CN116768996A - Application of a morphogen protein gene OsFH2 in plant breeding regulation - Google Patents

Abstract

The invention discloses the application of morphogen protein gene OsFH2 in plant breeding regulation. Belongs to the field of plant genetic engineering. Application of 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 Shown as IDNo:1. By over-expressing the morphogen protein gene OsFH2 in rice, the present invention makes the growth of the over-expressed plants under Cd stress significantly better than that of wild-type plants, and the transfer of Cd from roots to stems and leaves in the over-expressed plants is better than that of wild-type plants. The Cd content in grains is significantly reduced compared with wild-type plants, thus providing a new candidate gene resource for cultivating safe rice with low grain Cd, and also providing a potential repair method for treating Cd pollution in rice.

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 control.

Background technique

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 for human intake. If control and/or remedial measures are not taken, contaminated Cd 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 Cd intake of the human body. Using molecular breeding technology to cultivate low-Cd accumulation rice varieties is the most cost-effective and promising method to prevent the risk of Cd contamination in food. At present, genetic resources related to Cd tolerance or accumulation in rice are very limited. Therefore, the development of new Cd gene resources is of great significance for low-Cd molecular breeding of rice and Cd pollution remediation.

Under environmental heavy metal Cd stress, rice plants themselves have multiple tolerance mechanisms. For example, the antioxidant system in plants is activated under Cd stress to enhance the ability of cells to scavenge reactive oxygen species; the cell wall contains polysaccharides such as cellulose, pectin and other substances, which contain a large number of aldehyde groups, amino groups, carboxyl groups, and phosphate groups. The group 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, it can reduce the toxicity of Cd and limit the transport of Cd. Genes related to these processes can be used as candidate gene resources for molecular breeding related to Cd tolerance or accumulation regulation in rice.

Formin protein is mainly responsible for regulating the formation and homeostasis of the cytoskeleton in plant life activities, and plays an important role in intercellular material transport, cell growth, signal transduction, morphogenesis and other processes. In recent years, studies have shown that morphogen proteins may affect cell wall development by regulating the formation of the cytoskeleton, thereby playing an active role in Cd fixation of the cell wall. So far, there have been no reports on the role of morphogen proteins in the regulation of Cd resistance and Cd accumulation in rice.

Contents 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 the application of the morphogen protein gene OsFH2 in plant breeding regulation.

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

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 control refers to overexpressing the morphogen protein gene OsFH2 in plants to improve the plant's tolerance to Cd, increase rice biomass, and reduce the Cd content in plant grains.

The plant is preferably a gramineous plant; further preferably it is at least one of wheat, corn and rice; more preferably it is 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 plant expression vectors and host cells containing 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 grains includes the step of overexpressing the formin protein gene OsFH2.

Compared with the existing technology, 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 overexpressed plants under Cd stress is significantly better than that of wild-type plants, and the transfer ratio of Cd in overexpressed plants from roots to stems and leaves is The number of wild-type plants was significantly reduced, and the grain Cd content was significantly lower than that of wild-type plants. This provides a new candidate gene resource for creating safe rice with low grain Cd, and also provides a potential repair method for rice Cd pollution control.

(2) By overexpressing the morphogen protein gene OsFH2, the present invention can effectively improve the tolerance of rice to Cd, reduce the Cd content in rice grains, and provide a candidate gene for low-Cd molecular breeding of rice and Cd pollution control. Resources and potential fixes.

Description of drawings

Figure 1 is a structural information diagram of the morphogen protein gene OsFH2 and its encoded protein; (a) is a structural information diagram of the morphogen protein gene OsFH2; the rectangular box represents the exon, and the gray rectangular box represents the coding sequence (CDS). The white rectangular box represents the untranslated sequence; the solid black line represents the intron; (b) is the structural information diagram of the protein encoded by the morphogen protein gene OsFH2; the white rectangular box represents the transmembrane helical domain; the gray rectangular box represents the structure of FH1 and FH2 area.

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

Figure 3 shows the results of subcellular localization analysis of morphogen protein OsFH2 in rice protoplasts. p35S::OsFH2-GF represents the recombinant vector; p35S::GFP represents the empty vector; at the same time, the cytoplasmic membrane marker protein vector p35S::OsRac3-Mcherry is co-transferred into the protoplasts; the excitation wavelength and observation wavelength of GFP are 488nm and 507nm respectively. , emits green fluorescence; the excitation wavelength and observation wavelength of the plasma membrane marker protein OsRac3-Mcherry are 587nm and 610nm respectively, and emits red fluorescence. Scale bar is 30 μm.

Figure 4 shows the qRT-PCR detection results of morphogen protein gene OsFH2 expression in response to Cd stress; (a) shows the expression results of OsFH2 gene in roots under different CdCl ₂ concentrations; (b) shows the results of different CdCl ₂ concentrations The expression results of OsFH2 gene in stems and leaves under treatment; (c) is the expression result of OsFH2 gene in roots after culturing rice with CdCl ₂ solution with a final concentration of 10 μM for different times; (d) is the expression result of OsFH2 gene in roots with a final concentration of 10 μM CdCl ₂ The results of expression of OsFH2 gene in stems and leaves of rice grown in solution for different times; 3 independent biological replicates; materials from 3 strains were mixed into one replicate sample; data are mean ± SD; t test; * indicates P< 0.05.

Figure 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 Nipponbare rice wild type (WT); among them, (a) is the OsFH2 gene Phenotypic observation diagram of over-expressed independent stable homozygous lines (OE-1, OE-2) and Nipponbare rice wild type (WT); the ruler is 5 mm; (b)-(g) are the over-expressed OsFH2 genes respectively. (b) Plant height of independent stable homozygous lines (OE-1, OE-2) and Nipponbare rice wild type (WT); (c) root length; (d) fresh weight of stems and leaves; (e) stems and leaves Part dry weight; (f) Root fresh weight; (e) Statistical result diagram of root dry weight; 3 independent biological replicates; 30 plants were counted for each replicate; data are mean ± SD; t test; * indicates P< 0.05.

Figure 7 shows the results of Cd fluorescence staining of epidermal cells in the roots of rice plants; (a) shows the results of Cd fluorescence staining of epidermal cells in the roots of rice plants under no Cd treatment (control) conditions; (b) shows the overexpression of OsFH2 gene The results of Cd fluorescence staining of root epidermal cells of independent stable homozygous lines (OE-1, OE-2) and wild type (WT); the scale bar is 50 μm.

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

Figure 9 shows the phenotypic observation and yield trait statistical results of independent stable homozygous lines (OE-1, OE-2) and wild type (WT) overexpressing OsFH2 gene in Cd-contaminated soil cultivation experiments; among them, (a ) are the phenotypic observation results of independent stable homozygous lines (OE-1, OE-2) and wild type (WT) overexpressing the OsFH2 gene in Cd-contaminated soil cultivation experiments; the scale bar is 20cm. After the rice is fully mature, the plant height (b), dry weight of straw (c), number of effective tillers (d), seed setting rate (e), thousand-grain weight (f), and yield per plant (g) are measured and counted. . Data are the mean ± SD of 3 independent biological replicates, with 30 strains counted for each replicate; t test, * indicates P < 0.05.

Figure 10 shows the Cd content determination results of the independent stable homozygous lines (OE-1, OE-2) and wild type (WT) overexpressing the OsFH2 gene in the Cd-contaminated soil cultivation experiment. Picture (a) shows the Cd-contaminated soil cultivation experiment. Determination results of Cd content in brown rice of independent stable homozygous lines (OE-1, OE-2) and wild type (WT) overexpressing OsFH2 gene; (b) shows the results of independent stable homozygous lines overexpressing OsFH2 gene in Cd-contaminated soil cultivation experiments. Results of Cd content in roots, basal stems, upper stems, and leaves of stable homozygous lines (OE-1, OE-2) and wild type (WT); (c) is an independent stable homozygous line overexpressing the OsFH2 gene Determination results of essential elements (Fe, Mn, Zn, Cu) content in line (OE-1, OE-2) and wild-type (WT) brown rice; data are the mean ± SD of 3 independent biological replicates, every 10 Materials from strains were mixed into one replicate sample; t test, * indicates P<0.05.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Test methods that do not indicate specific experimental conditions in the following examples usually follow conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the vectors mentioned in the examples (including overexpression vectors and subcellular localization vectors) were constructed according to conventional operating methods in the field of genetic engineering. The backbone vector pCambia1300 sequence and promoter elements (including 35S, Ubi) were used. , reporter gene (including GFP) and other sequences, all of which 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 and reagents used are reagents and materials obtained from commercial sources unless otherwise specified. The chemical reagents used in the examples were all of imported or domestic analytical grade.

The pOx vector was provided by Academician Liu Yaoguang of the State Key Laboratory of Conservation and Utilization of Subtropical Agricultural Biological Resources at South China Agricultural University. The pOx vector has been used 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-1binduces adaxially rolled leaves by Disclosed in an article affecting leaf abaxial sclerenchymatous cell development in rice. Rice (N Y), 2019, 12 (1): 60.

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

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

Example 1:

(1) The accession 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, and its DNA length is 3306bp (such as SEQ ID No:1), containing 4 exons and 3 introns (shown in (a) in Figure 1). The mRNA length of the OsFH2 gene is 2994bp (as shown in SEQ ID No:2), the CDS length is 2502bp (as shown in SEQ ID No:3), and the amino acid sequence of the protein encoded by it is predicted (as shown in SEQ ID No:4) The length is 833 amino acids (shown in (b) in Figure 1).

(2) According to the conventional operating methods 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 the 35S promoter stored in our laboratory (as shown in the figure) (shown in 2), a recombinant vector driving the transient expression of OsFH2 gene driven by the 35S promoter was obtained, namely p35S::OsFH2-GFP. Transformation of rice protoplast cells. The results showed that when GFP was fused to the C-terminus of OsFH2 protein, the fluorescent signal was localized in the cell membrane and cytoplasm, and partially overlapped with the localization of the plasma membrane marker protein mCherry, while the GFP empty vector control was localized in various parts of the cell (as shown in the figure) shown in 3).

(3) Nipponbare rice seedlings were cultured hydroponically in an artificial climate chamber, and the Kimura B nutrient solution was replaced every 3 days. The culture conditions were 12 hours of light, 28°C/12 hours of darkness, 25°C, and a relative humidity of 60%. Treat 4-week-old rice seedlings with Cd (add CdCl ₂ solution to the hydroponic solution, the final concentrations of CdCl ₂ are 0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM respectively, and culture for 1 day; in addition, follow the same method with Rice seedlings were cultured with _a CdCl solution with a final concentration of 10 μM for 0h, 3h, 6h, 9h, 12h, 1d, 2d and 3d) respectively. Roots, stems and leaves were selected as materials, and conventional methods in the field of genetic engineering were used. According to the reagent company's Operation manual, use Trizol reagent from Invitrogen Company (USA) to extract RNA from rice tissue, use Vazayme Company (China) II qRT SuperMix kit was reverse transcribed to obtain cDNA, and then the SYBR GREEN Master Mix quantitative PCR kit from Biorad (USA) was used to detect the expression of the OsFH2 gene in roots, stems and leaves using qRT-PCR. The primers used for qRT-PCR detection are: 5'-TCGCACTTCTCCTACTCCGA-3' (forward primer) and 5'-GAGACGATCGCTCCCTGTTG-3' (reverse primer); the amplification program is: denaturation at 95°C for 3 minutes, then denaturation at 95°C for 15 seconds, Annealing at 60°C for 20 s and extension at 72°C for 20 s, 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'-GCGATAACAGTCCCTCTTGG-3' (reverse primer).

The results showed that when the Cd concentration reached 10 μM, the expression of the OsFH2 gene in the shoots of the seedlings was significantly up-regulated 1 day after treatment. When the Cd concentration reached 1 μM, the expression of the OsFH2 gene in the roots was significantly up-regulated; both in the roots and stems and leaves , as the concentration of Cd treatment increases, the expression of OsFH2 gene shows an upward trend (as shown in (a) and (b) in Figure 4). In addition, regardless of the stems, leaves or roots, when Cd was treated for 1 hour, the expression of OsFH2 gene was significantly increased, and the OsFH2 gene was significantly increased in the roots (shown in (c) in Figure 4), stems and leaves (Figure 4 (shown in (d)), the expression levels reached the highest at 12h and 6h after Cd treatment, respectively, and then their expression levels began to gradually decrease.

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

Select independent stable homozygous lines (OE-1, OE-2) with overexpression of OsFH2 gene (OE-1, OE-2) and Nipponbare rice wild-type (WT) seedlings grown in hydroponics for 4 weeks, and add Cd (add CdCl ₂ solution to water In the culture medium, the final concentrations of CdCl ₂ were 10 μM and 100 μM respectively) or in the hydroculture medium without Cd for 1 week. Observe the phenotypes of the independent stable homozygous lines overexpressing the OsFH2 gene (OE-1, OE-2) and Nipponbare rice wild type (WT), and determine the independent and stable homozygous lines overexpressing the OsFH2 gene (OE-1, OE-2). OE-2) and Nipponbare rice wild type (WT) growth traits, including plant height, root length, fresh weight, and dry weight.

The statistical results of the growth traits of rice seedlings show that under Cd-free conditions, the plant height (shown in (b) in Figure 6) and root length (shown in Figure 6) of the over-expression lines (OE-1, OE-2) (shown in (c) in Figure 6), fresh weight of stems and leaves (shown in (d) in Figure 6), dry weight of stems and leaves (shown in (e) in Figure 6), fresh weight of roots (shown in (e) in Figure 6) There was no significant difference between the wild type and the wild type. However, under Cd treatment, although the fresh weight and dry weight of the roots (shown in (f) and (g) in Figure 6) and root length (shown in (c) in Figure 6) were compared with those of the wild type, There is no significant difference, but the fresh weight of stems and leaves (shown in (d) in Figure 6) and the dry weight of stems and leaves (shown in (e) of Figure 6) of the over-expression line are significantly higher than those of the wild type. , among which the fresh weight and dry weight of the above-ground parts of the overexpression lines (OE-1, OE-2) increased by ≥63.41% and ≥40.80% respectively under 10 μM Cd treatment compared with the wild type, and compared with the wild type under 100 μM Cd treatment, respectively. Increase ≥53.70%, ≥28.28%. In addition, the seedling height of the overexpression lines (OE-1, OE-2) was significantly increased compared with the wild type under 10 μM Cd treatment (increase ≥14.89%). These results indicate that overexpression of OsFH2 gene improves the Cd tolerance of rice, thereby benefiting the growth of rice under Cd stress.

(5) Select independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene that are 4 weeks old and grown in hydroponics and Nipponbare rice wild-type (WT) seedlings, and add CdCl ₂ solution to the hydroponic solution. , so that the final concentration of _CdCl2 is 100 μM, and continue to culture for 1 week. Take the rice roots, wash them with ddH ₂ O, put them into a centrifuge tube containing 1 mL of 20mM Na ₂ -EDTA solution, and leave them at room temperature for 10 minutes. Take out the roots and wash them three times with ddH ₂ O to remove any remaining Na ₂ -EDTA. Add 50 μL DMSO to the Cd ion green fluorescent probe dye (Leadmium ^TM green AM dye, Invitrogen Company, USA), mix well, and then dilute the dye solution 20 times with 0.85% NaCl solution. Add the diluted dye solution into the centrifuge tube, submerge the root tissue, and react in the dark at 40°C for 2 to 3 hours. The root tissue was taken out, placed on a glass slide, and observed and photographed using a laser confocal scanning microscope (LSM710, ZEISS, Germany) (excitation and emission wavelengths were 488 nm and 515 nm, respectively). The results showed that without Cd treatment, there was no Cd-stained fluorescent signal in the root epidermal cells of all lines (as shown in (a) in Figure 7); under Cd treatment, the roots of wild-type (WT) plants Cd fluorescence signals can be observed in the cell wall and cytoplasm of epidermal cells, and the Cd fluorescence in the cell wall is stronger than that in the cytoplasm; in the OsFH2 gene overexpression homozygous lines (OE-1, OE-2), Cd fluorescence in root epidermal cells All were obviously concentrated on the cell wall, and the 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 binding or fixation to the cell wall.

The Cd concentration in each part (roots, stems and leaves) of the OsFH2 gene overexpression homozygous lines (OE-1, OE-2) and Nipponbare rice wild type (WT) treated with the above 100 μM CdCl ₂ solution for 1 week was measured. The method is as follows: wash the plant samples with tap water and dry them at 60°C for 3 to 4 days until constant weight. Weigh an appropriate amount of sample (generally weigh 0.3g of roots, 0.5g of stems, 1.0g of leaves, and 1.0g of seeds), put it into a boiling cup, and add 10 mL of mixed acid (concentrated nitric acid: perchloric acid = 87:13, volume ratio) , use a graphite digestion furnace for digestion, and end the digestion when the sample volume in the cup is ≤0.5mL. After the sample is cooled, add 5 mL of 5% dilute nitric acid, mix well, and pour into the volumetric flask; then rinse the digestion cup with ultrapure water 2 to 3 times, pour into the volumetric flask, and finally adjust the volume to 50 mL. Mix the sample evenly, filter impurities with filter paper, collect the solution, and transfer it to a 15 mL centrifuge tube. The Cd concentration was determined 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 lines overexpressing the OsFH2 gene (OE-1, OE-2) 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) (a decrease of ≥39.83%) (as shown in (a) in Figure 8). The transfer coefficient of Cd from roots to stems and leaves was calculated (Cd transfer coefficient from roots to stems and leaves = Cd concentration in stems and leaves/Cd concentration in roots). The results showed that after 1 week of Cd treatment, homozygous OsFH2 gene overexpression The Cd transfer coefficients in the strains were significantly lower than those in the wild type (WT) (a decrease of ≥63.94%) (as shown in (b) in Figure 8). The Cd concentration in the root cell wall was measured. The results showed that after 1 week of Cd treatment (100 μM), the Cd concentration in the cell wall of the independent stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene was higher than that of the wild type. (WT) increased significantly (increase ≥14.72%) (as shown in (c) in Figure 8).

The above results show that overexpression of OsFH2 gene increases the retention of Cd in rice roots and changes the distribution of Cd in cells, thereby reducing the transfer of Cd from roots to stems and leaves.

(6) Select independent stable homozygous lines (OE-1, OE-2) and wild-type (WT) Nipponbare rice seeds overexpressing the OsFH2 gene. After germination, cultivate them in a seedling field with sufficient water and fertilizer for 30 days, and then select seedlings with uniform growth. Transplanted into a Cd-contaminated experimental field (soil Cd content is 0.3 mg/kg; pH 5.02). Each rice material was planted in 3 independent plots (30 plants per plot, 20cm apart between plants). Carry out field management according to local rice cultivation methods. After the rice is fully mature, the phenotype of the plant is observed, and then the growth and yield traits (including plant height, number of effective tillers, thousand-grain weight, seed setting rate, and yield per plant) are measured and statistically analyzed. Randomly select 10 plant materials from each plot and mix them into a repeated sample, and measure the Cd content of each part of the rice and the essential element content of the grain.

The results showed that the growth of the two homozygous lines (OE-1 and OE-2) overexpressing the OsFH2 gene was stronger than that of the wild type. In particular, the number of tillers of the overexpressed line was significantly increased compared with the wild type (as shown in Figure 9 shown in (a)). The statistical results of growth and yield-related traits showed that they were consistent with the results of phenotypic observations. The dry weight of grass stems of the two overexpression lines (OE-1, OE-2) (as shown in (c) in Figure 9) , the effective tiller number (shown in (d) in Figure 9), and the yield per plant (shown in (g) in Figure 9) were significantly increased compared to the wild type (WT) (increased by ≥7.95% compared to the wild type respectively). , ≥11.98%, ≥18.68%); the plant height (shown in (b) in Figure 9), seed setting rate (shown in (e) in Figure 9) and thousand-grain weight (shown in Figure 9) of the over-expression line (shown in (f))) traits have no significant difference compared with the wild type.

The content analysis results of Cd and essential elements showed that the Cd content in the brown rice of the OsFH2 overexpression line was significantly lower than that of the wild type (≥36.84% lower than the wild type) (as shown in (a) in Figure 10). The measurement results of Cd content in the roots, basal stems, upper stems and leaf tissues of the over-expression line showed that compared with the wild type, the Cd content in the root tissue of the over-expression line increased significantly (increase ≥17.81%), and the Cd content in the basal stems of the over-expression line increased significantly (≥17.81%). The Cd content in the stems and upper stems was significantly reduced (respectively decreased by ≥15.26%, ≥9.84%), while the Cd content in the leaves was not significantly different from the wild type (as shown in (b) in Figure 10). In addition, although the content of essential elements Fe in brown rice is significantly lower than that of the wild type (a decrease of ≥31.91%), other essential elements such as Mn, Zn, and Cu have no significant difference compared with the wild type ((c) in Figure 10 shown).

The above results show 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 low-Cd molecular breeding of rice, thus providing benefits for rice food security production and production. Cd contamination remediation services.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. The application of the morphogenetic protein gene OsFH2 in plant breeding regulation is characterized in that the amino acid sequence of the morphogenetic protein gene OsFH2 encoding protein is shown as SEQ ID No. 4.

2. The use according to claim 1, wherein the nucleotide sequence of the morphogenic protein gene OsFH2 is shown in SEQ ID No. 1.

3. The use according to claim 1, wherein the plant breeding regulation means overexpression of the morphogenetic protein gene OsFH2 in plants, increasing the tolerance of plants to Cd, increasing rice biomass, and decreasing Cd content in plant kernels.

4. The use according to claim 1, wherein the plant is a gramineous plant.

5. The use according to claim 4, wherein the cereal is at least one of wheat, maize and rice.

6. The use according to claim 5, wherein the cereal plant is rice.

7. The application of the morphogenetic protein gene OsFH2 in improving the tolerance of plants to Cd and/or preparing/cultivating seed Cd low-accumulation plant varieties.

8. The application of the plant expression vector containing the morphogenic protein gene OsFH2 in improving the tolerance of plants to Cd and/or preparing/cultivating seed Cd low-accumulation plant varieties.

9. The application of a host cell containing the morphogenic protein gene OsFH2 in improving the tolerance of plants to Cd and/or preparing/cultivating seed Cd low-accumulation plant varieties.

10. A method for enhancing plant tolerance to Cd and/or reducing Cd content in plant kernels, comprising the step of overexpressing the morphogenic protein gene OsFH 2.