Application of MtCIPK12 gene in regulating plant tolerance to low iron stress and riboflavin synthesis

Abstract

The invention discloses an application of a MtCIPK12 gene in regulating plant resistance to low-iron stress and riboflavin synthesis, comprising: step one, obtaining a plant mutant by reducing the expression of a MtCIPK12 gene and a MtCIPK12 protein of a wild-type plant; the amino acid sequences of the MtCIPK12 gene and the MtCIPK12 protein are sequence 1 and sequence 2; step two, observing the expression and function of key enzyme genes in the riboflavin biosynthesis process of the plant mutant under iron deficiency; step three, increasing the accumulation of riboflavin in the plant mutant, and enhancing the stress resistance to low-iron stress; the MtCIPK12 gene mutant material has a stronger low-iron resistance, that is, exhibits lower leaf yellowing, higher leaf and root biomass, higher iron content, and higher riboflavin accumulation, proving that the MtCIPK12 gene mutation can improve the plant's resistance to low-iron stress and improve the plant's riboflavin content.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a MtCIPK12 gene in regulating plant low-iron stress tolerance and riboflavin synthesis. Background Art

[0002] Iron (Fe) is an essential mineral for humans. Eating iron-rich plants or animal foods is the main way for humans to obtain iron. On the other hand, iron is also an essential trace nutrient for plant growth and development, playing a key role in photosynthesis, respiration and other physiological processes (Hsieh & WMters, 2016). As the fourth most abundant element in the earth's crust, iron is mainly found in the soil, especially in neutral and alkaline soils, in the form of insoluble Fe 3+ In various forms, the lack of effective iron has become one of the main factors limiting plant growth (Wu H&Ling HQ, 2019).

[0003] After a long period of evolution, plants have gradually formed different iron absorption mechanisms in response to low iron stress, mainly including strategy I adopted by dicotyledons and non-gramineous monocotyledons and strategy II adopted by gramineous monocotyledons (Romheld & Marschner, 1986). In strategy I plants, iron acquisition is based on a reduction mechanism, which is characterized by the conversion of Fe to Fe by iron reductase in root cells. 3+ Reduction to Fe 2+ , then through Fe 2+ Transporter protein absorbs Fe 2+ (Santi & Schmidt, 2009). Strategy I plants can also respond to iron stress by accumulating and secreting compounds such as phenols, organic acids, flavonoids and riboflavin to promote iron acquisition (Jin et al., 2007; Cesco et al., 2010). Strategy II plants can secrete plant siderophores (PS) in the rhizosphere, PS-Fe 3+ The complex is transferred into root epidermal cells via specific transporters, ultimately promoting the acquisition of effective iron (Zhang et al., 2019).

[0004] Riboflavin, also known as vitamin B2, is a type of B vitamin. It is a component of the cofactor of flavin enzymes in the body (flavin enzymes play a role in transferring hydrogen in biological oxidation and reduction). It is a type of vitamin necessary for humans and animals. Riboflavin deficiency will affect the body's biological oxidation process and cause metabolic disorders. In plants, riboflavin mainly participates in redox reactions as cofactors in the form of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), thereby playing a role in a variety of physiological metabolic processes. Studies have shown that plants can synthesize riboflavin, but the content of riboflavin in different plants and different tissues varies greatly. At the same time, riboflavin can combine with a variety of polysaccharides in plants to form glycosylated riboflavin. There may be a mechanism for the redistribution of riboflavin in plants and the regulation of environmental stress responses (Song Tao, Nanjing Agricultural University, 2007). Its lesions are mostly manifested as inflammation in the mouth, eyes and external genitalia, so this product can be used to prevent and treat the above diseases. The storage of vitamin B2 in the body is very limited, so it must be provided by diet every day.

[0005] Alfalfa is known as the king of forage grasses and has the excellent characteristic of high protein content. In my country, alfalfa is mainly grown in Inner Mongolia, Xinjiang, Gansu, Heilongjiang and other regions. The soil in these regions is mostly neutral or alkaline, and the iron available to plants is relatively low, which seriously affects the yield and quality of alfalfa. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of the MtCIPK12 gene for improving the low iron tolerance of plants by promoting riboflavin synthesis in regulating the low iron tolerance of plants.

[0007] In order to solve the above technical problems, the technical solution of the present invention is: application of MtCIPK12 gene in regulating plant tolerance to low iron stress, comprising the following steps:

[0008] Step 1, preparing a mutant of a wild-type plant, and obtaining a plant mutant by reducing the expression of the MtCIPK12 gene and the MtCIPK12 protein of the wild-type plant;

[0009] The amino acid sequences of the MtCIPK12 gene and the MtCIPK12 protein are sequence 1 and sequence 2 in the sequence list;

[0010] Step 2: Observe the expression and function of key enzyme genes in the riboflavin biosynthesis process of plant mutants under iron deficiency. The specific enzymes are as follows: GTP cyclohydrolase II (GTPcII), 2,4-dioxotetrahydropyrimidine synthase (LS), and riboflavin synthase (RS);

[0011] Step three: the accumulation of riboflavin in the plant mutant increases, and the plant mutant has enhanced resistance to low iron stress.

[0012] As a preferred technical solution, in step one, the TNT1 sequence is inserted into the genome of a wild-type plant to obtain a plant mutant, and a homozygous cipk12 mutant is obtained by triple PCR screening based on the MtCIPK12 gene sequence and the TNT1 sequence.

[0013] As a preferred technical solution, the wild-type plant is a dicotyledonous plant.

[0014] As a preferred technical solution, the low iron means that the iron ion concentration contained in the culture matrix is ​​not higher than 100 μM.

[0015] Due to the adoption of the above technical scheme, the application of the MtCIPK12 gene in regulating the low iron stress tolerance of plants includes the following steps: step 1, making mutants of wild-type plants, and obtaining plant mutants by reducing the expression of the MtCIPK12 gene and the MtCIPK12 protein of the wild-type plants; the amino acid sequences of the MtCIPK12 gene and the MtCIPK12 protein are sequence 1 and sequence 2 in the sequence table; step 2, observing the expression and function of key enzyme genes in the riboflavin biosynthesis process of the plant mutants under iron deficiency, and the specific enzymes are as follows: GTP cyclohydrolase II (GTPcII) and 2,4-dioxotetrahydropyrimidine synthase (LS) and riboflavin synthase (RS ); Step three, the accumulation of riboflavin in the plant mutant increases, and the plant mutant has enhanced resistance to low iron stress; by comparing the MtCIPK12 gene mutant materials cipk12-1 (NF11558) and cipk12-2 (NF7022) with the wild type alfalfa truncatula R108, it was found that the MtCIPK12 gene mutant materials NF11558 and NF7022 materials have stronger low iron resistance, that is, they show lower leaf yellowing, higher leaf and root biomass, iron content, and higher riboflavin accumulation, which proves that the MtCIPK12 gene mutation can improve the plant's resistance to low iron stress and increase the plant's riboflavin content.

[0016] The ability of the above-mentioned gene mutant plant to resist low iron stress is greater than that of the wild-type plant, which is reflected in at least one of the following 1)-4):

[0017] 1) Under low iron stress, the leaf and root biomass of the gene mutant plant are higher than those of the wild-type plant;

[0018] 2) Under low iron stress, the chlorophyll content of the gene mutant plant is higher than that of the wild-type plant;

[0019] 3) Under low iron stress, the iron content of the gene mutant plant is higher than that of the wild-type plant;

[0020] 4) Under low iron stress, the root system trivalent iron reducing ability of the gene mutant plant is higher than that of the wild-type plant.

[0021] Another technical problem to be solved by the present invention is to provide an application of a MtCIPK12 gene capable of promoting riboflavin synthesis in regulating plant riboflavin synthesis.

[0022] In order to solve the above technical problems, the technical solution of the present invention is: application of MtCIPK12 gene in regulating plant riboflavin synthesis, comprising the following steps:

[0023] Step 1, preparing a mutant of a wild-type plant, and obtaining a plant mutant by reducing the expression of the MtCIPK12 gene and the MtCIPK12 protein of the wild-type plant;

[0024] The amino acid sequences of the MtCIPK12 gene and the MtCIPK12 protein are sequence 1 and sequence 2 in the sequence list;

[0025] Step 2: Observe the expression and function of key enzyme genes in the riboflavin biosynthesis process of plant mutants. The specific enzymes are as follows: GTP cyclohydrolase II (GTPcII), 2,4-dioxotetrahydropyrimidine synthase (LS), and riboflavin synthase (RS);

[0026] Step three, the accumulation of riboflavin in the plant mutant increases, and reducing the expression of the MtCIPK12 gene and the MtCIPK12 protein can increase riboflavin nuclear synthesis.

[0027] As a preferred technical solution, in step one, the TNT1 sequence is inserted into the genome of a wild-type plant to obtain a plant mutant, and a homozygous cipk12 mutant is obtained by triple PCR screening based on the MtCIPK12 gene sequence and the TNT1 sequence.

[0028] As a preferred technical solution, the wild-type plant is a dicotyledonous plant.

[0029] Due to the adoption of the above technical scheme, the application of MtCIPK12 gene in regulating riboflavin synthesis in plants, through comparative study of MtCIPK12 gene mutant materials NF11558 and NF7022 with wild-type alfalfa truncatula R108, found that MtCIPK12 gene mutant materials NF11558 and NF7022 had higher riboflavin accumulation, proving that the mutation of MtCIPK12 gene enhanced the accumulation of riboflavin in alfalfa truncatula, that is, inhibiting the expression of MtCIPK12 gene can promote riboflavin synthesis in plants.

[0030] The application of inhibiting the expression of MtCIPK12 gene and accumulating more riboflavin in reducing the ability of plants to resist low iron stress is also within the scope of protection of the present invention.

[0031] Experiments of the present invention prove that by comparing MtCIPK12 gene mutant materials NF11558 and NF7022 with their wild type alfalfa truncatula R108, the present invention finds that the MtCIPK12 gene mutant materials NF11558 and NF7022 have stronger low-iron resistance, that is, they show lower leaf yellowing, higher leaf and root biomass, iron content, and higher riboflavin accumulation, which proves that the MtCIPK12 gene mutation can improve the plant's resistance to low-iron stress and increase the plant's riboflavin content.

[0032] The beneficial effect of the present invention is that: it is found that the mutation of the MtCIPK12 gene enhances the accumulation of iron and improves the photosynthetic efficiency. At the same time, the mutation of the MtCIPK12 gene enhances the accumulation of riboflavin in Medicago truncatula, promotes the iron reduction activity that depends on the electron transport of riboflavin under iron deficiency conditions, thereby promoting iron absorption. The present invention provides a regulatory mechanism of CIPK12 protein kinase in riboflavin biosynthesis and plant iron deficiency response, and provides new technical support for improving agronomic traits such as tolerance, yield and quality of crops under iron deficiency environments through molecular breeding methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings are intended only to illustrate and explain the present invention, and do not limit the scope of the present invention.

[0034] Figure 1 is a comparison chart of phenotypic changes of the mtcipk12 mutant and the wild type under iron deficiency conditions in the embodiment of the present invention;

[0035] Figure 2 This is a comparison chart of the physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions in the embodiment of the present invention - single plant leaf dry weight;

[0036] Figure 3 This is a comparison chart of the physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions in the embodiment of the present invention - single plant root dry weight;

[0037] Figure 4 This is a comparison chart of physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions - chlorophyll content in an embodiment of the present invention;

[0038] Figure 5 It is a comparison chart of physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions-photosynthetic rate in the embodiment of the present invention;

[0039] Figure 6This is a comparison chart of the physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions in the embodiment of the present invention - leaf iron content;

[0040] Figure 7 This is a comparison chart of the physiological changes of the mtcipk12 mutant and the wild type under iron deficiency conditions in the embodiment of the present invention - root iron content;

[0041] Figure 8 This is an expression analysis of Medicago truncatula and riboflavin synthesis-related genes under iron deficiency conditions in the embodiment of the present invention - a comparison chart of MtGTPcII expression levels;

[0042] Fig. 9 This is an expression analysis of Medicago truncatula and riboflavin synthesis-related genes under iron deficiency conditions in the embodiment of the present invention - a comparison chart of MtLS expression levels;

[0043] Fig.10 This is an expression analysis of Medicago truncatula and riboflavin synthesis-related genes under iron deficiency conditions in the embodiment of the present invention - a comparison chart of MtRS expression levels;

[0044] Fig.11 It is a comparative diagram of the ferric iron reducing ability analysis of the wild type and mutant of Medicago truncatula under iron deficiency conditions in the embodiment of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and examples. In the following detailed description, certain exemplary embodiments of the present invention are described only by way of illustration. Needless to say, those of ordinary skill in the art will recognize that the described embodiments may be modified in various ways without departing from the spirit and scope of the present invention. Therefore, the drawings and description are illustrative in nature and are not intended to limit the scope of protection of the claims.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0047] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0048] In the following examples involving quantitative detection, the experiment was repeated three times and the results were averaged.

[0049] The specific method for obtaining the mtcipk12 gene mutant used in the following examples is as follows: The mutant of M. truncatula was obtained by inserting TNT1 into the genome of M. truncatula R108. By comparing the sequence of MtCIPK12 (Medtr8g086380), two mutants, mtcipk12-1 and mtcipk12-2, were identified.

[0050] The following method for obtaining Medicago truncatula mutants by inserting into the genome of M. truncatula R108 is recorded in the following reference: Tadege M, Wen JQ, He J, Tu HD, Kwak Y, Eschstruth A, Cayrel A, Endre G, Zhao PX, Chabaud M, et al. 2008. Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant Journal 54(2): 335-347.

[0051] In the following examples, the screening of homozygous mtcipk12 mutants was performed by triple PCR. The application of this method to mutant screening is recorded in the following references: Gao Qianrong, Cui Na, Bai Liping. 2008. PCR identification method for Arabidopsis thaliana T-DNA insertion mutant PP2A-C [J]. Chinese Horticulture Abstracts, 24(003): 36-37.

[0052] The plant materials and culture conditions used in the following examples are as follows: Medicago truncatula wild type R108 and mtcipk12 mutant materials NF11558 and NF7022 were used as materials, and the plant iron deficiency and normal culture were carried out by hydroponic culture. The normal alfalfa nutrient solution formula is: 0.5mM KH2PO4, 1mM MgSO4, 0.25mM CaCl2, 0.1mM Fe-Na2-EDTA, 0.25mM K2SO4, 1mM NH4NO3, 2.5mM KNO3, 30μM H3BO3, 5μM MnSO4, 1μM ZnSO4, 1μM CuSO4 and 0.7μM Na2MoO4. The iron deficiency treatment iron culture is basically the same as the normal iron culture, the difference is that the former Fe 3+ -EDTA concentration is only 0.5μM.

[0053] Example 1: Effect of MtCIPK12 gene mutation on plant phenotype

[0054] Soak the peeled alfalfa seeds in concentrated sulfuric acid for 8 minutes, shake several times, and rinse with tap water 3 to 5 times. Then spread them on a plate with agar, vernalize at 4℃ for 24 hours, and incubate them upside down in the dark at 22℃. When the roots grow to a suitable length (about 2cm), move them into a hydroponic bucket. The lighting conditions are: 26℃ during the day, 22℃ at night, 14 hours of light, and a light intensity of 220μmol / m 2 / s2 , 10 hours of darkness. The hydroponic bucket should be ventilated continuously with an air pump, and the nutrient solution should be replaced regularly. After culturing in 1 / 2 normal nutrient solution for 2 weeks, iron deficiency treatment was started, and the nutrient solution used was 1 / 2 iron-deficient nutrient solution.

[0055] The phenotypes of plants grown in low-iron or iron-sufficient medium for 5 days were observed and photographed for comparison.

[0056] The results are as follows Figure 1 As shown in the figure, under iron-sufficient conditions, the growth of the MtCIPK12 gene mutants cipk12-1 and cipk12-2 had no significant difference from that of the wild type; however, under iron-deficient conditions, the leaves of the wild type turned more yellow than those of the mutants, and the leaves became significantly smaller, while the leaves of the mutants did not change significantly, but the roots of the mutants accumulated significantly more riboflavin.

[0057] Example 2: Effects of MtCIPK12 gene mutation on plant physiology

[0058] 1) Determination of biomass

[0059] Biomass determination method: After 5 days of treatment with control and iron-deficient nutrient solution, leaf and root samples of 10 individual alfalfa seedlings were collected from the control and iron-deficient culture media, respectively, and weighed. They were then dried overnight at 80 degrees in an oven and weighed. The weight difference before and after drying was the measured biomass.

[0060] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 The dry weight of leaves per plant of different strains under low iron (-Fe) and sufficient iron (+Fe) conditions; Figure 3 The dry weight of individual roots of different strains under low iron and sufficient iron conditions. It can be seen that under sufficient iron conditions, the biomass of the MtCIPK12 gene mutant was not significantly different from that of the wild type; however, under iron deficiency conditions, the biomass of both the wild type and the mutant decreased significantly. However, compared with the wild type, the reduction in the mutant plant was smaller, and iron deficiency caused the biomass of the mutant to be higher than that of the wild type, indicating that MtCIPK12 is involved in plant iron nutrient absorption.

[0061] 2) Determination of chlorophyll content and photosynthetic rate

[0062] Method for determining chlorophyll content: Collect alfalfa seedlings cultured for 5 days under normal and iron-deficient conditions, dry the surface moisture with filter paper, weigh the fresh weight, put the entire aerial part of the seedling into a 5 mL centrifuge tube, add 1 mL of 95% ethanol solution, seal at 4°C overnight, and determine the chlorophyll content, where the chlorophyll concentration is expressed as the sum of chlorophyll a and chlorophyll b. The method for determining chlorophyll is referred to the following literature: Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidases in Beta vulgaris. Plant Physiology 24(1): 1-15.

[0063] Photosynthetic rate determination method: The photosynthetic rate was measured on newly expanded Medicago truncatula leaves using a Li-6800 portable photosynthetic system (Li-Cor, USA). The air flow rate and CO2 concentration were 750 μmol / s and 400 μmol / mol, respectively. Note that the best time for determination was between 9:00 am and 13:00 pm.

[0064] The results are as follows Figure 4 and Figure 5 As shown, Figure 4 is the chlorophyll content of different strains under low iron (-Fe) and iron sufficient (+Fe) conditions; Figure 5 is the photosynthetic rate of different strains under low iron and iron-sufficient conditions. It can be seen that under iron-sufficient conditions, the chlorophyll content and photosynthetic rate of the MtCIPK12 gene mutants cipk12-1 (NF11558) and cipk12-2 (NF7022) were not significantly different from those of the wild type. However, under iron-deficient conditions, the photosynthetic rates of both the wild type and the mutants decreased significantly. However, compared with the wild type, the reduction in the mutant plants was much smaller. The above results indicate that the mutation of MtCIPK12 improves the photosynthesis of Medicago truncatula under iron-deficient conditions, thereby improving the iron deficiency tolerance of Medicago truncatula.

[0065] 3) Determination of iron ion content in plants

[0066] The method for determining the iron ion content of plants is as follows: collect the leaves and roots of 7-day-old seedlings in different iron-treated media, take about 50 mg of the sample after drying, add 5 mL of concentrated nitric acid to the dry sample, shake well, leave overnight, add 2 mL of hydrogen peroxide the next morning, shake well, and then use a microwave system (MARS, CEM) to digest the sample. After transferring to a fixed volume, use ICP-OAS to determine the ion concentration.

[0067] The results are as follows Figure 6 and Figure 7 As shown, Figure 6The iron content of leaves of different strains under low iron (-Fe) and iron sufficient (+Fe) conditions; Figure 7 The iron content of the roots of different strains under low iron and iron-sufficient conditions. It can be seen that under iron-sufficient conditions, there is no significant difference in the iron content between the MtCIPK12 gene mutants cipk12-1 (NF11558) and cipk12-1 (NF7022) and the wild type, whether in the aboveground or underground parts. However, under iron-deficient conditions, the iron content of the aboveground and underground parts of the wild type and mutants decreased significantly. However, compared with the wild type, the reduction in mutant plants is much smaller, resulting in higher iron content in mutants than in wild types under iron-deficient conditions. The above results indicate that the mutation of MtCIPK12 increases the accumulation of plant iron in Medicago truncatula under iron-deficient conditions, thereby improving the iron deficiency tolerance of Medicago truncatula.

[0068] Example 3: Effect of MtCIPK12 gene mutation on riboflavin synthesis and accumulation in Medicago truncatula

[0069] The riboflavin content in the roots of wild-type and mutant Medicago truncatula under control and iron-deficient conditions was quantitatively analyzed by high-performance liquid chromatography (Shimadzu LC30) and QTOF (AB SciexTripleTOF 5600+). The riboflavin content was determined by the method of Rodriguez-Celma et al. (Rodriguez-Celma et al., 2011), as follows: The roots of Medicago truncatula R108 and cipk12 mutant materials NF11558 and NF7022 treated with iron deficiency for 5 days were used as materials, and about 0.1 g of the material was ground with 250 μL buffer containing 1 mM ammonium acetate and 5% methanol. Liquid phase separation was performed using a Waters Mters Xbridge C18 column of a high performance liquid chromatography system. The column box was maintained at 35°C, the flow rate was 300 μL / min, the mobile phase consisted of acetonitrile (A) and 0.1% acetic acid (B), and the injection volume of each sample was 200 μL. The gradient elution conditions were set as follows: 0-12 min, 5%-100% solvent A; 13-16 min, 0-95% solvent B. After the sample was separated by HPLC, a high-resolution tandem mass spectrometer was used to detect riboflavin eluted from the column. The QTOF was operated in positive and negative ion modes, with ion voltages of 5500 V and 4500 V, respectively. The MS2 spectrum was in high-sensitivity information-dependent acquisition (IDA) mode. The concentration of riboflavin was determined according to the corresponding standard curve.

[0070] The results are shown in Table 1: When grown on iron-sufficient medium, the riboflavin content of the cipk12 mutant was significantly increased compared with the wild-type plants. Under Fe-deficient conditions, the riboflavin concentration in the mutants was more significantly increased than that in the wild-type plants, especially in the two mutants, where the total riboflavin concentration in the roots of cipk12-1 and cipk12-2 was 7.7 and 10.5 times higher than that of the wild-type plants, respectively. These results indicate that CIPK12 negatively regulates riboflavin biosynthesis.

[0071] Table 1 Response of riboflavin synthesis and accumulation of Medicago truncatula to iron deficiency

[0072]

[0073] Note: Capital letters indicate the comparison between wild type and mutants under normal culture conditions; lowercase letters indicate the comparison between wild type and mutants under iron-deficient culture conditions; different letters indicate significant differences; asterisks indicate significant differences between the same material under different culture conditions.

[0074] Example 4: Analysis of related gene expression during riboflavin synthesis in Medicago truncatula

[0075] In order to explore the regulatory mechanism behind iron deficiency-induced riboflavin accumulation in the cipk12 mutant, we determined the expression levels of genes of three key enzymes in the riboflavin synthesis process, namely, GTP cyclohydrolase II gene (GTPcII), 2,4-dioxotetrahydropyrimidine synthase gene (LS) and riboflavin synthase gene (RS). The specific steps are as follows: First, RNA of plant materials was extracted, reversed into cDNA, and then expression levels were determined by real-time quantitative PCR (RT-qPCR) method.

[0076] 1) Extraction of alfalfa RNA

[0077] RNA was extracted using Takara's RNAiso Plus reagent. The specific steps are as follows: Take root samples of wild-type R108 and cipk12 mutant materials cipk12-1 and cipk12-2 at 0, 1, and 3 days after iron deficiency treatment, grind them into powder in liquid nitrogen; transfer the ground powder into a 1.5 mL centrifuge tube and add 1 mL Trizol, shake vigorously, and place at room temperature for 5 minutes; add 300 μL chloroform, shake vigorously for 15 seconds, place at room temperature for 2 minutes, and centrifuge at 12000rpm for 10 minutes at 4°C; take the supernatant (about 600 μL), add it to a 1.5ml centrifuge tube containing 500 μL isopropanol, mix gently (turn upside down 8 times), let it stand at room temperature for 10 minutes, and centrifuge at 12000rpm for 10 minutes (4°C); discard the supernatant, add 300 μL 75% ethanol, centrifuge at 7000rpm for 2 minutes (4°C), wash twice to thoroughly wash the salt in the sample, dry at room temperature for 5 minutes, blow dry the precipitate; dissolve the RNA in 30 μL RNase-free-H2O. Then take 2 μL RNA and use 1% agarose gel electrophoresis to detect the quality of RNA extraction by electrophoresis brightness. The sample is stored at -80°C for use.

[0078] 2) Synthesis of reverse transcribed cDNA

[0079] Reverse transcription was performed using PrimeScript from Takara TM RT reagent Kit with gDNA Eraser (Perfect Real Time) was used. It was divided into two parts: first removing genomic DNA and then reverse transcribing it into cDNA. The obtained product was stored at -20°C.

[0080] The procedure for removing genomic DNA is as follows: 5×gDNA Eraser Buffer 1.0μL, gDNA Eraser 0.5μL, RNA 1.0μL, RNase-free H2O 2.5μL, a total of 5μL system, mix and centrifuge, and place at room temperature for 5 minutes.

[0081] The 5 μL system for reverse transcription into cDNA is as follows: 5× Primer Script Buffer 2.0, Primer Script Enzyme Mix 0.5 μL, RT Primer Mix 2.0 μL, RNase-free H2O 0.5 μL.

[0082] 5 μL of the above two-step reaction system were mixed together, incubated in a water bath at 37°C for 30 min, then in a water bath at 85°C for 2 min, cooled on ice, and the obtained product was diluted 10 times and stored at -20°C for later use.

[0083] 3) Real-time quantitative PCR (RT-qPCR)

[0084] Real-time quantitative PCR (RT-qPCR) experiments used UltraSYBR Mixture (WithRox) from Kangwei Century, and the PCR instrument used ABI Stepone Plus I instrument. The relative expression level of genes was calculated using the comparative CT method. Two pairs of internal control primers were used in the qRT-PCR experiment. Gene-specific primers and internal control primers are listed in Table 2 below.

[0085] Table 2 Primer design for qRT-PCR

[0086] Gene Forward primer (5'-3') Reverse primer (5'-3') CIPK12 TTTGTGGTACACCTGCTTMt TMtCAAGAAGCCTAGTAAGA GTPcII MtTGTMtCTMtCGCTGACTT GTACCCTCACAAGAACMtC LS GGGGMtACAACTCACTMtGA MtTACCAGMtTTGCCACC RS CCCTCTTCGCMtCTTCCT CCCTCTTCGCMtCTTCCT Actin ACGAGCGTTTCAGMtG ACCTCCGMtCCAGACA GAPDH AAGGAGGAGTCTGAGGGC AACGGCTGCTAGGCTAMt

[0087] The PCR reaction system for RT-qPCR is as follows:

[0088] Reagents 10μL reaction system 2×UltraSYBR Mixture(With Rox) 5μL Forward Primer, 10 μM 0.3μL Reverse Primer, 10 μM 0.3μL TemplMte DNA 0.5μL

[0089] The PCR reaction procedure for RT-qPCR is as follows:

[0090]

[0091] Melting curve analysis:

[0092] 95℃ for 30s, 60℃ for 1min, 95℃ for 30s.

[0093] The results are as follows Figure 8 , Fig. 9 and Fig.10 As shown in the figure, when grown on Fe-sufficient medium, the expression levels of GTPcII and LS genes were higher than those of wild-type plant roots. In wild-type and mutant plants, GTPcII and LS were significantly upregulated due to iron deficiency, and the transcription levels of GTPCII, LS and RS in mutants were much higher than those in wild-type plants. The above results indicate that the mutation of mtcipk12 is beneficial to the expression of riboflavin synthesis-related genes under iron deficiency conditions.

[0094] Example 5: Analysis of ferric iron reduction activity

[0095] Alfalfa seedlings grown on normal culture medium for 3 weeks were transferred to nutrient solution containing 1 / 2 iron deficiency and cultured in water for 6 days before being used to determine the ferric iron reducing ability.

[0096] The determination protocol of ferric iron reducing capacity is described in the following literature: Waters BM, Lucena C, Romera FJ, Jester GG, Wynn AN, Rojas CL, Alcantara E, Perez-Vicente R. 2007. Ethylene involvement in the regulation of the H + -ATPase CsHA1 gene and of the newisolated ferric reductase CsFRO1 and iron transporter CsIRT1genes in cucumberplants. Plant Physiology and Biochemistry 45(5):293-301.

[0097] The ferric iron reducing capacity was determined as follows: the roots of the intact plants were placed in 8 mL of reaction solution (100 μM Fe 3+ -EDTA and 300 μM ferrozine, pH = 6.0) at 26°C for 1.5 hours, the roots were taken out, and the absorbance of the reaction solution was measured at 562 nm. 3+ The coefficient of the standard curve for -ferrozine was 36.252, and the ferric iron reductase activity was expressed in μmol Fe 2+ g -1 Fresh weight -1 Indicates (W Mters et al., 2007).

[0098] The results are as follows Fig.11 As shown in the figure, it can be seen that under normal iron supply conditions, the ferric iron reduction of the wild type and mutant of MtCIPK12 is basically the same; under iron deficiency conditions, the ferric iron reduction of the MtCIPK12 gene mutants cipk12-1 (NF11558) and cipk 12-2 (NF7022) is significantly higher than that of the wild type, indicating that the MtCIPK12 mutant has a stronger ferric iron reduction ability, which is beneficial to promote iron absorption. The above results show that CIPK12 regulates the biosynthesis of riboflavin and the ferric iron reduction in the roots of plants under iron deficiency conditions, thereby improving the tolerance of Medicago truncatula under iron deficiency conditions.

[0099] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents. Sequence Listing <110> Institute of Botany, Chinese Academy of Sciences <120> Application of MtCIPK12 gene in regulating plant resistance to low iron stress <130> 20213744 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1533 <212> DNA <213> Artificial sequence <400> 1 Atggcagttg tagctgctcc caagaagaac aactcaatga acaagaaaga taatccaaat 60 cttctattgg gacgttttga attaggaaaa cttcttggcc atggaacctt tgcaaaagtc 120 caccttgcca agaacctcaa aacaggtgaa tccgtagcta taaagatcat aagtaaagat 180 aaaatcctta aaagtggttt agtttcacat atcaaacgag aaatctccat tctgcgccgt 240 gttcgtcacc ccaacattgt tcaactcttt gaagtcatgg ctacaaagac aaagatttac 300 tttgtgatgg aatatgtacg aggtggtgag cttttcaaca aggttgctaa aggtaggttg 360 aaagaagaag ttgcaaggaa atattttcag cagttaatat gtgctgttgg atttgtcat 420 gctagaggtg ttttcatag agatctaaag cctgaaaatt tgttgcttga tgaaaaaggt 480 aaccttaaag tttcagattt tggtcttagt gctgtgtcgg atgaaattaa gcaagatggg 540 ttgtttcata cttttgtgg tacacctgct tatgttgctc ctgaggtttt gtctaggaaa 600 ggtatgatg gtgctaaggt tgatatttgg tcttgtgggg ttgttttgtt tgttttgatg 660 gctggttatt taccttttca tgatcctaat aatgttatgg ctatgtataa gaagatttat 720 aaaggtgaat ttaggtgtcc tagatggttt tcaccagaac ttgttagtct tcttactagg 780 cttcttgata ttaaacctca aactaggatt tctattcctg agattatgga gaatcgttgg 840 tttaagatag gttttaagca tattaaattt tatgttgagg atgatgttgt ttgtgatctt 900 gattcacttg atcttgatgg tgaggataat aataataagg tagtaaaggt cgatgatcat 960 cacgatgaag tactggaatc agtatcagaa aatgaatcgg attctgaggt tgtgaataga 1020 aggatcagga atcgtggttc attgccaagg cctgcaagtt tgaatgcttt tgatattata 1080 tcgttttcgc aaggttttga tctttctggt ttatttgagg aaaagggtga tgaggcaagg 1140 tttgtgtcta gtgcatcagt gccaaagata atatcaaaat tggaggaagt tggtcaaatg 1200 gttaggttca atgtgaggaa gaaagattgc aaggttagct tggagggttc aagagaaggg 1260 gcaaaagggc gattgactat tgctgccgag gtcttcgaat tgacaccttc tttggtggtg 1320 gttgaggtga agaaaaaagg aggggataag gttgagtatg ataaattctt aaacactgaa 1380 ttgaagcctg ctttgcatag tttaaccaag gaggaatctg caggttcttc aagtcaaact 1440 acaccagatg aatctttgca acaacgtgca ttttctgatt ctgccatcga caaacattca 1500 gatagtattg aatctctgaa cttagatacc tga 1533 <210> 2 <211> 510 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Val Val Ala Ala Pro Lys Lys Asn Asn Ser Met Asn Lys Lys 1 5 10 15 Asp Asn Pro Asn Leu Leu Leu Gly Arg Phe Glu Leu Gly Lys Leu Leu 20 25 30 Gly His Gly Thr Phe Ala Lys Val His Leu Ala Lys Asn Leu Lys Thr 35 40 45 Gly Glu Ser Val Ala Ile Lys Ile Ile Ser Lys Asp Lys Ile Leu Lys 50 55 60 Ser Gly Leu Val Ser His Ile Lys Arg Glu Ile Ser Ile Leu Arg Arg 65 70 75 80 Val Arg His Pro Asn Ile Val Gln Leu Phe Glu Val Met Met Thr Lys 85 90 95 Thr Lys Ile Tyr Phe Val Met Glu Tyr Val Arg Gly Gly Glu Leu Phe 100 105 110 Asn Lys Val Ala Lys Gly Arg Leu Lys Glu Glu Val Ala Arg Lys Tyr 115 120 125 Phe Gln Gln Leu Ile Cys Ala Val Gly Phe Cys His Ala Arg Gly Val 130 135 140 Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu Lys Gly 145 150 155 160 Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Glu Ile 165 170 175 Lys Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala Tyr Val 180 185 190 Ala Pro Glu Val Leu Ser Arg Lys Gly Tyr Asp Gly Ala Lys Val Asp 195 200 205 Ile Trp Ser Cys Gly Val Val Leu Phe Val Leu Met Ala Gly Tyr Leu 210 215 220 Pro Phe His Asp Pro Asn Asn Val Met Ala Met Tyr Lys Lys Ile Tyr 225 230 235 240 Lys Gly Glu Phe Arg Cys Pro Arg Trp Phe Ser Pro Glu Leu Val Ser 245 250 255 Leu Leu Thr Arg Leu Leu Asp Ile Lys Pro Gln Thr Arg Ile Ser Ile 260 265 270 Pro Glu Ile Met Glu Asn Arg Trp Phe Lys Ile Gly Phe Lys His Ile 275 280 285 Lys Phe Tyr Val Glu Asp Asp Val Val Cys Asp Leu Asp Ser Leu Asp 290 295 300 Leu Asp Gly Glu Asp Asn Asn Asn Lys Val Val Lys Val Asp Asp His 305 310 315 320 His Asp Glu Val Leu Glu Ser Val Ser Glu Asn Glu Ser Asp Ser Glu 325 330 335 Val Val Asn Arg Arg Ile Arg Asn Arg Gly Ser Leu Pro Arg Pro Ala 340 345 350 Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser Gln Gly Phe Asp Leu 355 360 365 Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu Ala Arg Phe Val Ser Ser 370 375 380 Ala Ser Val Pro Lys Ile Ile Ser Lys Leu Glu Glu Val Gly Gln Met 385 390 395 400 Val Arg Phe Asn Val Arg Lys Lys Asp Cys Lys Val Ser Leu Glu Gly 405 410 415 Ser Arg Glu Gly Ala Lys Gly Arg Leu Thr Ile Ala Ala Glu Val Phe 420 425 430 Glu Leu Thr Pro Ser Leu Val Val Val Glu Val Lys Lys Lys Gly Gly 435 440 445 Asp Lys Val Glu Tyr Asp Lys Phe Leu Asn Thr Glu Leu Lys Pro Ala 450 455 460 Leu His Ser Leu Thr Lys Glu Glu Ser Ala Gly Ser Ser Ser Gln Thr 465 470 475 480 Thr Pro Asp Glu Ser Leu Gln Gln Arg Ala Phe Ser Asp Ser Ala Ile 485 490 495 Asp Lys His Ser Asp Ser Ile Glu Ser Leu Asn Leu Asp Thr 500 505 510

Claims

1. MtCIPK12 The application of a gene in regulating plant tolerance to low iron stress is characterized in that: The steps include: Step 1: Create mutants of wild-type plants by reducing MtCIPK12 The expression of genes and MtCIPK12 proteins was used to obtain plant mutants; Said MtCIPK12 The amino acid sequences of the gene and MtCIPK12 protein are Sequence 1 and Sequence 2 in the sequence listing; Step 2: Observe the expression and function of key enzyme genes in the riboflavin biosynthesis process of plant mutants under iron deficiency. The specific enzymes are as follows: GTP cyclohydrolase II (GTPcII), 2,4-dioxotetrahydropyrimidine synthase (LS), and riboflavin synthase (RS); the iron deficiency environment is that the iron ion concentration contained in the culture medium is 0.5 μM; Step 3, the accumulation of riboflavin in the plant mutant increases, and the plant mutant has enhanced resistance to low iron stress; The wild plant is alfalfa.

2. MtCIPK12 The use of a gene in regulating riboflavin synthesis in plant tolerance to low iron stress is characterized in that: The steps include: Step 1: Create mutants of wild-type plants by reducing MtCIPK12 The expression of genes and MtCIPK12 proteins was used to obtain plant mutants; Said MtCIPK12 The amino acid sequences of the gene and MtCIPK12 protein are Sequence 1 and Sequence 2 in the sequence listing; Step 2: Observe the expression and function of key enzyme genes in the riboflavin biosynthesis process of plant mutants under iron deficiency. The specific enzymes are as follows: GTP cyclohydrolase II (GTPcII), 2,4-dioxotetrahydropyrimidine synthase (LS), and riboflavin synthase (RS); the iron deficiency environment is that the iron ion concentration contained in the culture medium is 0.5 μM; Step 3: the accumulation of riboflavin in the plant mutant increases and decreases MtCIPK12 The expression of the gene and MtCIPK12 protein can increase riboflavin nuclear synthesis; The wild plant is alfalfa.

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