CN116768996B - Application of a morphogen protein gene OsFH2 in plant breeding regulation - Google Patents

Abstract

The present invention discloses the application of morphogen protein gene OsFH2 in plant breeding regulation. It belongs to the field of plant genetic engineering. The application of morphogen protein gene OsFH2 in plant breeding regulation, the amino acid sequence of protein encoded by the morphogen protein gene OsFH2 is shown in SEQ ID No: 4; the nucleotide sequence of the morphogen protein gene OsFH2 is shown in SEQ ID No: 1. The present invention overexpresses the morphogen protein gene OsFH2 in rice, so that the growth of overexpressed plants under Cd stress is significantly better than that of wild-type plants, and the transfer of Cd from roots to stems and leaves in overexpressed plants is significantly reduced compared with wild-type plants, and the Cd content in grains is significantly lower than that in wild-type plants, thereby providing a new candidate gene resource for cultivating safe rice with low Cd in grains, and also providing a potential repair method for rice Cd pollution control.

Description

Application of a morphogen protein gene OsFH2 in plant breeding regulation

Technical Field

The invention belongs to the field of plant genetic engineering, and specifically relates to the application of a morphogen protein gene OsFH2 in plant breeding regulation.

Background Art

Cadmium (Cd) is a common heavy metal element in farmland soil pollution. Crops grown in Cd-contaminated soil will have a serious impact on food safety. Rice is the most important food crop in China and the main source of Cd intake by humans. If no control and/or remedial measures are taken, Cd contaminated in farmland may pose a permanent threat to human health through the food chain. Limiting the accumulation of Cd in rice can effectively reduce the intake of Cd by humans. Using molecular breeding technology to cultivate rice varieties with low Cd accumulation is the most cost-effective and promising method to prevent the risk of Cd contamination in food. At present, the genetic resources related to rice Cd tolerance or accumulation are very limited. Therefore, the development of new Cd genetic resources is of great significance for low Cd molecular breeding of rice and remediation of Cd pollution.

Under the stress of heavy metal Cd in the environment, rice plants have a variety of tolerance mechanisms. For example, the antioxidant system in the plant is activated under Cd stress to enhance the cell's ability to remove reactive oxygen; the cell wall contains polysaccharides such as cellulose and pectin, which contain a large number of aldehyde, amino, carboxyl and phosphate groups, which can adsorb and fix Cd in the cell wall, thereby preventing Cd from entering the cytoplasm; by chelating Cd and then transporting Cd to the vacuole or apoplast, the toxicity of Cd is reduced and the transport of Cd is restricted. Genes related to these processes can all be used as candidate gene resources for molecular breeding related to rice Cd tolerance or accumulation regulation.

In plant life, formin is mainly responsible for regulating the formation and homeostasis of the cytoskeleton, and plays an important role in intercellular material transport, cell growth, signal transduction, morphological construction, etc. In recent years, studies have shown that formin may affect the development of cell walls by regulating the formation of the cytoskeleton, thereby playing an active role in the fixation of Cd in the cell wall. So far, there has been no report on the role of formin in the regulation of rice resistance to Cd and Cd accumulation.

Summary of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide an application of the morphogen protein gene OsFH2 in plant breeding regulation.

The purpose of the present invention is achieved through the following technical solutions:

The application of the morphogen protein gene OsFH2 in plant breeding regulation, the amino acid sequence of the protein encoded by the morphogen protein gene OsFH2 is shown in SEQ ID No:4.

The nucleotide sequence of the morphogen protein gene OsFH2 is shown in SEQ ID No:1.

The plant breeding regulation refers to overexpressing the morphogen protein gene OsFH2 in plants, thereby improving the plant's tolerance to Cd, increasing rice biomass, and reducing the Cd content in plant seeds.

The plant is preferably a grass plant; more preferably at least one of wheat, corn and rice; more preferably rice.

Application of the morphogen protein gene OsFH2 in improving plant tolerance to Cd and/or preparing/cultivating plant varieties with low Cd accumulation in grains.

Application of a plant expression vector and a host cell comprising the morphogen protein gene OsFH2 in improving plant tolerance to Cd and/or preparing/cultivating plant varieties with low Cd accumulation in grains.

A method for enhancing plant tolerance to Cd and/or reducing Cd content in plant seeds, comprising the step of over-expressing the morphogen protein gene OsFH2.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention overexpresses the morphogen protein gene OsFH2 in rice, so that the growth of the overexpressing plants under Cd stress is significantly better than that of the wild-type plants. In addition, the transfer of Cd from the roots to the stems and leaves in the overexpressing plants is significantly reduced compared with the wild-type plants, and the Cd content in the grains is significantly lower than that in the wild-type plants. This provides a new candidate gene resource for creating safe rice with low Cd in the grains, and also provides a potential remediation method for the treatment of Cd pollution in rice.

(2) The present invention can effectively improve the tolerance of rice to Cd and reduce the Cd content in rice grains by overexpressing the morphogen protein gene OsFH2, thereby providing a candidate gene resource and a potential restoration method for low-Cd molecular breeding of rice and Cd pollution control.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a structural information diagram of the morphogenetic protein gene OsFH2 and its encoded protein; wherein, (a) is a structural information diagram of the morphogenetic protein gene OsFH2; the rectangular box represents exons, the gray rectangular box represents coding sequences (CDS), and the white rectangular box represents non-translated sequences; the black solid line represents introns; (b) is a structural information diagram of the protein encoded by the morphogenetic protein gene OsFH2; the white rectangular box represents the transmembrane helical domain; the gray rectangular box represents the FH1 and FH2 domains.

FIG. 2 is a schematic diagram of the protein subcellular localization GFP empty vector pYL322dl.

Figure 3 is a diagram showing the results of subcellular localization analysis of the morphogen protein OsFH2 in rice protoplasts. p35S::OsFH2-GF represents a recombinant vector; p35S::GFP represents an empty vector; the plasma membrane marker protein vector p35S::OsRac3-Mcherry was co-transfected into the protoplasts; the excitation wavelength and observation wavelength of GFP observation were 488nm and 507nm, respectively, emitting green fluorescence; the excitation wavelength and observation wavelength of the plasma membrane marker protein OsRac3-Mcherry were 587nm and 610nm, respectively, emitting red fluorescence. The scale bar is 30μm.

Figure 4 is a graph showing the qRT-PCR detection results of the expression of the morphogen protein gene OsFH2 in response to Cd stress; wherein, (a) is a graph showing the expression level of the OsFH2 gene in the root under different _CdCl2 concentrations; (b) is a graph showing the expression level of the OsFH2 gene in the stem and leaf under different _CdCl2 concentrations; (c) is a graph showing the expression level of the OsFH2 gene in the root after culturing rice in a _CdCl2 solution with a final concentration of 10 μM for different time periods; (d) is a graph showing the expression level of the OsFH2 gene in the stem and leaf after culturing rice in a _CdCl2 solution with a final concentration of 10 μM for different time periods; 3 independent biological replicates; materials from 3 strains were mixed as one replicate sample; data are mean ± SD; t test; * indicates P < 0.05.

FIG. 5 is a schematic diagram of the overexpression vector pOx.

Figure 6 shows the phenotypic observation and growth trait statistical results of independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT); wherein, (a) is the phenotypic observation diagram of the independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT); the scale bar is 5 mm; (b)-(g) are the independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) (b) plant height; (c) root length; (d) stem and leaf fresh weight; (e) stem and leaf dry weight; (f) root fresh weight; (e) root dry weight statistical results; 3 independent biological replicates; 30 plants were counted in each replicate; data are mean ± SD; t test; * indicates P < 0.05.

Figure 7 shows the Cd fluorescence staining observation results of root epidermal cells of rice plants; (a) shows the Cd fluorescence staining observation results of root epidermal cells of plants without Cd treatment (control); (b) shows the Cd fluorescence staining observation results of root epidermal cells of independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the wild type (WT); the scale bar is 50 μm.

Figure 8 shows the results of Cd content in various parts of independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) after being cultured in a _CdCl2 solution with a final concentration of 100 μM for one week; (a) shows the results of Cd content in the root (Root) and stem and leaf (Shoot) of the independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) after being cultured in a 100 μM _CdCl2 solution for one week; (b) shows the results of Cd transfer coefficient from root to stem and leaf in the independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) after being cultured in a 100 μM _CdCl2 solution for one week; (c) shows the results of Cd transfer coefficient from root to stem and leaf in the independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) after being cultured in a 100 μM CdCl2 solution ₂ Results of cell wall Cd concentration in independent stable homozygous strains (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) after one week of solution culture; 3 independent biological replicates; materials from 3 strains were mixed as one replicate sample; data are mean ± SD; t test; * indicates P < 0.05.

Figure 9 shows the phenotypic observation and yield trait statistics of independent stable homozygous strains (OE-1, OE-2) and wild type (WT) with overexpression of OsFH2 gene in Cd-contaminated soil cultivation experiment; (a) shows the phenotypic observation results of independent stable homozygous strains (OE-1, OE-2) and wild type (WT) with overexpression of OsFH2 gene in Cd-contaminated soil cultivation experiment; the scale is 20 cm. When the rice is fully mature, the plant height (b), grass stem dry weight (c), effective tiller number (d), fruit setting rate (e), thousand-grain weight (f), and yield per plant (g) are measured and counted. The data are the mean ± SD of 3 independent biological replicates, and 30 plants are counted in each replicate; t test, * indicates P < 0.05.

Figure 10 shows the results of Cd content determination in the independent stable homozygous strains (OE-1, OE-2) and wild type (WT) of overexpressed OsFH2 gene in the Cd contaminated soil cultivation experiment. Figure (a) shows the results of Cd content determination in brown rice of the independent stable homozygous strains (OE-1, OE-2) and wild type (WT) of overexpressed OsFH2 gene in the Cd contaminated soil cultivation experiment; (b) shows the results of Cd content determination in the independent stable homozygous strains (OE-1, OE-2) and wild type (WT) of overexpressed OsFH2 gene in the Cd contaminated soil cultivation experiment. (c) is the result of determination of essential elements (Fe, Mn, Zn, Cu) content in brown rice of independent stable homozygous strains (OE-1, OE-2) overexpressing OsFH2 gene and wild type (WT); data are the mean ± SD of 3 independent biological replicates, and materials of 10 strains were mixed into one replicate sample; t test, * indicates P < 0.05.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

The experimental methods in the following examples that do not specify specific experimental conditions are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the construction of the vectors (including overexpression vectors and subcellular localization vectors) mentioned in the examples is carried out according to the conventional genetic engineering field operation methods, and the backbone vector pCambia1300 sequence and promoter elements (including 35S, Ubi), reporter genes (including GFP) and other sequences used are all sequences that have been published on relevant websites or databases in the field of genetic engineering (https://cambia.org and https://www.ncbi.nlm.nih.gov). The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial sources. The chemical reagents used in the examples are all imported or domestic analytically pure.

The pOx vector was provided by Academician Liu Yaoguang from the State Key Laboratory of Conservation and Utilization of Subtropical Agricultural Bioresources, South China Agricultural University. The pOx vector has been published in Li YH, Yang YQ, Liu Y, Li CX, Zhao YH, Li ZJ, Liu Y, Jiang DG, LiJ, Zhou H, Chen JH, Zhuang CX, Liu ZL. Overexpression of OsAGOE-1 binds to adaxially rolled leaves by affecting leaf abaxial sclerenchymatous cell development in rice. Rice (N Y), 2019, 12(1): 60.

The pYL322dl vector was provided by Academician Liu Yaoguang from the State Key Laboratory of Subtropical Agricultural Bioresources Conservation and Utilization, South China Agricultural University. The pYL322dl vector has been published in Han JL, Ma K, Li HL, Su J, Zhou L, Tang JT, Zhang SJ, Hou YK, Chen LT, Liu YG, Zhu QL. All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants. Plant Biotechnol J, 2022, 20(6): 1098-1109.

Unless otherwise specified in the examples, the rice used was wild-type Nipponbare rice, which was commercially available.

Embodiment 1:

(1) The retrieval number of the rice morphogen protein gene OsFH2 in the GenBank database (https://www.ncbi.nlm.nih.gov) is Os04g38810. The gene is located on chromosome 4 of rice, with a DNA length of 3306 bp (as shown in SEQ ID No: 1), containing 4 exons and 3 introns (as shown in (a) in Figure 1). The mRNA length of the OsFH2 gene is 2994 bp (as shown in SEQ ID No: 2), the CDS length is 2502 bp (as shown in SEQ ID No: 3), and the amino acid sequence of the protein encoded by it (as shown in SEQ ID No: 4) is predicted to be 833 amino acids in length (as shown in (b) in Figure 1).

(2) According to the conventional operation method in the field of genetic engineering, green fluorescent protein (GFP) was fused to the C-terminus of OsFH2 protein and inserted into the protein subcellular localization GFP empty vector pYL322dl vector containing 35S promoter stored in our laboratory (as shown in Figure 2), and the recombinant vector driven by 35S promoter for transient expression of OsFH2 gene was obtained, namely p35S::OsFH2-GFP. Rice protoplast cells were transformed. The results showed that when GFP was fused to the C-terminus of OsFH2 protein, the fluorescence signal was localized to the cell membrane and cytoplasm, and partially overlapped with the localization of plasma membrane marker protein mCherry, while the GFP empty vector control was localized to various parts of the cell (as shown in Figure 3).

(3) Nipponbare rice seedlings were cultured hydroponically in an artificial climate chamber. Kimura B nutrient solution was replaced every 3 days. The culture conditions were 12 h light, 28°C/12 h dark, 25°C, and 60% relative humidity. Four-week-old rice seedlings were treated with Cd (CdCl ₂ solution was added to the hydroponic solution. The final concentrations of CdCl ₂ were 0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM, respectively, and cultured for 1 day. In addition, the rice seedlings were cultured with CdCl ₂ solution with a final concentration of 10 μM for 0 h, 3 h, 6 h, 9 h, 12 h, 1 day, 2 days, and 3 days, respectively). Roots, stems, and leaves were selected as materials. The conventional methods in the field of genetic engineering were used to extract RNA from rice tissues using Trizol reagent from Invitrogen (USA) according to the operating manual of the reagent company. RNA from rice tissues was extracted using Trizol reagent from Vazayme (China). cDNA was obtained by reverse transcription using the SYBR GREEN Master Mix quantitative PCR kit from Biorad (USA). The expression of OsFH2 gene in roots, stems and leaves was detected by qRT-PCR using the SYBR GREEN Master Mix quantitative PCR kit from Biorad (USA). The primers used for qRT-PCR detection were: 5'-TCGCACTTCTCCTACTCCGA-3' (forward primer) and 5'-GAGACGATCGCTCCCTGTTG-3' (reverse primer); the amplification program was: denaturation at 95℃ for 3min, then denaturation at 95℃ for 15s, annealing at 60℃ for 20s, and extension at 72℃ for 20s, for a total of 30 cycles. The rice Actin gene (Os10g0510000) was used as the internal standard, and the primers used were 5'-CACATTCCAGCAGATGTGGA-3' (forward primer) and 5'-GCGATAACAGCTCCTCTTGG-3' (reverse primer).

The results showed that when the Cd concentration reached 10 μM, the expression of OsFH2 gene in the aboveground part of the seedlings was significantly upregulated 1 day after treatment, and when the Cd concentration reached 1 μM, the expression of OsFH2 gene in the root was significantly upregulated; whether in the root or stem and leaf, as the Cd treatment concentration increased, the expression of OsFH2 gene showed an upward trend (as shown in (a) and (b) in Figure 4). In addition, whether in the stem and leaf or root, the expression of OsFH2 gene was significantly upregulated after 1 hour of Cd treatment, and the expression of OsFH2 gene in the root (as shown in (c) in Figure 4) and the stem and leaf (as shown in (d) in Figure 4) reached the highest level at 12 hours and 6 hours of Cd treatment, respectively, and then its expression began to gradually decrease.

(4) Using conventional methods in the field of genetic engineering, RNA from rice leaf tissue was extracted and reverse transcribed to obtain single-stranded cDNA according to the above method, and then high-fidelity enzymes from Vazyme (China) were used to Max Super-Fidelity DNA Polymerase was used to amplify a double-stranded cDNA fragment containing the full-length CDS sequence of the OsFH2 gene. The PCR amplification program was denaturation at 95°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles, and finally extension at 72°C for a total of 5 min. The primers used in PCR were: 5'-ACATGCAACAATGCCGTCAC-3' (forward primer) and 5'-TGTCAAGAACAATCCGAAGCCA-3' (reverse primer). After sequencing verification, the double-stranded cDNA fragment containing the full-length CDS sequence of the OsFH2 gene was inserted into the overexpression vector pOx containing the Ubi promoter preserved in our laboratory (as shown in Figure 5) according to the conventional operation in the field of genetic engineering, and the OsFH2 gene overexpression vector driven by the Ubi promoter, namely pUbi::OsFH2, was obtained, and then transformed into Nipponbare rice. The transformed seedlings and their progeny were planted and identified, and independent stable homozygous strains (OE-1 and OE-2) with overexpression of the OsFH2 gene were obtained.

Seedlings of 4-week-old independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) were selected and cultured in a hydroponic solution with or without Cd (CdCl ₂ solution was added to the hydroponic solution, and the final concentration of CdCl ₂ was 10 μM and 100 μM, respectively) for 1 week. The phenotypes of the independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) were observed, and the growth traits of the independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene and the Nipponbare rice wild type (WT) were measured, including plant height, root length, fresh weight, and dry weight.

The statistical results of the growth traits of rice seedlings showed that under Cd-free conditions, there were no significant differences in plant height (as shown in Figure 6 (b)), root length (as shown in Figure 6 (c)), stem and leaf fresh weight (as shown in Figure 6 (d)), stem and leaf dry weight (as shown in Figure 6 (e)), root fresh weight (as shown in Figure 6 (f)), and root dry weight (as shown in Figure 6 (g)) of the overexpression lines (OE-1 and OE-2) compared with the wild type. However, under Cd treatment, although the fresh weight and dry weight of the roots (as shown in (f) and (g) in Figure 6) and the root length (as shown in (c) in Figure 6) were not significantly different from those of the wild type, the fresh weight of the stems and leaves (as shown in (d) in Figure 6) and the dry weight of the stems and leaves (as shown in (e) in Figure 6) of the overexpression lines were significantly higher than those of the wild type, and the fresh weight and dry weight of the aerial parts of the overexpression lines (OE-1 and OE-2) increased by ≥63.41% and ≥40.80% respectively under 10 μM Cd treatment, and increased by ≥53.70% and ≥28.28% respectively under 100 μM Cd treatment. In addition, the seedling height of the overexpression lines (OE-1 and OE-2) was significantly increased (≥14.89%) compared with the wild type under 10 μM Cd treatment. These results indicate that overexpression of the OsFH2 gene improves Cd tolerance in rice, thereby facilitating its growth under Cd stress.

(5) Selected 4-week-old hydroponic OsFH2 gene overexpression independent stable homozygous lines (OE-1, OE-2) and wild-type (WT) seedlings of Nipponbare rice, added CdCl ₂ solution to the hydroponic solution to make the final concentration of CdCl ₂ 100 μM, and continued to culture for 1 week. The rice roots were taken, washed with ddH ₂ O, placed in a centrifuge tube containing 1 mL of 20 mM Na ₂ -EDTA solution, and placed at room temperature for 10 min. The roots were taken out and washed with ddH ₂ O three times to remove the residual Na ₂ -EDTA. 50 μL DMSO was added to the Cd ion green fluorescent probe dye (Leadmium ^TM Green AM dye, Invitrogen, USA), mixed, and then the dye solution was diluted 20 times with 0.85% NaCl solution. The diluted dye solution was added to the centrifuge tube to submerge the root tissue and reacted at 40°C in the dark for 2 to 3 h. The root tissue was removed and placed on a glass slide, and observed and photographed using a laser confocal scanning microscope (LSM710, ZEISS, Germany) (excitation wavelength and emission wavelength were 488nm and 515nm, respectively). The results showed that under no Cd treatment, there was no fluorescence signal of Cd staining in the root epidermal cells of all strains (as shown in (a) in Figure 7); under Cd treatment, Cd fluorescence signals could be observed in the cell wall and cytoplasm of the root epidermal cells of the wild-type (WT) plants, and the Cd fluorescence of the cell wall was stronger than that of the cytoplasm; while in the OsFH2 gene overexpression homozygous strains (OE-1 and OE-2), the Cd fluorescence in the root epidermal cells was obviously concentrated in the cell wall, and its cell wall Cd fluorescence was significantly enhanced compared with the wild type (WT) (as shown in (b) in Figure 7). These results indicate that overexpression of the morphogen protein gene OsFH2 promotes more Cd to bind or fix to the cell wall.

The Cd concentration in various parts (roots, stems and leaves) of the homozygous strains overexpressing the OsFH2 gene (OE-1, OE-2) and the wild type (WT) of Nipponbare rice treated with the above 100 μM CdCl ₂ solution for one week was determined as follows: the plant samples were washed with tap water and dried at 60°C for 3-4 days until constant weight. An appropriate amount of sample (generally 0.3 g of root, 0.5 g of stem, 1.0 g of leaf, and 1.0 g of grain) was weighed and placed in a digestion cup, and 10 mL of mixed acid (concentrated nitric acid: perchloric acid = 87:13, volume ratio) was added, and digestion was performed using a graphite digestion furnace until the sample volume in the cup was ≤0.5 mL. After the sample cooled, 5 mL of 5% dilute nitric acid was added, mixed, and poured into a volumetric flask; the digestion cup was rinsed with ultrapure water 2-3 times, and also poured into the volumetric flask, and finally the volume was fixed to 50 mL. The sample was mixed, impurities were filtered out with filter paper, and the solution was collected and transferred to a 15 mL centrifuge tube. The Cd concentration was measured using an inductively coupled plasma optical emission spectrometer (ICP-OES, PerkinElmer, USA).

The results showed that after 1 week of Cd treatment (100 μM), the Cd concentration in the roots of the homozygous strains (OE-1 and OE-2) with overexpression of the OsFH2 gene was significantly higher than that of the wild type (WT) (increase ≥ 65.65%), while the Cd concentration in the stems and leaves was significantly lower than that of the wild type (WT) (decrease ≥ 39.83%) (as shown in (a) in Figure 8). The transfer coefficient of Cd from roots to stems and leaves (Cd transfer coefficient from roots to stems and leaves = Cd concentration in stems and leaves/Cd concentration in roots) was calculated, and the results showed that after 1 week of Cd treatment, the Cd transfer coefficient in the homozygous strains with overexpression of the OsFH2 gene was significantly lower than that of the wild type (WT) (decrease ≥ 63.94%) (as shown in (b) in Figure 8). The Cd concentration in the root cell wall was measured, and the results showed that after one week of Cd treatment (100 μM), the Cd concentration in the cell wall of independent stable homozygous strains (OE-1 and OE-2) overexpressing the OsFH2 gene was significantly increased (increased by ≥14.72%) compared with the wild type (WT) (as shown in (c) in Figure 8).

These results indicated that overexpression of the OsFH2 gene increased the retention of Cd in rice roots and altered the distribution of Cd in cells, thereby reducing the transfer of Cd from roots to stems and leaves.

(6) Selected independent stable homozygous strains (OE-1, OE-2) with overexpression of OsFH2 gene and wild type (WT) Nipponbare rice seeds, cultured in a nursery field with sufficient water and fertilizer for 30 days after germination, and selected seedlings with uniform growth to be transplanted into the Cd pollution experimental field (soil Cd content of 0.3 mg/kg; pH 5.02). Each rice material was planted in three independent plots (30 plants per plot, 20 cm between plants). Field management was carried out according to the local rice planting method. When the rice was fully mature, the phenotype of the plant was observed, and then the growth and yield traits (including plant height, number of effective tillers, 1000-grain weight, fruit setting rate, and single plant yield) were measured and statistically analyzed. Ten materials were randomly selected from each plot and mixed as a replicate sample, and the Cd content of various parts of the rice and the content of essential elements in the grain were measured.

The results showed that the growth of the two homozygous strains (OE-1 and OE-2) with overexpression of the OsFH2 gene was stronger than that of the wild type, especially the tiller number of the overexpression strain was significantly higher than that of the wild type (as shown in (a) in Figure 9). The statistical results of growth and yield-related traits showed that, consistent with the results of phenotypic observation, the dry weight of the grass stem (as shown in (c) in Figure 9), the number of effective tillers (as shown in (d) in Figure 9), and the yield per plant (as shown in (g) in Figure 9) of the two overexpression strains (OE-1 and OE-2) were significantly higher than those of the wild type (WT) (≥7.95%, ≥11.98%, and ≥18.68% respectively). There were no significant differences in plant height (as shown in (b) in Figure 9), fruit setting rate (as shown in (e) in Figure 9), and 1000-grain weight (as shown in (f) in Figure 9) between the overexpression strains and the wild type.

The results of the analysis of the content of Cd and essential elements showed that the content of Cd in the brown rice of the OsFH2 overexpression strain was significantly lower than that of the wild type (reduced by ≥36.84% compared with the wild type) (as shown in (a) of Figure 10). The results of the determination of the content of Cd in the root, base stem, upper stem and leaf tissue of the overexpression strain showed that compared with the wild type, the Cd content in the root tissue of the overexpression strain increased significantly (increased by ≥17.81%), the Cd content in the base stem and upper stem decreased significantly (reduced by ≥15.26% and ≥9.84%, respectively), and the Cd content in the leaf was not significantly different from that of the wild type (as shown in (b) of Figure 10). In addition, although the content of Fe, an essential element in brown rice, was significantly lower than that of the wild type (reduced by ≥31.91%), there were no significant differences in other essential elements such as Mn, Zn and Cu compared with the wild type (as shown in (c) of Figure 10).

The above results indicate that overexpression of the morphogen protein gene OsFH2 not only helps to reduce the Cd content in rice grains, but also helps to increase rice yield. It can be used as a candidate gene for molecular breeding of low-Cd rice, thus serving the safe production of rice food and remediation of Cd pollution.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. The application of the morphogenic protein gene OsFH in rice breeding regulation and control is characterized in that the amino acid sequence of the morphogenic protein gene OsFH2 coding protein is shown as SEQ ID No. 4;

the rice breeding regulation refers to over-expression of the morphogenetic protein gene OsFH in rice, so that the tolerance of the rice to Cd is improved, the biomass of the rice is increased, and the Cd content in rice grains is reduced.

2. The use according to claim 1, wherein the nucleotide sequence of the morphogenic protein gene OsFH is shown in SEQ ID No. 1.

3. Application of the morphogenetic protein gene OsFH in improving the tolerance of rice to Cd and/or preparing/cultivating rice varieties with low accumulation of seed Cd;

The amino acid sequence of the protein coded by the morphogenic protein gene OsFH is shown as SEQ ID No. 4.

4. The use according to claim 3, wherein the nucleotide sequence of the morphogenic protein gene OsFH is shown in SEQ ID No. 1.

5. Application of rice expression vector containing morphogenic protein gene OsFH in improving rice tolerance to Cd and/or preparing/cultivating rice varieties with low accumulation of seed Cd;

The amino acid sequence of the protein coded by the morphogenic protein gene OsFH is shown as SEQ ID No. 4.

6. The use according to claim 5, wherein the nucleotide sequence of the morphogenic protein gene OsFH is shown in SEQ ID No. 1.

7. Application of host cells containing morphogenic protein gene OsFH in improving rice tolerance to Cd and/or preparing/cultivating rice varieties with low accumulation of seed Cd;

The amino acid sequence of the protein coded by the morphogenic protein gene OsFH is shown as SEQ ID No. 4.

8. The use according to claim 7, wherein the nucleotide sequence of the morphogenic protein gene OsFH is shown in SEQ ID No. 1.

9. A method for enhancing the tolerance of rice to Cd and/or reducing the Cd content in rice grains, comprising the step of overexpressing a morphogenic protein gene OsFH;

The amino acid sequence of the protein coded by the morphogenic protein gene OsFH is shown as SEQ ID No. 4.

10. The method of claim 9, wherein the nucleotide sequence of the morphogenic protein gene OsFH is shown in SEQ ID No. 1.