Application of rice CSLF6 gene and / or coded protein in improvement of rice cadmium stress tolerance in rice breeding regulation

Abstract

The invention relates to the field of gene engineering, in particular to application of a CSLF6 gene and / or coded protein to improvement of rice cadmium stress tolerance in rice breeding regulation. CSLF6 gene overexpression can be used for cultivating rice with improved cadmium stress tolerance and cadmium accumulation, higher-level growth of the rice is facilitated, and the rice can be used for repairing soil cadmium pollution and has excellent application value. The CSLF6 gene mutant can effectively reduce the accumulation of cadmium in rice grains.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to rice CSLF6 Application of genes and / or encoded proteins in rice breeding regulation to improve rice cadmium stress tolerance. Background Art

[0002] In recent years, with the development of industry and agriculture, especially the increase in the discharge of industrial "three wastes", the pollution of heavy metal cadmium (Cd) in my country's soil has become increasingly severe. Rice has a strong ability to absorb Cd. Cd pollution not only leads to a reduction in rice production, but also accumulates in rice, enters the human body through the food chain, and threatens human health. After entering the human body, Cd accumulates in the kidneys, harms the bones and respiratory system, and causes a series of diseases. The World Health Organization has classified Cd as a first-class carcinogen. Therefore, improving rice resistance to heavy metal stress through genetic engineering technology to reduce the accumulation of heavy metals in rice is of great significance to improving the ecological environment of cultivated fields and achieving sustainable development of agricultural ecology and food production.

[0003] Plant cell walls are mainly composed of a complex of cellulose, hemicellulose, lignin, pectin and a small amount of protein. As the first barrier for heavy metal ions to enter plant cells, it not only supports and protects cells, but is also the site of functional signals that respond to heavy metal stress. When plants are subjected to adverse stress, they sense and initiate downstream stress responses in the first place, and play an important role in the accumulation and transport of Cd. The cell wall can isolate heavy metal ions outside the cell, actively participate in the plant's response to heavy metal stress, and thus reduce the number of heavy metal ions entering the protoplast. Cellulose synthase-like F (CSLF) is an important enzyme involved in the synthesis of cellulose, thereby affecting the composition of plant cell walls.

[0004] CSLF6 It is an important gene in plants and belongs to the plant cell wall synthase family. Cellulose synthase CESA and cellulose synthase-like CSLF are key enzymes in the cellulose synthesis process. CSLF6 The gene encodes an enzyme responsible for the synthesis of (1,3;1,4)-β-glucan, a mixed-chain glucan (MLG) found in high concentrations in cereals such as wheat, barley, and rice. This polysaccharide is considered a valuable dietary fiber due to its high solubility in water. CSLF6 Gene mutants help us understand the role of the gene in plant growth and development as well as stress response. Summary of the invention

[0005] In order to analyze the molecular physiological mechanism of rice response to cadmium stress and provide a basis for breeding rice varieties with high cadmium tolerance, the present invention provides CSLF6 The application of genes and / or encoded proteins in rice breeding regulation and the exploration of the regulatory mechanism of rice response to cadmium stress are of great significance for the breeding of new rice varieties.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: Rice CSLF6 Application of genes and / or encoded proteins in rice breeding regulation, wherein the rice breeding regulation is overexpression in rice CSLF6 Gene, improves rice cadmium stress tolerance and increases cadmium accumulation; CSLF6 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No: 1; the amino acid sequence of the encoded protein is shown in SEQ ID No: 2.

[0007] Furthermore, the present invention also discloses a rice CSLF6 Application of gene transformation vector in rice breeding regulation, wherein the rice breeding regulation is to overexpress in rice CSLF6 Gene, improves rice cadmium stress tolerance and increases cadmium accumulation.

[0008] Furthermore, the present invention also discloses the use of a host cell containing the transformation vector in rice breeding regulation, wherein the rice breeding regulation is to overexpress CSLF6 Gene, improves rice cadmium stress tolerance and increases cadmium accumulation.

[0009] Preferably, rice overexpresses CSLF6 Gene regulation OsNramp1 , OsNramp5 , OsLCD The expression of increases the absorption of cadmium in rice.

[0010] Preferably, rice overexpresses CSLF6 Gene regulation OsHMA2、OsHMA3 , OsLCT1 The expression of cadmium affects the transport of cadmium in rice.

[0011] Furthermore, the present invention also discloses a breeding method for enhancing rice tolerance to cadmium stress, the method comprising over-expressing rice CSLF6 Gene steps, the rice CSLF6 The nucleotide sequence of the gene is shown in SEQ ID No: 1.

[0012] Preferably, the method comprises the following steps: 1) Extract DNA from rice and amplify the rice CSLF6 The coding sequence of a gene; 2) The PCR-amplified CSLF6 gene fragment was cut with restriction endonucleases and inserted into a plant expression vector containing a strong promoter to drive efficient gene expression; 3) Select rice varieties suitable for gene transformation, transfer the constructed vector into Agrobacterium, and culture the Agrobacterium strain under suitable conditions; 4) Surface disinfection of rice seeds, followed by culture in an induction medium to form embryonic callus; mixing the embryonic callus with Agrobacterium for co-cultivation; 5) Screening the transformed callus with medium containing antibiotics to remove untransformed cells; 6) The selected callus tissue is transferred to the regeneration medium to induce the formation of complete rice plants.

[0013] Furthermore, the present invention also discloses an overexpression CSLF6 A method for identifying a rice plant containing a gene, the method comprising the following steps: 1) Extraction of rice RNA; 2) Reverse transcription of total RNA; 3) Fluorescence quantitative PCR detection, the primer sequences are: F: 5'-ACCAGCACAACCAAACGAACA-3' R: 5'-GCGGACACGGTCAACGACT-3'.

[0014] Furthermore, the present invention also discloses a method for identifying overexpression CSLF6 The fluorescent quantitative PCR detection primers for rice plants expressing the gene, the nucleotide sequences of the primers are: F: 5'-ACCAGCACAACCAAACGAACA-3' R: 5'-GCGGACACGGTCAACGACT-3'.

[0015] The present invention has found CSLF6 Application of genes and / or encoded proteins in rice breeding regulation. CSLF6 Gene overexpression can be used to cultivate rice with improved tolerance to cadmium stress and increased cadmium accumulation, which helps the rice grow to a higher level. The rice can be used to repair soil cadmium pollution and has excellent application value. CSLF6 Mutants of the gene can effectively reduce the accumulation of cadmium in rice grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for CSLF6 Results of temporal expression patterns of genes in tissues.

[0017] Figure 2 Figure 2 shows the growth of rice after Cd stress for different time periods. CSLF6 Expression characteristics of (A: root, B: leaf).

[0018] Figure 3 for CSLF6 Quantitative results of overexpressing rice plants (A) and CSLF6 Analysis of mutation sites in mutant-positive plants (B).

[0019] Figure 4 for CSLF6 Phenotypic diagram of overexpression and wild-type rice after Cd treatment and the length of rice aerial part and root.

[0020] Figure 5 for CSLF6 Phenotypes of mutant and wild-type rice after Cd treatment and the lengths of the aboveground and roots of rice.

[0021] Figure 6 for CSLF6 Overexpression, CSLF6 Phenotypes of seedlings and internodes and plant height of mutant and wild-type rice at maturity.

[0022] Figure 7 For 4-week-old rice, 10 μM CdCl 2 Distribution of Cd content in different tissues after 4 weeks of treatment (A: roots, B: aerial parts, C: leaves, D: stems).

[0023] Figure 8 For 4 weeks of culture CSLF6 Overexpression, CSLF6 Mutants and wild-type rice were treated with 10 μM CdCl 2 Subcellular distribution of Cd in roots and shoots after 4 weeks of treatment.

[0024] Fig. 9 is the Cd content in rice grains.

[0025] Fig.10 For WT, CSLF6 Overexpression and mutant plants were grown in 10 μM CdCl 2 Cellulose content in the second internode and flag leaf after 4 weeks of treatment, A is the second internode, B is the flag leaf.

[0026] Fig.11 for OsNramp5, OsNramp1, OsHMA2, OsHMA3, OsLCT1, OsLCD In 10 μM CdCl 2 WT after four weeks of treatment, CSLF6 Expression analysis in roots and leaves of overexpression and mutant rice (left column for roots, right column for leaves).

[0027] Fig.12 Agarose gel electrophoresis. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 Breeding method of rice with overexpression of CSLF6 gene 1. Materials and Reagents Preparation Rice variety: Zhonghua 11.

[0030] CSLF6 gene: The nucleotide sequence of the CDS region is shown in SEQ ID No: 1.

[0031] Gene cloning reagents: including PCR amplification reagents, restriction endonucleases, ligases, etc.

[0032] Transformation vector: Select the expression vector pCAMBIA1301 suitable for plant transformation, which carries the resistance selection marker kanamycin resistance gene.

[0033] Agrobacterium strains: Commonly used Agrobacterium strains, such as EHA105, are used to mediate rice transformation.

[0034] Instruments and equipment: PCR instrument, fluorescence quantitative PCR instrument, clean bench, autoclave, incubator, microscope, etc.

[0035] 2. Cloning of CSLF6 gene and vector construction 1) Gene extraction and amplification: Genomic DNA was extracted from rice leaf tissue. CSLF6 gene was amplified by PCR using specific primers. The amplification conditions were as follows: Initial denaturation: 95°C for 5 min; Cycle: 95°C for 30 seconds, annealing temperature Tm°C (depending on primer) for 30 seconds, extension at 72°C for 1 minute, 35 cycles; Final extension: 72°C for 10 min.

[0036] Check the correctness of the PCR product and confirm the size of the target fragment by agarose gel electrophoresis, such as Fig.12 shown.

[0037] 2) Vector construction: The plant expression vector pCAMBIA1301 containing the strong promoter of CaMV 35S was selected to be used for the expression of CSLF6 gene. The PCR product and the vector EcoRI and XbaI were cut with restriction endonucleases to recycle the product after the enzyme cutting. The CSLF6 gene fragment after cutting was connected to the vector using T4 DNA ligase, and the ligation reaction was incubated at 16°C for 1 hour. The ligation product was transformed into Escherichia coli competent cells, positive clones were screened by kanamycin, and the correct insertion and sequence integrity of the CSLF6 gene were verified by sequencing.

[0038] 3. Agrobacterium-mediated rice transformation 1) Induction of rice callus: The sterilized rice seeds are cultured in a callus induction medium (such as 2,4-D medium) to form embryonic callus.

[0039] 2) Co-culture: The constructed vector was transformed into Agrobacterium EHA105 strain.

[0040] The rice callus was mixed with Agrobacterium and co-cultured in a co-culture solution (acetosyringone was added to the liquid induction medium) for 2-3 days to promote the infection of Agrobacterium.

[0041] 3) Screening and regeneration: The co-cultured callus tissue is transferred to a culture medium containing a selection antibiotic (such as kanamycin), and the untransformed tissue is removed and cultured for 2-4 weeks. The selected callus tissue is transferred to a regeneration medium, and the callus tissue is allowed to differentiate and sprout to form plantlets. The plantlets are transferred to a root induction medium to induce root growth, and finally a complete regenerated plant is obtained.

[0042] Example 2 CSLF6 Overexpression in rice and identification of its cadmium tolerance under cadmium stress 1 Materials and methods 1.1 Experimental Materials Rice materials ( Oryza sativa L. subsp. japonica) CSLF6 Overexpression of rice, CSLF6 Mutant and wild-type rice Zhonghua11 (WT).

[0043] 1.2 Rice cultivation and Cd treatment Select plump rice seeds in 0.1% diluted HNO 3 After 12 hours of dormancy breaking, disinfect with 0.5% NaClO solution for 20-25 minutes, ddH 2After rinsing with O, the cells were cultured in the dark at 30°C and placed in an artificial climate chamber. The nutrient solution from the International Rice Research Institute was used and the pH was adjusted to 5.2-5.5. After 4 weeks of culture, 10 μM CdCl 2 After 4 weeks of treatment, the rice phenotype was observed, the root length and aboveground length were measured, and samples were taken for subsequent experiments.

[0044] 1.2 Gene expression detection 2-week-old rice was treated with 50 μM CdCl 2 After treatment, roots and leaves were taken for CSLF6 Expression detection. RNA was extracted from the roots and aerial parts of rice using RNA extraction kit (CW0581M). Total RNA was reverse transcribed using RNA reverse transcription kit PrimeScriptTM RT reagent Kit with gDNA Eraser. Fluorescence quantitative PCR was performed using SYBR Premix exTaqTM (Takara, Japan) kit, and β-actin was used as an internal reference. CSLF6 The primer sequences were: F: 5'-ACCAGCACAACCAAACGAACA-3' R: 5'-GCGGACACGGTCAACGACT-3'. The data were obtained using 2 -∆∆Ct Method analysis.

[0045] 1.3 CSLF6 Analysis of temporal expression patterns of genes in tissues Roots, stems, leaves, flag leaves, nodes and panicles (only roots, stems and leaves were collected at seedling and tillering stages) of wild-type rice ZH11 at different developmental stages (seedling, tillering, heading, filling and maturity) were collected and analyzed by qRT-PCR. CSLF6 Analysis of tissue temporal expression patterns of genes.

[0046] 1.4 Subcellular fractionation According to the method of Lozano-Rodríguez, the roots and aerial parts of rice plants were separated into three parts: cell wall, organelles, and cell sap + membrane system. 3 g of roots and 10 g of aerial parts were taken in a mortar and added with 50 mL of extraction buffer, and the pH was adjusted to 7.5 with KOH and then homogenized. After obtaining the homogenate, the various cell components in the roots and aerial parts were separated. The residue and precipitate components were evaporated and 8 mL of HNO 3 After standing for 2 h, use a microwave digestion instrument to digest, remove the acid to 1 mL and dilute to 50 mL with ultrapure water.

[0047] 1.5 Cd content determination Four-week-old rice plants were treated with 10 μM CdCl 2 Samples were taken 4 weeks after treatment, and ddH2 O Wash the rice roots, and sterilize the roots, stems, leaves and aboveground tissues of the rice plants at 105℃ for 2 h. Weigh 0.1g of roots, 0.3g of stems, leaves and aboveground tissues and place them in a digestion tube. Add 8 mL of nitric acid and let stand for 2 h. Then place them symmetrically in a microwave digestion instrument for digestion. 2 O was diluted to 50 mL and the sample concentration was determined by flame continuous method using atomic absorption spectrometer. Excel and GraphPad Prism software were used for data processing, and the data were expressed as mean ± SD.

[0048] 1.6 Determination of Cd content in rice grains All rice samples were rinsed with tap water, then washed with distilled water, dried in an oven, hulled with a huller, and brown rice was obtained. It was ground into powder with a grinder and then bagged and stored in a dry place for later use. Weigh 0.3 g of the previously treated rice sample into a digestion tube, add 8 mL of nitric acid and let stand for 2 h, then symmetrically put into a microwave digestion instrument for digestion, and ddH 2 O was diluted to 50 mL and the sample concentration was determined by flame continuous method using atomic absorption spectrometer. Excel and GraphPad Prism software were used for data processing, and the data were expressed as mean ± SD.

[0049] 1.7 Determination of cellulose and hemicellulose content Take the flag leaf and the second internode of rice in the tillering stage, use the solarbio kit, and soak 0.3 g of the dried sample in 80% ethanol, 90°C water bath for 20 minutes, and then centrifuge at 6000g and 25°C for 10 minutes. The precipitate was washed twice with acetone and 80% ethanol, then soaked in chloroform-methanol (1:1, v:v) for 15 hours, centrifuged at 6000g and 25°C for 10 minutes, and the precipitate was washed twice in distilled water, 6000g, 25°C, centrifuged for 10 minutes, and dried at 80°C to obtain cell wall material. Take 5 mg of cell wall material, add 0.5 mL of distilled water to fully homogenize, transfer the homogenate to an EP tube, and dilute to 0.5 mL with distilled water. Place the homogenate after distillation in an ice-water mixture, slowly add 0.75 mL of concentrated sulfuric acid, slowly mix, and stand in an ice-water bath for 30 minutes. The mixture was centrifuged at 8000 g and 4 °C for 10 min. The supernatant was taken and diluted 20 times with distilled water. The wavelength was adjusted to 620 nm. The OD value was measured under an ultraviolet spectrophotometer to calculate the cellulose content.

[0050] 2. Experimental results and analysis 2.1 CSLF6 Bioinformatics analysis and tissue temporal expression analysis Through the search of basic information of genes and proteins, it is known that CSLF6 has 952 amino acids, which are mainly located outside the membrane. There are 9 transmembrane domains, 1 coil region and 2 low-complexity regions, and its transmembrane structure is located at the N-terminus. There are 72 sites where CSLF6 protein may be phosphorylated, and there is no signal peptide.

[0051] Roots, stems, leaves, flag leaves, nodes and panicles (only roots, stems and leaves were collected at seedling and tillering stages) of wild-type rice ZH11 at different developmental stages (seedling, tillering, heading, filling and maturity) were collected and analyzed by qRT-PCR. CSLF6 Analysis of the temporal expression pattern of genes in tissues. Figure 1 It can be seen that CSLF6 The expression patterns were similar in the seedling and tillering stages, mainly expressed in roots and stems. The expression patterns were similar in the heading, filling and maturity stages, mainly expressed at higher levels in roots, nodes and ears. CSLF6 The expression in roots was significantly higher than that in other organs at other stages.

[0052] 2.2 Cd treatment CSLF6 Expression analysis 2-week-old rice was treated with 50 μM CdCl 2 After treatment, real-time fluorescence quantitative PCR was used to detect CSLF6 Expression characteristics under different Cd stress periods. Figure 2 As shown in Figure 2, under Cd treatment, the root CSLF6 The expression level of α began to rise continuously, reaching a peak at 3 h, and then continued to decline until 12 h, reaching the lowest value, and then continued to rise. CSLF6 The expression level of α continued to decrease, and increased after 24 h of treatment. CSLF6 Related to Cd stress response of rice, especially roots.

[0053] 2.3 CSLF6 Identification of overexpression and mutant plants The overexpression of different strains was analyzed by qRT-PCR. CSLF6 The plants were tested and the results were as follows Figure 3 As shown in A. The experimental results showed that the expression levels of different strains were increased to varying degrees compared with the wild type WT, proving that the overexpression was successful. Three strains with relatively high expression levels, OE-1, OE-4 and OE-5, were selected for subsequent phenotypic analysis.

[0054] Sequences were selected about 150 bp on both sides of the target sequence as identification CSLF6 Primers for mutant plants, extraction CSLF6PCR and sequencing were performed on the mutant rice leaf DNA. DNAMAN and chromas software were used to analyze the mutation types of transgenic rice plants. Two homozygous mutant plants were selected for subsequent research, namely: CSLF6J-4 44 bases are missing; CSLF6 -8 1 base inserted.

[0055] 2.4 Rice under Cd treatment CSLF6 Phenotypic and biomass analysis of overexpression and mutant plants 2.4.1 4-week-old rice seedlings Wild-type rice (WT) cultured for 4 weeks, CSLF6 Overexpression and mutant rice were treated with 10 μM CdCl 2 After 4 weeks of treatment, the rice phenotypes were observed and photographed, and biomass indicators such as rice plant height and root length were measured and recorded. Figure 4 As shown, compared with WT, the overexpression rice plants under Cd treatment grew better and more vigorously, and the plant height and root length were significantly higher than those of WT. Figure 5 As shown in the figure, compared with WT, the mutant rice plants grew poorly, had a deeper yellowing degree, and had significantly lower plant height and root length than WT. These results indicate that CSLF6 Overexpression can improve rice tolerance to Cd to a certain extent. CSLF6 The mutation reduced the tolerance of rice to Cd.

[0056] 2.4.2 Rice cultivation at maturity After 4 months of cultivation, rice entered the mature stage. CSLF6 The phenotypic differences between overexpressing plants and mutant plants, such as Figure 6 As shown, compared with WT plants, the overexpression plants grew significantly better, with longer stem internodes and higher cellulose content; while the mutant plants grew poorly and were significantly shorter in height than WT plants.

[0057] 2.5 Tissue and subcellular distribution of Cd Rice grown for 4 weeks was treated with 10 μM CdCl 2 The Cd content was measured after 4 weeks of treatment. Figure 7As shown, the Cd content in the root of rice is much higher than that in the aboveground part, and the Cd content in the overexpression plant is significantly higher than that in the other two groups. From this we can conclude that the accumulation of Cd in various parts of rice is ranked as root>stem>bud>leaf, and the root is the most direct and main organ stressed by heavy metals. Morphologically, the toxic symptoms of Cd on plants include inhibition of root elongation growth and enlargement of the root tip, which are caused by the interaction between Cd and the root tip, leading to the inhibition of root development. The Cd content in the roots and aboveground parts of the overexpression plants is significantly higher than that of the WT plants, and the WT plants are significantly higher than the mutant plants. Explanation CSLF6 Overexpression increased the absorption of Cd by rice and its transport to the aboveground parts. CSLF6 Mutations are the opposite.

[0058] The subcellular components of rice were separated and the subcellular distribution of Cd was analyzed. Figure 8 It can be seen that the Cd content in the root is mainly concentrated in the cell wall, and in the aboveground part, it is concentrated in the cell wall, cell sap and membrane system. Heavy metal transporters involved in the transport of various heavy metals are not only present on the cell membrane, but also widely distributed on the plasma membrane of organelles such as chloroplasts, Golgi bodies, vacuoles, and endothelial cells. This shows that Cd is mainly accumulated in the cell wall and vacuole. The Cd content in the overexpression plant was significantly higher than that in the WT, and the WT plant was significantly higher than the mutant, and the Cd accumulated in the cell wall was higher.

[0059] Rice was cultured in 1 mg kg-1 cadmium-contaminated soil, and the Cd content in the grains was determined, such as Fig. 9 As shown in the figure, the Cd content in the overexpression plants was significantly higher than that in the WT plants, and the WT plants were significantly higher than that in the mutants, indicating that CSLF6 Overexpression of will lead to Cd enrichment in rice grains, while mutant plants can reduce Cd accumulation in rice grains.

[0060] 2.6 Determination of cellulose and hemicellulose content After the rice plants were treated with 10 μM CdCl2 for 4 weeks, the flag leaves and second internodes of WT, CSLF6 overexpression and mutant plants at the tillering stage were taken to determine the cellulose and hemicellulose contents, as shown in Fig.10 As shown in the figure, in the flag leaf of rice, compared with the wild type, there was no significant difference in the hemicellulose content of the overexpression and mutant rice, while the cellulose content of the overexpression rice was significantly increased, and the cellulose content of the mutant rice was significantly reduced; in the second internode of rice, compared with the wild type, the cellulose and hemicellulose contents of the overexpression rice were significantly increased, while the cellulose and hemicellulose contents of the mutant rice were significantly reduced. This indicates that in the second internode and flag leaf of rice, the CSLF6 gene may enhance the resistance of rice to heavy metals by regulating the synthesis of cellulose.

[0061] 2.7 CSLF6Expression analysis of genes related to Cd accumulation and transport: OsNramp5 It is mainly expressed in the epidermis and cortex of the roots and is closely related to the absorption and transport of Fe and Cd. OsNramp5 It helps rice roots absorb Cd from the soil. Fig.11 As can be seen from A, after Cd stress, the roots of overexpressing plants OsNramp5 The expression level of was significantly higher than that of wild-type plants, while there was no significant difference between wild-type plants and mutant plants. OsNramp1 As OsNramp5 Homologous genes of Fig.11 B and OsNramp5 The expression pattern of OsNramp1 The expression level of α was significantly higher than that of wild-type plants, while there was no significant difference between wild-type plants and mutant plants. OsNramp5 and OsNramp1 exist CSLF6 The overexpression of β-actin in the roots of rice plants increased the expression of β-actin, thereby promoting the absorption of Cd by rice roots, which explains the CSLF6 The phenomenon of high Cd accumulation in the roots of overexpressing plants.

[0062] OsHMA2 The loss-of-function mutants with mutations in the C-terminal region can restrict the transport of Cd from roots to shoots. OsHMA3 It is a cytoplasm-localized transporter that regulates the transport of Cd from roots to stems in rice; Fig.11 C. 11D is OsHMA2 and OsHMA3 Expression characteristics in rice roots. As can be seen from the figure, OsHMA2 and OsHMA3 The expression patterns of the overexpression genes were similar to those of the mutant genes. After Cd stress, the expression levels in the roots of the overexpression plants were significantly higher than those in the wild-type plants, and the wild-type plants were significantly higher than those in the mutant plants. OsHMA3 exist CSLF6 The high expression in the overexpression plants indicates CSLF6 Overexpression may mediate more Cd storage in root vacuoles, while OsHMA2 exist CSLF6 The high expression in the roots of overexpression plants suggests that it may promote the transport of Cd to the aboveground parts, explaining CSLF6 The phenomenon of high accumulation of Cd in the aboveground parts of overexpressing plants.

[0063] OsLCT1 It can affect the pathway of phloem-dependent Cd transport. Its deletion mutant greatly reduces the accumulation of Cd in rice grains by reducing phloem-mediated Cd transport. Fig.11 E overexpression plants OsLCT1The expression level in leaves was significantly higher than that in wild-type plants, and that in wild-type plants was significantly higher than that in mutant rice plants. OsLCT1 The up-regulation of expression may affect the transport of Cd in the phloem, explaining CSLF6 The phenomenon of high accumulation of Cd in leaves and grains of overexpression mutants. OsLCD The gene was mainly expressed in the root vascular tissue and phloem cells of leaves and was involved in the low accumulation of Cd in rice. Fig.11 F shows that after Cd treatment, the root of the overexpressing plants OsLCD The expression level of the mutant plant was significantly higher than that of the wild-type plant, and the expression level of the mutant plant was significantly lower than that of the wild-type plant. It may be that the expression of this gene increased Cd absorption, explaining CSLF6 The phenomenon of high accumulation of Cd in the roots of overexpressing plants.

[0064] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. Rice CSLF6 Application of gene in rice breeding regulation, wherein the rice breeding regulation is overexpression in rice CSLF6 Gene, improves rice cadmium stress tolerance and increases cadmium accumulation; CSLF6 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID No:

1.

2. The rice according to claim 1 CSLF6 The application of the protein encoded by the gene in rice breeding regulation, wherein the rice breeding regulation is used to improve the cadmium stress tolerance of rice and increase cadmium accumulation. The amino acid sequence of the protein is shown in SEQ ID No:

2.

3. The rice according to claim 1 CSLF6 Application of gene transformation vector in rice breeding regulation, wherein the rice breeding regulation is to overexpress in rice CSLF6 Gene, improves rice cadmium stress tolerance and increases cadmium accumulation.

4. Use of a host cell comprising the transformation vector according to claim 3 in rice breeding regulation, wherein the rice breeding regulation is to overexpress the CSLF6 gene in rice, improve the cadmium stress tolerance of rice, and increase cadmium accumulation.

5. The use according to any one of claims 1 to 4, characterized in that: Rice overexpression CSLF6 Gene regulation OsNramp1 , OsNramp5 , OsLCD The expression of increases the absorption of cadmium in rice.

6. The use according to any one of claims 1 to 4, characterized in that: Rice overexpression CSLF6 Gene regulation OsHMA2、OsHMA3 , OsLCT1 The expression of cadmium affects the transport of cadmium in rice.

7. A breeding method for enhancing rice tolerance to cadmium stress, characterized in that: The method comprises overexpressing a rice CSLF6 Gene steps, the rice CSLF6 The nucleotide sequence of the gene is shown in SEQ ID No:

1.

8. The method according to claim 7, characterized in that The method comprises the following steps: 1) Extract DNA from rice and amplify the rice CSLF6 The coding sequence of a gene; 2) PCR amplified rice CSLF6 The gene fragment is cut by restriction endonucleases and inserted into a plant expression vector, which contains a strong promoter to drive efficient expression of the gene; 3) Select rice varieties suitable for gene transformation, transfer the constructed vector into Agrobacterium, and culture the Agrobacterium strain under suitable conditions; 4) Surface sterilization of rice seeds, followed by culture in induction medium to form embryonic callus; The embryonic callus is mixed with Agrobacterium for co-cultivation; 5) Screening the transformed callus with medium containing antibiotics to remove untransformed cells; 6) The selected callus tissue is transferred to the regeneration medium to induce the formation of complete rice plants.

9. An overexpression CSLF6 A method for identifying a rice plant having a gene, characterized in that The method comprises the following steps: 1) Extraction of rice RNA; 2) Reverse transcription of total RNA; 3) Fluorescence quantitative PCR detection, the nucleotide sequences of PCR primers are: F: 5'-ACCAGCACAACCAAACGAACA-3' R: 5'-GCGGACACGGTCAACGACT-3'.

10. Identification of Overexpression CSLF6 The fluorescent quantitative PCR detection primers for rice plants expressing the gene, the nucleotide sequences of the primers are: F: 5'-ACCAGCACAACCAAACGAACA-3' R: 5'-GCGGACACGGTCAACGACT-3'.

Images