CN116768996A - Application of morphogenic protein gene OsFH2 in plant breeding regulation and control - Google Patents
Application of morphogenic protein gene OsFH2 in plant breeding regulation and control Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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Abstract
The invention discloses an application of a morphogenic protein gene OsFH2 in plant breeding regulation and control. Belonging to the field of plant genetic engineering. The application of the morphogenetic protein gene OsFH2 in plant breeding regulation and control is characterized in that the amino acid sequence of the morphogenetic protein gene OsFH2 encoding protein is shown as SEQ ID No. 4; the nucleotide sequence of the morphogenic protein gene OsFH2 is shown as SEQ ID No. 1. The invention enables the growth of the over-expressed plant under Cd stress to be obviously better than that of a wild plant by over-expressing the formed protein gene OsFH2 in rice, and the transfer of Cd in the over-expressed plant from root to stem and leaf is obviously reduced compared with that of the wild plant, and the Cd content of seeds is obviously reduced compared with that of the wild plant, thereby providing a new candidate gene resource for cultivating safe rice with low Cd seeds and a potential restoration method for rice Cd pollution treatment.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a morphogenetic protein gene OsFH2 in plant breeding regulation.
Background
Cadmium (Cd) is a common heavy metal element in farmland soil pollution, and crops planted in Cd polluted soil can have serious influence on food safety. Rice is the most important grain crop in china and is also the main source of Cd intake by humans, and if no control and/or remedial measures are taken, contaminated Cd in farmlands may pose a permanent threat to human health through the food chain. The accumulation of Cd in rice is limited, and the intake of Cd in human body can be effectively reduced. The molecular breeding technology is utilized to cultivate rice varieties with low Cd accumulation, and the method is the most cost-effective and promising method for preventing the risk of Cd pollution in foods. At present, the Cd tolerance of rice or the related gene resources of accumulation are very limited, so that the development of new Cd gene resources has great significance for low-Cd molecular breeding of rice and Cd pollution repair.
Under the stress of environmental heavy metal Cd, rice plants themselves have various tolerance mechanisms. For example, in vivo antioxidant systems are activated under Cd stress to enhance the ability of cells to scavenge reactive oxygen species; the cell wall contains polysaccharide such as cellulose, pectin and the like, and contains a large number of aldehyde groups, amino groups, carboxyl groups, phosphate groups and the like, so that Cd can be adsorbed and fixed in the cell wall, and then the Cd is prevented from entering cytoplasm; by sequestering Cd, then transporting Cd into vacuoles or aposomes, toxicity of Cd is reduced and transportation of Cd is limited. The genes related to the processes can be used as candidate gene resources for rice Cd tolerance or accumulation regulation related molecular breeding.
The morphogenic protein (formin) is mainly responsible for regulating the formation and homeostasis of cytoskeleton in plant life activities, and plays an important role in the processes of intercellular substance transport, cell growth, signal transduction, morphogenesis and the like. In recent years, studies have shown that morphogenic proteins may affect cell wall development by modulating cytoskeletal formation, thereby playing a positive role in Cd fixation of the cell wall. To date, no report has been made on the role of morphogenic proteins in regulation of rice anti-Cd and Cd accumulation.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the primary aim of the invention is to provide the application of the morphogenic protein gene OsFH2 in plant breeding regulation.
The aim of the invention is achieved by the following technical scheme:
the application of the morphogenetic protein gene OsFH2 in plant breeding regulation and control is characterized in that the amino acid sequence of the morphogenetic protein gene OsFH2 encoding protein is shown as SEQ ID No. 4.
The nucleotide sequence of the morphogenic protein gene OsFH2 is shown as SEQ ID No. 1.
The plant breeding regulation refers to over-expression of the morphogenetic protein gene OsFH2 in plants, so that the tolerance of the plants to Cd is improved, the biomass of rice is increased, and the Cd content in plant seeds is reduced.
The plant is preferably a gramineous plant; further preferably at least one of wheat, corn and rice; more preferably rice.
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.
The application of a plant expression vector containing the morphogenic protein gene OsFH2 and a host cell in improving the tolerance of plants to Cd and/or preparing/cultivating seed Cd low-accumulation plant varieties.
A method of enhancing plant tolerance to Cd and/or reducing Cd content in plant kernels comprising the step of overexpressing the morphogenic protein gene OsFH 2.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention enables the growth of the over-expressed plant under Cd stress to be obviously better than that of a wild plant by over-expressing the formed protein gene OsFH2 in rice, and the transfer of Cd in the over-expressed plant from root to stem and leaf is obviously reduced compared with that of the wild plant, and the Cd content of seeds is obviously reduced compared with that of the wild plant, thereby providing a new candidate gene resource for creating safe rice with low Cd seeds and a potential restoration method for rice Cd pollution treatment.
(2) The invention can effectively improve the tolerance of rice to Cd by over-expressing the protein gene OsFH2, reduce the Cd content in rice grains, and provide a candidate gene resource and potential restoration method for rice low-Cd molecular breeding and Cd pollution treatment.
Drawings
FIG. 1 is a diagram showing structural information of a morphogenic protein gene OsFH2 and a protein encoded by the morphogenic protein gene OsFH 2; wherein, (a) is a structural information diagram of the forming element protein gene OsFH 2; rectangular boxes represent exons, grey rectangular boxes represent coding sequences (CDS), white rectangular boxes represent untranslated sequences; the black solid line represents the intron; (b) A structural information diagram for forming a protein encoded by the plain protein gene OsFH 2; white rectangular boxes represent transmembrane helical domains; gray rectangle boxes represent FH1 and FH2 domains.
FIG. 2 is a schematic representation of the protein subcellular localization of GFP empty vector pYL322dl vector.
FIG. 3 is a graph showing the results of subcellular localization analysis of the morphogenic protein OsFH2 in rice protoplasts. p35S is that OsFH2-GF represents recombinant vector; GFP represents empty vector; simultaneously, the cytoplasmic membrane marker protein carrier p35S is transferred into protoplast together with OsRac 3-Mcherry; the excitation wavelength and the observation wavelength of GFP are 488nm and 507nm respectively, and green fluorescence is generated; the excitation wavelength and the observation wavelength of the plasma membrane marker protein OsRac 3-Mchery are 587nm and 610nm respectively, and red fluorescence is generated. The scale is 30 μm.
FIG. 4 shows response of morphogenic protein gene OsFH2 expression to Cd stressqRT-PCR detection result diagram; wherein (a) is a different CdCl 2 An expression level result diagram of root OsFH2 gene under concentration treatment; (b) For different CdCl 2 An expression level result diagram of the OsFH2 gene of the stem and leaf part under concentration treatment; (c) CdCl at a final concentration of 10. Mu.M 2 Carrying out solution culture on the expression level result graph of the root OsFH2 gene after different times of rice; (d) CdCl at a final concentration of 10. Mu.M 2 The expression level result diagram of the stem and leaf OsFH2 gene after different times of solution culture of rice; 3 independent biological replicates; mixing materials from 3 strains into a repeated sample; data are mean ± SD; t testing; * Representing P<0.05。
FIG. 5 is a schematic diagram of an overexpression vector pOx.
FIG. 6 is a graph showing the phenotypic observation and growth trait statistics of independent stable homozygous lines (OE-1, OE-2) and wild-type Nipponbare (WT) for OsFH2 gene overexpression; wherein, (a) is a phenotype observation diagram of an independent stable homozygous strain (OE-1, OE-2) with over-expressed OsFH2 gene and Wild Type (WT) of Nippon Rice; the scale is 5mm; (b) - (g) independently stable homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene, respectively, (b) strain height of wild-type Nippon Rice (WT); (c) root length; (d) fresh weight of stem and leaf; (e) stem and leaf dry weight; (f) root fresh weight; (e) a plot of statistical results of root dry weight; 3 independent biological replicates; counting 30 strains in each repetition; data are mean ± SD; t testing; * Represents P <0.05.
FIG. 7 is a graph showing the result of Cd fluorescent staining of epidermal cells at the root of rice plants; wherein, (a) is a Cd fluorescent staining observation result diagram of the plant root epidermal cells under the condition of no Cd treatment (control); (b) Cd fluorescent staining observations of independent stable homozygous lines (OE-1, OE-2) and Wild Type (WT) root epidermal cells over-expressed for OsFH2 gene; the scale is 50. Mu.m.
FIG. 8 shows CdCl at a final concentration of 100. Mu.M 2 Independent stable homozygous lines (OE-1, OE-2) and Cd content results of all parts of wild type Nipponbare (WT) of OsFH2 gene over-expression after 1 week of solution culture; wherein (a) is 100. Mu.M CdCl 2 Independent stable homozygous lines (OE-1, OE-2) and Nipponbare for overexpression of the OsFH2 gene after 1 week of hydroponic cultureRoot (Root) and stem and leaf (Shoot) Cd content results of Wild Type (WT) of rice are shown; (b) CdCl of 100. Mu.M 2 The transfer coefficient results of Cd in independent stable homozygous lines (OE-1, OE-2) and wild type Nipponbare (WT) of OsFH2 gene over-expression after 1 week of solution culture are shown; (c) CdCl of 100. Mu.M 2 Cell wall Cd concentration results of independent stable homozygous lines (OE-1, OE-2) and wild type Nipponbare (WT) with overexpression of the OsFH2 gene after 1 week of solution culture; 3 independent biological replicates; mixing 3 strains of materials into a repeated sample; data are mean ± SD; t testing; * Representing P<0.05。
FIG. 9 is a graph showing phenotypic observations and yield trait statistics of independent stable homozygous lines (OE-1, OE-2) and Wild Type (WT) for overexpression of the OsFH2 gene in a Cd contaminated soil cultivation experiment; wherein, (a) is a diagram of phenotype observation results of independent stable homozygous lines (OE-1, OE-2) and Wild Type (WT) of the overexpression of the OsFH2 gene in the Cd contaminated soil cultivation experiment; the scale is 20cm. After the rice is fully mature, the plant height (b), the dry weight (c) of the straw, the effective tillering number (d), the fruiting rate (e), the thousand seed weight (f) and the single plant yield (g) are measured and counted. Data are mean ± SD of 3 independent biological replicates, each replicate counting 30 strains; t-test, P <0.05.
FIG. 10 is a graph showing the measurement results of the content of the independent stable homozygous lines (OE-1, OE-2) and the Wild Type (WT) Cd in the overexpression of the OsFH2 gene in the Cd contaminated soil cultivation experiment, and the graph showing the measurement results of the content of Cd in the independent stable homozygous lines (OE-1, OE-2) and the Wild Type (WT) brown rice in the overexpression of the OsFH2 gene in the Cd contaminated soil cultivation experiment; (b) The result diagram of Cd content in roots, basal stems, upper stems and leaves of independent stable homozygous lines (OE-1, OE-2) and Wild Type (WT) of the overexpression of the OsFH2 gene in the Cd contaminated soil cultivation experiment; (c) An independent stable homozygous strain (OE-1, OE-2) for over-expression of the OsFH2 gene and essential element (Fe, mn, zn, cu) content measurement result diagram in Wild Type (WT) brown rice; data are mean ± SD of 3 independent biological replicates, with each 10 strains of material mixed as one replicate; t-test, P <0.05.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The construction of the vectors (including the over-expression vectors and the subcellular localization vectors) mentioned in the examples, unless otherwise specified, was carried out according to the methods of operation in the conventional genetic engineering field, using the backbone vector pCambia1300 sequence and the sequences of promoter elements (including 35S, ubi), reporter genes (including GFP) and the like, which are all sequences disclosed in the genetic engineering field in the relevant websites or databases (https:// cammbi. Org and https:// www.ncbi.nlm.nih.gov). The materials, reagents and the like used, unless otherwise specified, are those obtained commercially. The chemical reagents used in the examples were all imported or homemade analytically pure.
pOx the vector is provided by the national emphasis laboratory Liu Yaoguang institution of agricultural resource protection and utilization of subtropical agriculture at the university of south China. pOx vectors are disclosed in Li YH, yang YQ, liu Y, li CX, zhao YH, li ZJ, liu Y, jiang DG, li J, zhou H, chen JH, zhuang CX, liu ZL. Overexpression of OsAGOE-1binduces adaxially rolled leaves by affecting leaf abaxial sclerenchymatous cell development in rice.Rice (N Y), 2019,12 (1): 60.
pYL322dl carrier is provided by national emphasis laboratory Liu Yaoguang institution at the university of agricultural in south China for subtropical agricultural biological resource protection and utilization. pYL322dl vectors are disclosed in Han JL, maK, li HL, su J, zhou L, tang JT, zhang SJ, hou YK, chen LT, chu YG, zhu QL.all-in-one a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants Biotechnol J,2022,20 (6): 1098-1109.
In the examples, unless otherwise specified, the rice used was wild type Nippon Rice, which is commercially available.
Example 1:
(1) The rice morphogenic protein gene OsFH2 has the search number Os04g38810 in GenBank database (https:// www.ncbi.nlm.nih.gov), is located on chromosome 4 of rice, has the DNA length of 3306bp (shown as SEQ ID No: 1), and contains 4 exons and 3 introns (shown as (a) in FIG. 1). The mRNA length of the OsFH2 gene is 2994bp (shown as SEQ ID No: 2), the CDS length is 2502bp (shown as SEQ ID No: 3), and the predicted encoded protein amino acid sequence (shown as SEQ ID No: 4) is 833 amino acids (shown as (b) in FIG. 1).
(2) According to a conventional operation method in the genetic engineering field, green Fluorescent Protein (GFP) is fused to the C end of OsFH2 protein and is inserted into a protein subcellular localization GFP empty vector pYL dl vector (shown in figure 2) containing a 35S promoter stored in the research laboratory, so as to obtain a recombinant vector which drives the instantaneous expression of an OsFH2 gene by the 35S promoter, namely p35S:: osFH2-GFP. Transforming rice protoplast cells. The results showed that when GFP was fused to the C-terminus of OsFH2 protein, the fluorescent signal was localized to the cell membrane and cytoplasm and there was partial overlap with the localization of plasma membrane marker protein mCherry, whereas GFP empty vector controls were localized to various sites of the cells (as shown in figure 3).
(3) The seedlings of Nippon Rice are cultivated in a water culture way in a manual climate chamber, and Kimura B nutrient solution is replaced every 3d, wherein the cultivation conditions are 12h illumination, 28 ℃/12h darkness, 25 ℃ and relative humidity of 60 percent. Cd treatment was performed on seedlings of rice cultured for 4 weeks (CdCl was added 2 Adding the solution into the water culture solution, cdCl 2 The final concentration of (2) was 0. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M, respectively, and cultured for 1d; in addition, cdCl with a final concentration of 10. Mu.M was used in the same manner 2 Solution-culturing young rice 0h, 3h, 6h, 9h, 12h, 1d, 2d and 3d respectively, selecting root and stem as materials, extracting RNA of rice tissue by Trizol reagent of Invitrogen company (USA) according to the manual of reagent company by using Vazayme company (China) by using conventional method in genetic engineering fieldReverse transcription of II qRT Supermix kit to obtain cDNA, and qRT-PCR of the expression level of OsFH2 gene in root and stem and leaf was performed using SYBR GREEN Master Mix quantitative PCR kit from Biorad Co (USA)And (5) detecting. The primers used for qRT-PCR detection are as follows: 5'-TCGCACTTCTCCTACTCCGA-3' (forward primer) and 5'-GAGACGATCGCTCCCTGTTG-3' (reverse primer); the amplification procedure was: denaturation at 95℃for 3min, then 15s at 95℃and annealing at 60℃for 20s and extension at 72℃for 30 cycles. The rice action gene (Os 10g 0510000) is used as an internal standard, and the primers used are 5'-CACATTCCAGCAGATGTGGA-3' (forward primer) and 5'-GCGATAACAGCTCCTCTTGG-3' (reverse primer).
The results show that when the Cd concentration reaches 10 mu M, the expression level of the OsFH2 gene at the upper part of the seedlings is obviously up-regulated after 1 day of treatment, and when the Cd concentration reaches 1 mu M, the expression level of the OsFH2 gene at the root part is obviously up-regulated; the expression level of the OsFH2 gene increased with increasing Cd treatment concentration, regardless of the root or the stem and leaf (as shown in (a) and (b) of fig. 4). In addition, the expression level of the OsFH2 gene was significantly up-regulated after 1h of Cd treatment, and the expression levels of the OsFH2 gene in the root (as shown in (c) of fig. 4) and the stem (as shown in (d) of fig. 4) were respectively highest at 12h and 6h of Cd treatment, and then the expression levels began to gradually decrease.
(4) Extracting RNA of rice leaf tissue by using conventional operation method in gene engineering field, reverse transcribing to obtain single-stranded cDNA, and using high-fidelity enzyme of Vazyme companyMax Super-Fidelity DNA Polymerase, amplified to contain the OsFH2 gene full-length CDS sequence of double-stranded cDNA fragments. The PCR amplification procedure was 95℃denaturation for 5min, then 95℃denaturation for 30s,58℃annealing for 30s,72℃extension for 30s, total 35 cycles, and finally 72℃total extension for 5min. The primers used for PCR were: 5'-ACATGCAACAATGCCGTCAC-3' (forward primer) and 5'-TGTCAAGAACAATCCGAAGCCA-3' (reverse primer). After the sequencing verification is correct, the double-stranded cDNA fragment containing the full-length CDS sequence of the OsFH2 gene is inserted into an overexpression vector pOx vector (shown in figure 5) containing a Ubi promoter stored in the research laboratory according to the conventional operation in the genetic engineering field, so as to obtain the OsFH2 gene overexpression vector driven by the Ubi promoter, namely pUbi:: osFH2, and thenTransforming Nippon Rice. Planting and identifying the transformed seedlings and offspring to obtain independent stable homozygous lines (OE-1 and OE-2) with over-expressed OsFH2 genes.
Selecting independent stable homozygous lines (OE-1, OE-2) with 4 weeks old OsFH2 gene overexpression grown by hydroponics and seedlings of wild type Japanese sunny rice (WT), adding Cd (CdCl) 2 Adding the solution into the water culture solution, cdCl 2 Final concentration of 10. Mu.M, 100. Mu.M) or in water culture without Cd for 1 week. The phenotype of the independent stable homozygous lines (OE-1 and OE-2) with over-expressed OsFH2 genes and the wild type of Nipponbare (WT) is observed, and the growth characters of the independent stable homozygous lines (OE-1 and OE-2) with over-expressed OsFH2 genes and the wild type of Nipponbare (WT) are measured, wherein the growth characters comprise plant height, root length, fresh weight and dry weight.
Statistical results of growth traits of rice seedlings show that under the condition of no Cd, the plant heights (shown as (b) in fig. 6), root lengths (shown as (c) in fig. 6), stem leaf fresh weights (shown as (d) in fig. 6), stem leaf dry weights (shown as (e) in fig. 6), root fresh weights (shown as (f) in fig. 6) and root dry weights (shown as (g) in fig. 6) of the over-expressed strain (OE-1, OE-2) are not significantly different from those of the wild type. However, under Cd treatment, although there was no significant difference between the fresh weight and dry weight of the roots (as shown in (f) and (g) in fig. 6), and the root length (as shown in (c) in fig. 6) compared to the wild type, the fresh weight of the stem and leaf of the overexpressed strain (as shown in (d) in fig. 6), and the dry weight of the stem and leaf (as shown in (e) in fig. 6) were all significantly higher than that of the wild type, with the fresh and dry weights of the aerial parts of the overexpressed strain (OE-1, OE-2) increased by ≡63.41% and ≡ 40.80% respectively under 10 μm Cd treatment, and by ≡ 53.70% and ≡28.28% respectively under 100 μm Cd treatment. In addition, the seedling height of the over-expressed lines (OE-1, OE-2) increased significantly (by > 14.89%) over the wild-type with 10. Mu.M Cd. These results indicate that overexpression of the OsFH2 gene improves the Cd tolerance of rice, thereby being beneficial to the growth of rice under Cd stress.
(5) Selecting independent stable homozygous lines (OE-1, OE-2) with 4 weeks old OsFH2 gene overexpression and Wild Type (WT) seedlings of Nippon Rice, adding C into the hydroponic solutiondCl 2 Solution of CdCl 2 The final concentration of (2) was 100. Mu.M, and the culture was continued for 1 week. Taking rice roots, using ddH 2 O the roots were washed and put into a solution containing 1mL of 20mM Na 2 Placing in a centrifuge tube of EDTA solution at room temperature for 10min, taking out root, and adding ddH 2 O is washed 3 times to remove residual Na 2 EDTA. Cd ion green fluorescent probe dye (Leadmium) TM Green AM dye, invitrogen, USA) was added to 50 μl LDMSO, mixed well, and the dye was diluted 20-fold with 0.85% nacl solution. Adding diluted dye liquor into a centrifuge tube, submerging root tissues, and reacting for 2-3 h under the dark condition at 40 ℃. Root tissue was removed, placed on a glass slide, and observed and photographed (excitation wavelength and emission wavelength were 488nm, 515nm, respectively) using a confocal laser scanning microscope (LSM 710, ZEISS, germany). The results showed that under Cd-free treatment, the root epidermal cells of all strains were free of Cd-stained fluorescent signals (as shown in (a) of fig. 7); under the treatment of Cd, cd fluorescent signals can be observed in the cell wall and cytoplasm of root epidermal cells of Wild Type (WT) plants, and Cd fluorescence of the cell wall is stronger than that of cytoplasm; in the case of the homozygous strain (OE-1, OE-2) overexpressing the OsFH2 gene, cd fluorescence in the root epidermal cells was significantly concentrated on the cell wall, and the Cd fluorescence on the cell wall was significantly enhanced as compared with that of the wild-type strain (WT) (as shown in FIG. 7 (b)). These results indicate that the morphogenic protein gene OsFH2 when overexpressed promotes more Cd binding or immobilization to the cell wall.
For the above 100. Mu.M CdCl 2 The concentration of Cd in each part (root and stem) of wild type Nippon Rice (WT) of the homozygous strain (OE-1, OE-2) overexpressed by the OsFH2 gene after 1 week of solution treatment was measured as follows: the plant sample is washed by tap water and dried for 3-4 d at 60 ℃ until the weight is constant. Weighing a proper amount of samples (generally weighing 0.3g of roots, 0.5g of stems, 1.0g of leaves and 1.0g of seeds), putting the samples into a digestion cup, adding 10mL of mixed acid (concentrated nitric acid: perchloric acid=87:13, volume ratio), and digesting by using a graphite digestion furnace until the volume of the samples in the cup is less than or equal to 0.5mL, and ending digestion. After the sample is cooled, 5mL of 5% dilute nitric acid is added, mixed evenly and poured into a volumetric flask; rinsing the boiled cup with ultrapure water for 2-3 times, pouring into a volumetric flask, and finally determiningTo 50mL. The samples were mixed well, the impurities were filtered with filter paper, the solution was collected and transferred to a 15mL centrifuge tube. The Cd concentration was determined using an inductively coupled plasma emission spectrometer (ICP-OES, perkinElmer, usa).
The results showed that, after 1 week of Cd treatment (100. Mu.M), the root Cd concentration of the homozygous lines (OE-1, OE-2) overexpressed by the OsFH2 gene was significantly higher than that of the wild-type (WT) (rise. Gtoreq.65%) and the stem and leaf Cd concentration was significantly lower than that of the wild-type (WT) (drop. Gtoreq. 39.83%) (as shown in (a) of FIG. 8). The transfer coefficient of Cd from root to stem and leaf was calculated (transfer coefficient of Cd from root to stem and leaf=stem and leaf Cd concentration/root Cd concentration), and the result showed that Cd transfer coefficient was significantly reduced (decrease: 63.94%) compared to Wild Type (WT) in homozygous lines in which OsFH2 gene was overexpressed after 1 week of Cd treatment (as shown in (b) of fig. 8). Measurement of the Cd concentration in the root cell wall revealed that the cell wall Cd concentration was significantly increased (increased. Gtoreq.14.72%) in each of the independent stable homozygous lines (OE-1, OE-2) in which the OsFH2 gene was overexpressed after 1 week of Cd treatment (100. Mu.M) (as shown in (c) of FIG. 8).
The above results show that the overexpression of the OsFH2 gene increases the retention of Cd in the root of rice and changes the distribution of Cd in cells, thereby reducing the transfer of Cd from the root to the stem and leaf.
(6) Independent stable homozygous lines (OE-1 and OE-2) and Wild (WT) Nippon rice seeds with over-expressed OsFH2 genes are selected, after germination, seedlings with uniform growth vigor are selected to be transplanted into a Cd pollution experimental field (the Cd content of soil is 0.3mg/kg; pH 5.02) after being cultured in a seedling bed with sufficient water and fertilizer for 30 d. 3 independent cells (30 plants per cell, with a spacing of 20 cm) were planted for each rice material. And performing field management according to a local rice planting mode. After the rice is fully mature, observing the phenotype of the plant, and then measuring and statistically analyzing the growth and yield traits (including plant height, effective tillering number, thousand grain weight, fruiting rate and single plant yield). 10 strains of materials are randomly taken from each cell and uniformly mixed into a repeated sample, and the Cd content of each part of the rice and the content of essential elements of seeds are measured.
The results show that both homozygous lines (OE-1, OE-2) overexpressing the OsFH2 gene grew stronger than the wild-type, especially the overexpressing lines had significantly increased tillering numbers than the wild-type (as shown in FIG. 9 (a)). Statistical results of growth and yield-related traits showed that, consistent with the results of phenotypic observations, the straw dry weights (as shown in (c) of FIG. 9), the effective tillers (as shown in (d) of FIG. 9), the individual yields (as shown in (g) of FIG. 9) were all significantly increased over Wild Type (WT) (7.95%, > 11.98%, > 18.68% over wild type, respectively); the plant height (as shown in (b) of fig. 9), the setting rate (as shown in (e) of fig. 9) and the thousand kernel weight (as shown in (f) of fig. 9) of the overexpressing line all have no significant differences from the wild-type.
The analysis results of the content of Cd and essential elements show that the content of Cd in the brown rice of the OsFH2 over-expression strain is obviously reduced compared with the wild type (the Cd content is reduced by more than or equal to 36.84 percent compared with the wild type) (as shown in (a) in fig. 10). The measurement results of Cd content in roots, basal stems, upper stems and leaf tissues of the over-expression strain show that the Cd content in root tissues of the over-expression strain is remarkably increased (increased by more than or equal to 17.81%) compared with the wild type, the Cd content in the basal stems and the upper stems is remarkably reduced (respectively reduced by more than or equal to 15.26% and more than or equal to 9.84%), and the Cd content in the leaf is not remarkably different from the wild type (as shown in (b) of FIG. 10). In addition, although the content of Fe in brown rice is significantly reduced (reduced by 31.91%) as compared with the wild type, other essential elements such as Mn, zn, cu are not significantly different from the wild type (as shown in (c) of FIG. 10).
The result shows that the over-expression of the formed protein gene OsFH2 is beneficial to reducing the Cd content in rice grains, is beneficial to improving the rice yield, and can be used as a candidate gene for rice low-Cd molecular breeding, thereby serving the safety production of rice grains and the restoration of Cd pollution.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are 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.
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