICP-MS), and the total S and Se content of shoots and roots of the wild type rice and the mutants is calculated according to a sample amount and a dilution factor.

[0047] A result of the present embodiment shows that compared with the wide type rice, more total S is concentrated on the shoots of the osshm4 mutant materials instead of the roots (FIG. 1A), and the total Se content of the shoots of the osshm4 mutant materials is also greatly improved without influencing the Se content of the roots (FIG. 1B).

[0048] Embodiment 3

[0049] Comparison on absorbing capacities of wide type rice and osshm4 mutant materials to selenate specifically includes the following implementation process.

[0050] 1) Wild type rice seeds and osshm4 mutant seeds are soaked at 37° C. for 48 hours, rinsed with tap water, sown on a suspended plastic small black mesh, and cultured with a 0.5 mM CaCl.sub.2 solution for 1 week, and the solution is changed every three days.

[0051] 2) Wild type rice seedlings and osshm4 mutant seedlings are transferred to 4 L plastic black barrels for culture and cultured with ½ KimuraB (pH 4.5-5.0) for two weeks for standby.

[0052] 3) During treatment, the wild type rice seedlings and the osshm4 mutant seedlings grown for about three weeks are transferred into a 1 L container and treated with 1 L of sodium selenate solution for 20 minutes respectively, and concentrations are 0, 1, 2, 5, 10, 20 and 60 μM respectively. The water temperature is 28° C. and the room temperature is 28° C. Three biological replicates are set for each concentration, and one biological replicate is three strains of wild type rice and three strains of osshm4 mutant materials in the 1 L container.

[0053] 4) After treatment for 20 minutes, only roots are taken, then washed three times with deionized water to remove adsorbed ions and impurities, and placed in a 65° C. oven to be dried for 3 days for digestion.

[0054] 5) Root samples are weighed to be put into a graphite digestion tube. 5 mL of mixed acid solution (85%:15%, V/V, nitric acid: perchloric acid, guaranteed reagent) is added. Then the graphite digestion tube is placed in a graphite digestion furnace for sample digestion, and the temperature control conditions are as follows: 60° C. for 1 hour, 90° C. for 1 hour, 120° C. for l hour and 150° C. for 1 hour. Then the residual acid solution in the tube is completely drained off under the 190° C. condition. The volume is made up to 25 mL with a 2% nitric acid solution. The solution is mixed well and then transferred to a 50 mL plastic centrifuge tube.

[0055] 6) The content of Se absorbed by roots of the wild type rice and the content of Se absorbed by roots of the mutants are finally calculated according to a sample amount and a dilution factor after the content of Se in the sample solution is determined by an inductively coupled plasma mass spectrometry (ICP-MS).

[0056] The present embodiment shows that compared with the wide type rice, the absorbing capacity of the roots of the osshm4 mutant materials to sodium selenate [Se(V)] is greatly improved (FIG. 2), After linear fitting, the Km value (WT, 9.4±4.6 μM vs osshm4, 9.0±0.8 μM) has no change while the Vmax value (WT, 70.6±12.1 mg kg.sup.−1 h.sup.−1 vs cdtl, 164.3±5.6 mg kg.sup.−1 h.sup.−1) of the osshm4 mutant materials is remarkably improved, which shows that the Se(V) absorbing capacity of the roots of the osshm4 mutants is remarkably improved.

[0057] Embodiment 4

[0058] Comparison on total S and Se concentrations in brown rice of wide type rice and osshm4 mutant materials in a field experiment specifically includes the following implementation process.

[0059] 1) Wild type rice seeds and osshm4 mutant seeds are sown in a seedling raising field for seedling raising. Seedlings are transferred into rice fields when three or four leaves grow up. One plant is arranged in each hole. The interval between every two plants is 10 cm. and the interval between rows is 15 cm. During the period, normal water and nutrient management control and pest control are carried out.

[0060] 2) The seedlings are planted in rice fields at different sites (Nanjing, Xiangtan and Changxing), and soils are sampled to determine pH values and Se contents.

[0061] 3) After the rice is mature, brown rice of four single plants is mixed into a sample. Three biological repeats are arranged. The total S and Se content of the brown rice of the wild type rice and the osshm4 mutants is determined.

[0062] 4) After the rice is mature, ten single rice plants in the middle of each row are selected to determine agronomic traits of the wild type rice and the osshm4 mutants, and the plants are shorn off at contact positions of shoots and soil during sampling.

[0063] 5) The total S and Se content of brown rice is determined, specifically, 0.25 g of dried brown rice is weighed to be placed into a microwave digestion tube. 5 ml of concentrated nitric acid (guaranteed reagent) is added for microwave digestion. Then the residual acid solution is removed at 160° C., and after acid removal is completed, the volume is made up to 10 mL with 5% nitric acid, and then the solution is transferred to a 15 mL centrifuge tube for storage. The S content in brown rice samples is determined through ICP-MS, and the Se content in the brown rice samples is determined through a liquid chromatography-atomic fluorescence spectrometer (LC-AFS8500).

[0064] 6) The value of a soil sample is determined, specifically, 10 g of ground dried rice field soil is weighed. 25 mL of deionized water is added. The mixture stands after being mixed evenly, and the pH value of upper-layer liquid is determined by a pH meter after the soil completely settles.

[0065] 7) The total Se content of soil is determined, specifically, 0.2 g of ground dried rice field soil is weighed to be put into a graphite digestion tube. 4 ml of concentrated hydrochloric acid (guaranteed reagent) is added. Then 1 ml of concentrated nitric acid (guaranteed reagent) is added, and the digestion temperature of the soil sample is controlled as follows:

TABLE-US-00001 Stage Temperature control (° C.) Time (minute) 1 0-35 35 2 35 180 3 35-60  25 4 60 180 5 60-105 20 6 105  60 7 105-125  10 8 125  120 Overnight (opening a cover and adding 5 ml of 20% nitric acid through a pipette) 1 0-80 40 2 80 30

[0066] 8) After digestion is finished, the volume is made up to 25 mL with 2% nitric acid, vortexed and allowed to stand, and supernatant is poured into a 50 mL centrifuge tube for storage. A supernatant solution is taken in a centrifugal mode to be detected, and the determination method is the same as the determination of Se in brown rice.

[0067] The result of the present embodiment shows that when the wild type rice and the osshm4 mutants are planted in rice fields at different sites, compared with the wide type rice, more S and more Se are enriched in brown rice of the osshm4 mutants (FIG. 3A) and (FIG. 3B), and an agronomic trait does not change much (Table 1). In general, the pH value of soil in each rice field is meta-acid and the Se content of the soil is low and below 1 mg kg.sup.−1 (Table 2).

TABLE-US-00002 TABLE 1 Agronomic trait indexes of wide type rice and OsSHM4 mutant materials under different planting sites Experimental Plant Effective tiller Kernels Maturing Thousand seed Yield per sites Sample height(cm) number(plant.sup.−1) per spike rate(100%) weight(g) plant(g) Nanjing WT 85.2 ± 3.7a 10 ± 2a 149 ± 14a 78 ± 3a 20.6 ± 0.7a 26.0 ± 4.7a osshm4 81.6 ± 4.9a 10 ± 2a 126 ± 11b 67 ± 5b 19.5 ± 0.5b 20.8 ± 5.2b Xiangtan WT 79.4 ± 1.4a 16 ± 3a 101 ± 15a 68 ± 7a 20.0 ± 0.8a 25.3 ± 5.7a osshm4 80.3 ± 2.9a 16 ± 3a 111 ± 12a 69 ± 5a 19.1 ± 0.5b 25.9 ± 7.2a Changxing WT 82.3 ± 5.0a 13 ± 2a 128 ± 30a 84 ± 9a 23.2 ± 0.7a 29.3 ± 3.4a osshm4 78.4 ± 2.5b 15 ± 3a 116 ± 23a 80 ± 8a 23.9 ± 0.9a 25.8 ± 7.1a

TABLE-US-00003 TABLE 2 pH value and total Se content office field soil samples under different planting sites Experimental site pH value Se content of soil (mg kg.sup.−1 DW) Nanjing 7.66 ± 0.04 0.61 ± 0.03 Xiangtan 4.84 ± 0.09 0.93 ± 0.03 Changxing 6.44 ± 0.03 0.40 ± 0.05

[0068] Embodiment 5

[0069] Wide type serine hydroxymethyltransferase coded genes OsSHM4 are complemented to mutants osshm4, whether mutation phenotypes of high S and Se concentrations of shoots are complemented successfully or not is verified, and the specific implementation process is as follows:

[0070] An OsSHM4 gene sequence is amplified by a PrimeSTAR® GXL DNA Polymerase (Takara) high fidelity enzyme by using a wild type rice genome DNA as a template and a complementation primer sequence as shown below and is inserted between enzyme cutting sites PstI and KpnI of a plant double-source expression vector pZH2B by using a homologous recombination mode (FIG. 4). After the construction is finished, sequencing verification is carried out, and plasmids are stored for later use.

[0071] Complementation primers are as follows:

TABLE-US-00004 (SEQ ID NO. 5) OsSHM4-Com-F: AGAGGATCCCCGGGTACCTTGTTTGCTAGGATTGGGTT OsSHM4-Com-R: (SEQ ID NO. 6) ATTGTTAATTAAGAATTCGATGCTAGACAGAGGCCGAGT

[0072] The constructed plasmids are sent to Wuhan BioRun Biological Company to complete the transgenic work of mutant rice, and the rice mutant osshm4 is used as the transgenic background.

[0073] The DNA of a single plant leaf of a positive transgenic strain is extracted. The fragments are amplified by using the primers to carry out sequencing verification, and finally a positive transgenic complemented strain is selected, and self breeding is continuously carried out to obtain T3-generation homozygous materials to be used in a subsequent complementing phenotype verification experiment.

[0074] Seeds of wild type rice, osshm4 mutants and three independent transgenic complemented strains are soaked at 37° C. for 48 hours, rinsed with tap water, sown on a suspended plastic mesh, and cultured with a 0.5 mM CaCl.sub.2 solution for 1 week, and the solution is changed every three days. Then seedlings are transferred to 4 L plastic black barrels to be cultured with ½ Kimura B (pH 4.5-5.0) for two weeks.

[0075] Four strains of wide type rice, four strains of osshm4 mutants and four strains of each of three independent transgenic complemented strains are planted in the 4 L plastic black barrels respectively. A total of three barrels serve as three biological replicates, and then the seedlings are treated with a sodium selenite [Se(VI)] nutrient solution with a final concentration of 5 mM for 72 hours.

[0076] Shoot and root samples are collected, washed three times with deionized water to remove ions and impurities adsorbed on the surface, and placed in a 65° C. oven to be dried for 3 days for digestion. All the shoot or root samples are weighed to be put into a graphite digestion tube. 5 mL of mixed acid solution (85%:15%, V/V, nitric acid: perchloric acid, guaranteed reagent) is added. Then the graphite digestion tube is placed in a graphite digestion furnace for sample digestion, and the temperature control conditions are as follows: 60° C. for 1 hour, 90° C. for 1 hour, 120° C. for 1 hour and 150° C. for 1 hour. Then the residual acid solution in the tube is completely drained off under the 190° C. condition. The volume is made up to 25 mL with a 2% nitric acid solution. The solution is mixed well and then transferred to a 50 mL plastic centrifuge tube.

[0077] After S and Se content in the sample solution is determined by an inductively coupled plasma mass spectrometry (ICP-MS), the total S and Se content of shoots and roots is calculated according to a sample amount and a dilution factor.

[0078] A result of the present embodiment (FIG. 5) shows that the total S and Se content of the shoots of the wild type gene OsSHM4 transgenic complemented strains has been restored to the normal level of wild type rice, which demonstrates that the serine hydroxymethyltransferase coded genes OsSHM4 influence the high S and Se concentration phenotypes of the shoots of the rice mutants.

[0079] Embodiment 6

[0080] A rice OsSHM4 gene point mutation material is constructed and verified through a crisper gene knockout technology, and the specific implementation process is as follows.

[0081] 1) Knockout targets sgRNAs on OsSHM4 genes are designed according to a rice OsSHM4 genome sequence by using Broad Institute (http://www.broadinstitute.org/rnai/public/analysis-tools/sgma-design), and are compounded by primers:

TABLE-US-00005 sgRNA-F(5′-3′): (SEQ ID NO. 7) GGCAAGGCTCATTGCTGATAAGTG sgRNA-R(5′-3′): (SEQ ID NO. 8) AAACCACTTATCAGCAATGAGCCT

[0082] 2) The sgRNA-F and sgRNA-R fragments are inserted into a BsaI enzyme cutting site of a linearized vector pOS-sgRNA by T4 ligase. Then an intermediate vector is fused with a final pOS-cas9 by LR reaction in a Gateway method, and a vector spectrum is shown in FIG. 6. After sequencing verification, the genes are sent to Wuhan BioRun Biological Company for rice transgenosis, and the OsSHM4 genes are knocked out by a crispr technology on the background of japonica rice Zhonghua 11 to obtain the point mutation material.

[0083] 3) A hygromycin marker is configured to screen positive transgenic seedlings. A TO generation single plant leaf DNA is used as a template. A forward primer is F(5′-3′): GCCCAAGGACCGTATCAT (SEQ ID NO. 9). A reverse primer is R(5′-3′): GCATTGTGATGTTGAATGGT (SEQ ID NO. 10). A 250 bp sequence upstream and downstream of the knockout targets sgRNA is amplified by PCR. A knockout result of the OsSHM4 genes is verified by sequencing verification, and the knockout targets of crisper knockout are a 999-1019 bp knockout target sequence (5′-AGGCTCATTGCTGATAAGTG-3′ (SEQ ID NO. 11)) of a first exon of the OsSHM4 genes.

[0084] 4) The seedlings are strictly related to T2 generations, and homozygous osshm4 knockout mutants are used for phenotype analysis.

[0085] 5) A result of the present embodiment shows that the total S and Se content of shoots of OsSHM4 point transgenic strains is significantly higher than that of wild type rice, which demonstrates that the loss of functions of the serine hydroxymethyltransferase coded genes OsSHM4 results in high S and Se concentration of the shoots of rice.

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