CN117535312A - Application of vacuole membrane anion channel protein in regulation and control of carbon-nitrogen balance of corn - Google Patents
Application of vacuole membrane anion channel protein in regulation and control of carbon-nitrogen balance of corn Download PDFInfo
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- CN117535312A CN117535312A CN202410032296.9A CN202410032296A CN117535312A CN 117535312 A CN117535312 A CN 117535312A CN 202410032296 A CN202410032296 A CN 202410032296A CN 117535312 A CN117535312 A CN 117535312A
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Abstract
The invention belongs to the technical field of botanics, and particularly relates to application of a vacuole membrane anion channel protein in regulation and control of carbon-nitrogen balance of corn. The invention takes wild corn, zmALMT9 mutant and ZmALMT9 overexpressing plants as materials, and detects the responses of different materials to nitrogen: the photosynthetic efficiency, chlorophyll content and Fv/Fm of zmalmt9 mutant are all obviously smaller than those of wild type under the condition of high nitrogen; overexpression of zmammt 9 did not affect plant growth, but resulted in increased SPAD and malic acid concentration in plants under high nitrogen conditions. ZmALMT9 is transported into redundant malic acid to enter vacuoles for storage under the condition of high nitrogen, once the process is lost, the malic acid metabolism in cells is disturbed, photosynthesis and amino acid metabolism are affected, and the carbon and nitrogen utilization capacity of plants is reduced. ZmALMT9 regulates plant carbon and nitrogen metabolism by maintaining malic acid steady state to participate in the physiological function of carbon and nitrogen utilization.
Description
Technical Field
The invention belongs to the technical field of botanics, and particularly relates to application of a vacuole membrane anion channel protein in regulation and control of carbon-nitrogen balance of corn.
Background
Carbon and nitrogen are main nutritional elements required for plant growth and development, and are important for synthesizing biomolecules such as proteins, nucleic acids, amino acids and the like. Carbon metabolism and nitrogen metabolism are two most basic metabolic processes of plants, carbon metabolism provides carbon sources and energy for nitrogen metabolism, and nitrogen metabolism provides enzymes and photosynthetic pigments for carbon metabolism, which have a common carbon skeleton, ATP and reducing power. However, most plants cannot directly utilize nitrogen in the air, although nitrogen is present in large amounts in the atmosphere. Instead, they generally acquire the required nitrogen nutrients by absorbing nitrogen compounds in the soil, and the adaptability of plants to nitrogen mainly involves three main aspects of nitrogen absorption, assimilation and redistribution, and regulation of plant carbon-nitrogen metabolism is of great significance to the growth and development of plants.
As early as 2004, sasaki et al (Sasaki T, yamamoto Y, ezaki B, et al A wheat gene encoding an aluminum-activated malate transporter [ J ]]The Plant Journal, 2004, 37 (5): 645-653) the ion channel protein TaALMT1, which is an Aluminum-induced malate secretion, was isolated for the first time from the root tip of an anti-Aluminum wheat variety (ET 8) by inhibition of differential hybridization techniques, and was named Aluminum ion activated malate transporter (Aluminum-activated malate transporter, ALMT) by researchers as well, which also determined the name of the whole protein family, since it was able to mediate tolerance of plants to Aluminum ions in soil, by sequestering Aluminum ions in soil by efflux malate anions. ALMT family is a specific anion transporter for plant, and is mainly used for transporting organic acids such as malic acid and fumaric acid or Cl - 、NO 3 - And SO 4 2- An isopoly anion (Delhaize E, gruber B D, ryan P R. The roles of organic anion permeases in aluminium resistance and min)eral nutrition[J]Febs Letters, 2007, 581 (12): 2255-2262), to date only a small portion has been shown to be involved in the detoxification of aluminum, after which studies have found that the physiological effects of ALMTs far outweigh those of aluminum (Shalma S, dietz K J, mimura T Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants [ J]. Plant, Cell &Environmental, 2016, 39 (5): 1112-1126.). Different members of the ALMT family participate in different physiological processes and play different roles, in part because of the nature of ALMT as an anion channel, which can transport organic and inorganic anions, however, the prior art does not describe the regulation of plant carbon-nitrogen metabolism by the zmarmt 9 gene.
Disclosure of Invention
The invention aims to regulate and control carbon and nitrogen metabolism of plants, especially corns, promote carbon and nitrogen metabolism of plants and improve the carbon and nitrogen utilization capacity of the plants.
The invention provides application of a corn ZmALMT9 gene or a coded protein thereof in regulating and controlling plant carbon nitrogen metabolism.
Preferably, the regulating carbon nitrogen metabolism of the plant comprises: the corn ZmALMT9 gene or the coded protein thereof regulates photosynthesis and amino acid metabolism of plants.
Preferably, the regulating carbon nitrogen metabolism of the plant comprises: the corn ZmALMT9 gene or the encoding protein thereof regulates and controls the malic acid metabolism of plants.
Preferably, the modulating malic acid metabolism of the plant comprises: and under the condition of 4 mM nitrogen stress, the malic acid metabolism of the plants is regulated.
Preferably, the nitrogen comprises an ammonium salt and/or a nitrate salt.
Preferably, the regulating carbon nitrogen metabolism of the plant comprises: upregulating expression of the maize zmarmt 9 gene promotes carbon-nitrogen metabolism of the plant.
Preferably, the plant comprises maize and/or tobacco.
The beneficial effects are that:
according to the invention, subcellular localization, in-situ hybridization and electrophysiological technology are combined to ensure that ZmALMT9 is mainly located on a vacuole membrane both in corn and tobacco, then the expression mode of ZmALMT9 is further ensured by inquiring chip data and combining RT-qPCR and in-situ hybridization results, namely, the expression of the aerial part is stronger than that of the underground part, wherein the expression is mainly carried out in a leaf vascular bundle system and air holes of epidermal cells, and patch clamp technology is utilized to ensure that the ZmALMT9 mediates various anions on the vacuole membrane to enter the vacuole cavity so as to participate in carbon and nitrogen utilization by maintaining malic acid steady state. The invention also quantitatively detects the response condition of ZmALMT9 to nitrogen, and through carrying out growth phenotype identification under different nitrogen levels by means of transgenic materials, combining the measurement of physiological indexes such as ion content and the like and analysis by combining transcriptome and metabonomics, the invention reveals that the deficiency of the ZmALMT9 gene leads to the imbalance of malic acid steady state of corn plants under high nitrogen conditions, further influences the intracellular malic acid metabolism and carbon nitrogen metabolism processes, and plants over-expressing the ZmALMT9 lead to the increase of SPAD of plants, the increase of malic acid concentration, and the corn ZmALMT9 gene or the coded protein thereof can regulate and control the carbon nitrogen metabolism of plants under high nitrogen conditions.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the basic information analysis results of zein ZmALMT 9; wherein A is a evolutionary tree; b is a gene expression heat map; c is a membrane protein structure prediction result;
FIG. 2 shows the subcellular analysis of ZmALMT 9; wherein A is the subcellular analysis result of ZmALMT9 transformed by corn leaf protoplast; b is the subcellular analysis result of the transient transformation ZmALMT9 of the tobacco;
FIG. 3 shows the results of tissue expression analysis of ZmALMT 9; wherein A is the analysis result of the chip data; b is a real-time fluorescence quantitative PCR detection result, and different lower case letters show significant differences; c is the in situ hybridization detection result;
FIG. 4 shows the results of ion-selective analysis of the vacuolar membrane anion channel ZmALMT 9; wherein a is a localized single vacuole; b is a program set by patch clamp experiments; C-E is the result of current intensity detection, and different lower case letters represent significant differences;
FIG. 5 shows the results of analysis of the transport characteristics of ZmALMT9 ion channel with respect to substrate concentration (for example, nitrate);
FIG. 6 shows the results of expression analysis of ZmALMT9 response to nitrogen; wherein A is the relative expression quantity of ZmALMT9 at different times of nitrogen deficiency; b is the relative expression quantity of ZmALMT9 of the corn plants after nitrogen deficiency treatment under different time periods of supplying ammonium salt and nitrate respectively; different lowercase letters represent significant differences;
FIG. 7 is the identification of mutants of ZmALMT9 and over-expressed transgenic material; wherein A is the sequence difference between the mutant material of ZmALMT9 and ND 101; b is the semi-quantitative detection result of the ZmALMT9 over-expressed transgenic material; c is the relative amount of expression of zmammt 9 in the transgenic material and ND101, p < 0.0001, p < 0.001, p < 0.01, p < 0.05;
FIG. 8 is a graph showing the phenotypic and agronomic trait measurements of zmalmt9 mutants at different nitrogen concentrations; wherein a is a growth phenotype; B-G is a physiological trait, p < 0.0001, p < 0.001, p < 0.01, p < 0.05;
FIG. 9 shows the results of the detection of the growth phenotype and physiological properties of ZmALMT9 overexpressing plants at different nitrogen concentrations; wherein A is a phenotype; B-G is a physiological trait, p < 0.0001, p < 0.001, p < 0.01, p < 0.05;
FIG. 10 is the results of a phenotype recovery experiment for mutant zmalmt 9; wherein A is the phenotype of the plant under different treatment modes; b is the dry weight of plants on the ground under different treatment modes, and different lowercase letters show significant differences;
FIG. 11 shows the results of transcriptomic analyses of wild-type ND101 and mutant zmalmt9-1 under high nitrogen treatment; wherein A is the number of up-regulated genes and down-regulated genes; b is KEGG enrichment analysis result; c is a heat map of differential gene expression;
FIG. 12 shows the results of a wide target metabonomics analysis of wild type ND101 and mutant zmalmt9-1 under high nitrogen treatment; wherein, A is a super-cluster heat map and a VIP bar map of the differential metabolite; b is KEGG enrichment analysis result; c is the metabolic process in which the differential metabolite participates;
FIG. 13 is a graph showing the results of analysis of photosynthetic capacity and amino acid content of zmalmt9 mutant; wherein A is photosynthetic utilization efficiency; b is chlorophyll content; c is Fv/Fm; D-E is the amino acid concentration; * P < 0.0001, p < 0.001, p < 0.01, and p < 0.05.
Detailed Description
The invention provides application of a corn ZmALMT9 gene or a coded protein thereof in regulating and controlling plant carbon nitrogen metabolism. In the invention, the accession number information of the corn ZmALMT9 gene is Zm00001d045696.
The invention constructs a ZmALMT9 gene expression vector, converts protoplasts, confirms that ZmALMT9 is mainly positioned on a vacuole membrane in corn and tobacco through subcellular localization, further confirms that the expression of ZmALMT9 is stronger than underground part in the overground part of plants through inquiring chip data and combining RT-qPCR and in situ hybridization results, wherein the expression is mainly performed in a leaf vascular bundle system and pores of epidermal cells, confirms that the ZmALMT9 mediates various anions on the vacuole membrane to enter the vacuole cavity by utilizing a patch clamp technology, and participates in carbon and nitrogen utilization by maintaining malic acid steady state. According to the invention, wild corn ND101, zmALMT9 mutant and ZmALMT9 overexpressed plants are used as materials, and response conditions of different materials to nitrogen are detected, so that photosynthetic efficiency, chlorophyll content and Fv/Fm of the ZmALMT9 mutant are obviously smaller than those of the wild ND101 under high nitrogen conditions; overexpression of zmammt 9 did not affect plant growth, but resulted in increased SPAD and malic acid concentration in plants under high nitrogen conditions. ZmALMT9 is mainly responsible for transporting excessive malic acid into vacuoles for storage, and once the process is lost, the malic acid metabolism in cells is disturbed, photosynthesis and amino acid metabolism are affected, so that the carbon and nitrogen utilization capacity of plants is reduced.
Based on the differences of wild corn ND101, zmALMT9 mutant and ZmALMT9 over-expression plants, the application of corn ZmALMT9 gene or encoded protein thereof in regulating photosynthesis and amino acid metabolism of plants also belongs to the protection scope of the invention.
Based on the differences of wild corn ND101, zmALMT9 mutant and ZmALMT9 over-expression plants, the application of corn ZmALMT9 gene or encoded protein thereof in regulating and controlling malic acid metabolism of plants also belongs to the protection scope of the invention. In the present invention, the regulation of malic acid metabolism of a plant preferably comprises: and under the condition of 4 mM nitrogen stress, the malic acid metabolism of the plants is regulated. The nitrogen element according to the invention preferably comprises an ammonium salt and/or a nitrate salt.
In the present invention, the regulation of carbon nitrogen metabolism in plants preferably comprises: upregulating expression of the maize zmarmt 9 gene promotes carbon-nitrogen metabolism of the plant. The plant of the invention is preferably maize and/or tobacco, more preferably maize.
For further explanation of the present invention, the application of the tonoplast anion channel protein provided by the present invention in regulating carbon-nitrogen balance of corn will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Basic information of zein zmammt 9
The AtALMT1 genes in Arabidopsis are taken as baits, and the amino acid sequences of 14 Arabidopsis AtALMTs genes and 9 corn ZmALMTs genes are found in NCBI (National Cenetr for Biotechnology List). Alignment was performed by Clustal-W method, and the evolutionary tree was drawn by the neighbor-joining method of MEGA9 (FIG. 1A), and they were named according to the relationship. Members of the same group have similar expression localization and physiological functions. The arabidopsis gene members AtALMT3, atALMT4, atALMT5, atALMT6, atALMT9 and maize gene Zm00001d045696 and gene Zm00001d017928 are assigned to the fourth branch, with 75% amino acid sequence similarity. Zm00001d045696 was named zmammt 9 according to the evolutionary far-near relationship, where monocot maize and dicot arabidopsis were in turn clearly distinguished, suggesting that members in the same branch may have functional differentiation in monocot and dicot (a in fig. 1). In order to comprehensively understand the expression mode of each member of the family, the invention collects transcriptome data of 9 ALMTs genes in corn under the normal growth condition in the background of B73 in a MaizeGDB database (https:// www.maizegdb.org), and establishes gene expression heat map analysis by integrating data of 13 different tissue parts of corn under the background of B73, and the result shows that the expression modes of 9 genes are different, the expression quantity of most genes is relatively low, the expression condition of ZmALMT9 is strongest in the whole family, but the expression parts cover each tissue and are constitutive expression. Wherein another maize gene located in branch four (Zm 00001d 017928) was only expressed to some extent in anthers and maize silk (B in fig. 1), suggesting that it may be functional in maize kernel building. The ALMT protein encoded by ZmALMT9 consists of 616 amino acids, and its homologous protein has been reported to have various anion transport activities in Arabidopsis thaliana, and the amino acid sequence of ZmALMT9 was predicted using the membrane protein structure prediction website (http:// wlab. Etz. Ch/protein/start /), which showed that ZmALMT9 has 5 consecutive transmembrane domains, which is a typical membrane transporter (C in FIG. 1). The above results suggest that zmarmt 9 may be a major gene in the ALMTs family, suggesting that it has an important function, for which the gene zmarmt 9 is of great concern.
Example 2
1. Materials and reagents
1.1 test materials
1.1.1 plant Material
Corn (Zea mays L.) ND101 (supplied by the chinese agricultural university crop functional genomics and molecular breeding research center, consistent with the inbred lines of the material parts in articles Cao H, liu Z, guo J, jia Z, shi Y, kang K, peng W, wang Z, chen L, neuhaeuser B, wang Y, liu X, hao D, yuan L, zmnrt1.1b (zmnpf6.6) determines nitrogen use efficiency via regulation of nitrate transport and signalling in maze, plant Biotechnol j.2023 Oct 2) and tobacco;
1.1.2 species
Coli DH 5-alpha (purchased from Norvezan, beijing, inc.); agrobacterium GV3101 (purchased from Beijing Bomaide Co.).
1.1.3 vectors
P1300-GFP vector; P1300-mCherry vector.
1.2. Related reagents
1.2.1 tool enzymes and related biochemical reagents and related uses are shown in Table 1.
Table 1 tool enzymes and related reagents
1.2.2 primer Synthesis and sequencing
The synthesis of the related primers and DNA sequencing were completed by Beijing qingke company, and specific information is shown in Table 2.
TABLE 2 primer list
Construction and transformation of GFP-ZmALMT9 vector
2.1 plant cultivation
(1) Sterilizing seeds of corn ND101, mutant material zmalmt9-1 and mutant material zmalmt9-2 with 10% hydrogen peroxide (or 2% sodium hypochlorite) for 30 min, respectively, and saturated CaSO 4 Soaking overnight (10 h-16 h), spreading on wet filter paper, and germinating in darkness, wherein when embryo and radicle happen downward and contact with the filter paper for germination, water should be reduced as much as possible, and water should be supplemented in the middle, so as to improve germination rate of seeds and reduce bacterial infection rate. Seeds with buds (about 1cm embryo and rolled seedlings) are rolled into filter paper and vertically stand in deionized water (the seeds are flush with the water surface and are most preferably 2-3 cm away from the top end of the filter paper), are cultivated in darkness for about 2-3 days, and then are cultivated in visible light (far from a high-pressure sodium lamp to avoid scorching of leaf tips) for one leaf and one heart (about 5 days). Removing endosperm, rolling with sponge seedling (1 cm upwards at the root of the first layer or 1.5 and cm at the junction between the upper part and the lower part), and culturing in 1/2 nutrient solution for two days (half nutrient solution culture, reducing illumination time and care intensity as much as possible, and avoiding charring of leaf tip). And starting full nutrient solution culture on the third day. The nutrient solution was changed every 2-3 days and cultured to the 5-leaf stage (about 2 weeks from the time of the transfer of the nutrient solution to the 5-leaf stage). KOH/H for nutrient solution 2 SO 4 The pH was adjusted to 5.8. The total nutrient solution contains 0.3 mM KH 2 PO 4 ,0.6 mM MgSO 4 , 0.5 mM K 2 SO 4 ,0.5 mM CaCl 2 ,1 μM H 3 BO 3 ,0.5 mM MnSO 4 、0.5 mM ZnSO 4 、0.2 mM CuSO 4 、0.07 mM Na 2 MoO 4 0.05M EDTA-FeNa with nitrogen of 1 mM (NH) 4 ) 2 SO 4 And 2 mM KNO 3 . Ventilation is necessary during the water culture to avoid root tip and root necrosis, and the size of bubbles is determined according to personal preference and the number of seedlings. The photoperiod of the plant cultivation room is 14 hours with a daytime period of 8:00-22:00, the photoperiod is 10 hours with a night period of 22:00-the next day of 8:00, and the illumination intensity is 500 mu mol m -2 s -1 The humidity is 60%, all hydroponic plants are best collected at about 2-6 pm, and the same time collection is ensured as much as possible.
(2) Tobacco seeds were directly sown in the prepared culture medium and cultured in a 26℃long sunlight greenhouse (16 h light, 8 h dark 100% humidity for 3 days, and then normally cultured in ambient humidity for 2 weeks.
2.2. Molecular cloning (homologous recombination method)
(1) PCR amplification of the target gene:
extracting genome DNA of corn plants, and carrying out PCR amplification by taking the extracted genome DNA as a template, wherein the PCR amplification system and the PCR amplification program are shown in tables 3 and 4.
TABLE 3 PCR amplification reaction System
TABLE 4 amplification Gene reaction procedure
(2) Vector enzyme tangential (double enzyme digestion), enzyme digestion system and conditions are shown in Table 5.
TABLE 5 cleavage reaction System and conditions
(3) Recovering the enzyme digestion product in the step (2) for recombination reaction, wherein the reaction system and the conditions are shown in table 6 (preparation on ice):
TABLE 6 fragment and vector recombination reaction systems and conditions
(4) Conversion (Advance of the Water bath to 42 ℃ C.)
Thawing DH5 alpha competence on ice; adding 5 μl of recombinant product into competence, mixing with light bullet wall (without shaking), and standing on ice for 15 min; heat shock 100 s at 42deg.C, immediately cooling on ice for 2-3 min; 1ml LB medium (without antibiotics) is added, shaking is carried out at 37 ℃ and 180-220 rpm for 1 h (the maximum time is not more than one half hour); the resistant plate is preheated, centrifuged at 5000 rpm for 5 min, plated at about 100. Mu.l and cultured upside down for 12-16 h.
(5) Colony PCR, systems and procedures are shown in table 7.
TABLE 7 colony PCR reaction System and procedure
(6) Shaking bacteria (adding corresponding antibiotics), extracting plasmid and sequencing
(7) The electric shock is transformed into agrobacterium (the electric shock cup is precooled on ice), and the specific steps are as follows:
cleaning the electric shock cup, airing, and preparing plasmids to be shocked; thawing Agrobacterium on ice to get competent (efficiency is higher than 25. Mu.l), adding 0.5-1. Mu.l of plasmid (less than 100 ng), mixing with gun head, transferring to pre-cooled electric shock cup, inserting electric shock cup into electric shock tank, selecting proper electric shock parameters to start electric shock, immediately adding 1 ml LB/YEB/SOC liquid culture medium into electric shock cup after electric shock is successful (no explosion), repeatedly sucking, transferring to new 1.5 ml centrifuge tube, 28 deg.C, 220 rpm, shake culturing for 2 h, directly taking 50-100. Mu.l without centrifuging, and culturing for 2 d at 28deg.C in incubator to obtain transformed colony.
2.3. Plasmid small handle (Nuowenzan kit)
(1) Picking single colony, placing into a test tube with 1-5 mL LB+antibiotics, shaking at 37 ℃ and 220 rpm for 12-16 h;
(2) Taking 1-5 mL bacterial liquid, 12000 rpm,1min, discarding supernatant (if bacterial liquid is more, the steps can be repeated);
(3) 250. Mu.L of Solution I/RNase A (stored in a refrigerator at 4 ℃) was added and the pellet resuspended by vortexing;
(4) Adding 250 μL Solution II, mixing, standing for 3 min;
(5) Adding 350 mu L of Solution III, and shaking vigorously;
(6) After 10 min at 13500 rpm, the supernatant was drawn into a filter column (note that no pellet was drawn);
(7) 13500 rpm,1 min later, the filtrate in the recovery column is poured off (the recovery column can be reused);
(8) Add 500. Mu.L of HBC buffer to the filter column (ensure buffer has been isopropanol loaded);
(9) 13500 rpm, and 1 min later, the filtrate is poured off (the recovery column can be reused);
(10) 700 μ L DNA wash buffer (absolute ethanol has been added previously) was added;
(11) 13500 rpm,10 min later, the filtrate is poured off (the recovery column can be reused);
(12) Repeating the steps (10) - (11), namely cleaning twice;
(13) 13500 rpm,2 min air separation, placing the filter column into a new 1.5 mL centrifuge tube, adding 30 μl of the solution Buffer (pre-heating at 65deg.C);
(14) Standing for 3-5 min, 13500 rpm for 1 min, collecting, measuring concentration, labeling, and storing at-20deg.C.
2.4. Plasmid big dipper (Nuowenzan kit)
(1) Sucking the preserved bacterial liquid into 200 mL liquid LB culture medium containing corresponding antibiotics one day before plasmid extraction, and culturing at 37 ℃ and 220 rpm overnight;
(2) Pre-column balancing, adding 2.5. 2.5 mL balancing solution BL into CP6 adsorption column, centrifuging at 8000 rpm for 2 min, pouring out liquid, and collecting the adsorption column;
(3) The thalli are collected by a 50 mL centrifuge tube for a plurality of times, and are centrifuged for 3 min at 8000 rpm each time, and the supernatant is discarded;
(4) Adding 10 mL solution P1 into the collected thalli, and shaking to thoroughly suspend the thalli cells;
(5) Adding 10 mL solution P2, turning over gently for 6-8 times, and standing at room temperature for 5 min;
(6) Adding 10 mL solution P4 into the centrifuge tube, gently turning over for 6-8 times, and standing at room temperature for 10 min;
(7) Centrifuging at 8000 rpm for 15 min, and filtering the supernatant with a filter CS1 into a clean 50 mL centrifuge tube;
(8) Adding 3 mL to the centrifuge tube to remove endotoxin solution ER, and reversing and uniformly mixing until the mixture is uniform and transparent yellow;
(9) Adding 0.3 times of isopropanol into the solution, reversing and uniformly mixing, and transferring to a pre-balanced CP6 adsorption column;
(10) Centrifuging at 8000 rpm for 2 min, discarding the waste liquid, and placing the adsorption column back into the centrifuge tube;
(11) Sequentially rinsing with 10 mL buffer ED and 10 mL rinsing liquid PW for 2 times, and centrifuging at 8000 rpm for 2 min;
(12) Air drying the adsorption column CP6 at room temperature for 5 min, adding 1 mL eluting buffer TB, standing at room temperature for 5 min, centrifuging at 8000 rpm for 2 min, and collecting the eluate for subsequent experiment or preservation at-20deg.C in refrigerator.
3. Corn protoplast preparation and transformation
(1) Only the middle part of the first true leaf of the maize yellowing seedling is cut into strips of 1mm, and the rest leaves are not used;
(2) Adding 20 mL enzymolysis solution (0.15 g Cellulase onomra RS, 0.04g Macerozy)Me R10, 6.25 mL 0.8M Manitol, 1.0mL of 0.2M MES (pH 5.7) and 0.1 mL of 2M KCl were mixed, sterilized water was added to make up to 10 mL,55℃for 10 min, cooled to room temperature, and 100. Mu.L of 1mM CaCl was added 2 And 100. Mu.L of 10% BSA), 3. Mu.L of 5 mM ME (mercaptoethanol) was added before and mixed well, and sterilized by suction with a pre-bacterial filter;
(3) Vacuum filtering for 30 min;
(4) Horizontal shaking table, 40 rpm,4 h,26 ℃, 60-80 rpm before taking out;
(5) The solution was filtered (pre-rinsed with W5) through a 35 μm nylon filter to a 50 mL round bottom centrifuge tube, and the residue was rinsed with a small amount of W5;
(6) 100 g (2. 2 g L2 g) for 5 min, sedimentation of protoplasts to the bottom of the tube, pipetting the supernatant; washing with precooled W5 for 2 times, 100 g (2. 2 g L.2. 2 g) for 2 min;
(7) Re-suspending the protoplast with a proper amount of pre-chilled W5, and standing on ice for 30 min (the period can be examined by microscopy); 100 g (2. 2 g up to 2. 2 g down) for 1 min, pipetting the supernatant and collecting protoplasts;
(8) MMG was resuspended (5 mL 0.8M mannitol, 0.2mL 0.2M Mes, 0.15mL 1M MgCl) 2 And 4.65mL dH 2 O) protoplasts to a final concentration of 5X 10 5 individual/mL; 15. Mu.g of plasmid and 200. Mu.L of protoplast were added to a 2mL centrifuge tube and gently mixed (scissor head);
(9) 220. Mu.L PEG (4 g PEG 4000, 2.5 mL 0.8M mannitol, 1 mL 1M CaCl) 2 And 3 mL dH 2 O) lightly mixing; incubating for 18 min at room temperature in dark; adding 800 mu L W5, and gently mixing to terminate the reaction;
(10) 100 g (2. 2 g up to 2. 2 g) for 2 min, discarding the supernatant;
(11) 1 mL of W5 was added, gently mixed, 100 g (L2 g ), 2 min, and the supernatant discarded;
(12) 1 mL WI and 1 mL W5 were added; 25. at the temperature of 12-18 ℃ and h in the dark.
4. Transient transformation of tobacco leaves and extraction of original plastids
4.1 transient transformation of tobacco leaves
(1) Picking agrobacterium containing target plasmid and agrobacterium containing P19 vector into 3-5 mL LB liquid culture medium containing corresponding antibiotics, shaking the shaking table at 200 rpm at 28 ℃ for overnight (about 16-20 h);
(2) Centrifuging at 4000 rpm for 10 min, removing supernatant, and collecting thallus;
(3) Infection with 5 ml buffer (10 mM MES, 200. Mu.M Ace,10 mM MgCl) 2 ) Re-suspending the thalli, adding an equal volume of infection buffer solution, uniformly mixing, centrifuging at 4000 rpm for 10 min, removing the supernatant, and collecting thalli;
(4) Re-suspending the bacterial cells by using a proper amount of infection buffer solution, detecting the OD600 light absorption value of the re-suspended bacterial cells by using the infection buffer solution as a control, and adjusting the OD600 value of each bacterial cell to be 0.4;
(5) Placing the prepared suspension on a horizontal shaking table, culturing in a dark place for 2 h, and injecting tobacco leaves;
(6) Selecting tobacco with good growth vigor in the 2-week-old period, mixing the suspension bacteria liquid to be co-transformed in equal volume, removing the needle head of a 1mL syringe to inject the back of tobacco leaves, and marking the injection position and the injection content;
(7) After 1 day of dark light culture of the injected tobacco, the tobacco is cultured for 2 days at normal light temperature and can be used for sampling and observation.
4.2 extraction of protoplast from tobacco leaves
(1) Immediately after gently rubbing the lower epidermis of the tobacco leaf with 1000 mesh sandpaper, the rubbed surface was placed flat into an enzymatic hydrolysate containing 0.3% (w/v) R-10,0.03% (w/v) Y-23,1 mM CaCl2, 500 mM sorbitol, 10 mM MES-KOH (ph=5.3), 28 ℃,1 h.
(2) Protoplasts were collected at 4℃and 100 g (2. 2 g L.2. 2 g L.) and washed twice with washing solution containing 1 mM CaCl 2 500, mM sorbitol, 10 mM MES-KOH (pH=5.3).
(3) Placed on ice for 30 min for subsequent experiments.
Subcellular localization of zmammt 9
Step 3 after preparation and transformation of corn protoplasts, by performing high penetration treatment on the protoplasts after transformation incubation, the results of vacuole release observation show that zmammt 9 is mainly localized on the vacuole membrane, while even GFP is only expressed in cytoplasm when empty (fig. 2 a);
CDS of ZmALMT9 is constructed on a p1300-GFP vector to obtain 35s of GFP-ZmALMT9; TPK, a potassium ion transporter typically known to localize on the vacuolar membrane, was constructed onto the p1300-mCherry vector to give 35s by TPK-mCherry co-infection of tobacco, and the results show that co-localization of the two proteins occurs (FIG. 2B), indicating that the maize gene remains consistent with the localization results of maize protoplasts in tobacco protoplasts. ZmALMT9 is mainly localized to the vacuole membrane, and its localization is consistent with that of the Arabidopsis homologous gene AtALMT 9.
Tissue expression of ZmALMT9
5.1 review of chip data (MaizeGDB website: https:// www.maizegdb.org /) found that ZmALMT9 was expressed in both the aerial and subsurface parts of maize, with aerial expression being stronger than in the subsurface part (FIG. 3A);
5.2 real-time fluorescent quantitative PCR
Wild-type corn tissue was taken for RNA extraction, and total RNA was extracted from plant samples using RNAiso Plus Kit (Takara). The cDNA was generated by PrimeScript RT kit (Takara). The RT-qPCR uses a primer and a Renzan SYBR Premix Ex TaqTM II kit. The primer sequences used in this step are shown in Table 2. The fluorescence quantification system is as follows: 2. Mu.L of template, 1. Mu.L of 10. Mu.M upstream primer, 2 XSYBR 10. Mu.L of TaqTM II, ddH 2 O was made up to 20. Mu.L. The reaction process comprises the following steps: 95. 5 min at the temperature; 95. ct values were recorded for 30 s 40 cycles at 15 s,60℃for subsequent quantitative analysis. The results show that ZmALMT9 in various tissues and organs (root, stem, leaf, sheath and seed) of the maize seedling stage is expressed higher in the leaf and the seed, the expression multiple of the overground part is nearly 8 times of the root expression (B in FIG. 3), and the real-time fluorescence quantitative PCR is consistent with the chip data result.
5.3 in order to further clarify the expression in the tissue, the experiment was carried out by depending on the principle of the combination of the biological sciences of the Nuo, and by observing the red light target signal region, it was further confirmed that the expression of ZmALMT9 in the aerial part was stronger than that in the underground part, and that the gene was strongly expressed in the aerial vascular bundle system, and in addition, it was found that there was strong expression in the stomata of the epidermis, and that there was a certain expression in the root cortex, and that the expression in the aerial part was stronger than that in the root (C in FIG. 3).
The above results indicate that zmammt 9 is expressed more strongly above the ground than below and is mainly expressed in the above-ground vascular bundle system and stomata.
Transport substrate analysis of ZmALMT9
Analysis of transport substrates for ZmALMT9 based on patch clamp experiments
Two weeks of greenhouse-grown tobacco plants were transiently transformed with agrobacterium tumefaciens (GV 3101) containing GFP-zmammt 9 construct by agrobacterium infiltration, after 48 hours, tobacco protoplasts were extracted and vacuoles of the tobacco protoplasts were observed using an inverted fluorescence microscope (Nikon Ti 2). Referring to the prior art, ion channel current was recorded using an outside-out approach in patch clamp technology. Briefly, patch pipettes were prepared using a P-97 micropipette puller (Sutter Instruments), a resistance of 5-6M Ω was obtained, the seal resistance was above 2G Ω, and the current and Axon Digidate 1550B were recorded using an Axon 200B patch clamp amplifier. Data were analyzed using a Clampfit 11. The extracellular fluid contains 100 mM malic acid and 3 mM MgCl 2 、0.1 mM CaCl 2 And the pH was adjusted to 7.5 with BTP and the osmotic pressure was adjusted to 500 mOsm with sorbitol. The intracellular fluid contained 100 mM malate, adjusted to pH 6, and the osmotic pressure was adjusted to 550 mOsm using sorbitol. According to previous studies (Neher E. Correction for liquid junction potentials in patch clamp experiments [ J) ]Methods enzymes, 1992, 207:123-31.) liquid junction potentials were measured and corrected beyond +2 mV. Sequential priming of cytoplasmic side buffers was performed using a custom gravity driven priming system. Experiments show that a single vacuole which can be positioned correctly can be found under a fluorescence microscope, at the moment, a glass electrode filled with the vacuole inner liquid is used for clamping a vacuole membrane (A in fig. 4) by adopting an everting and external stabilizing clamp, clamping is carried out between +60 mV and-120 mV, the voltage gradient is set to be 20 mV, and then the operation is carried outRow current intensity detection (B in fig. 4). In the experiment, tobacco vacuoles expressing empty plasmids were used as a control group, and the current results showed that the ZmALMT 9-expressing vacuole membrane pair Mal was compared with the control group 2- 、NO 3 - And Cl - Has obvious current with the order of Mal 2- >NO 3 - >Cl - ZmALMT9 translocates Mal under conditions of-120 mV 2- The average current of the (E) is-1.7 nA +/-0.31 nA, and NO is transported 3 - The current average size of (a) is-0.6 nA +/-0.17 nA; to transport Cl - Only-0.37 nA.+ -. 0.12 nA, while under the same conditions, the control group failed to record significant current, indicating that ZmALMT9 is capable of transporting at least Mal on the vacuole membrane 2- 、NO 3 - And Cl - These three anions. The ability of zmarmt 9 to mediate the excretion of these three anions into the vacuole lumen was demonstrated by scaling the transport capacity of each anion relative to the control, with zmarmt 9 having a greater capacity to transport malate ions relative to chloride and nitrate (C-E in fig. 4). The above results indicate that zmarmt 9 is an anion channel protein, not a specific transporter, which can transport at least Mal 2- 、NO 3 - And Cl - Into the vacuole lumen, where Mal is transported 2- Is the most powerful. .
In order to determine the relation between the transport capacity and the substrate concentration of ZmALMT9, the invention realizes the replacement of a cytoplasmic solution under the condition of clamping the same vacuole by means of a tobacco transient expression system and a gravity perfusion system. This approach allows for more accurate recording of the changes in zmarmt 9 transport for different concentrations of substrate. Taking here the example of changing different nitrate ions, it was found that the current intensity of zmammt 9 decreases with decreasing nitrate concentration, and the same rule is also shown from the graph (fig. 5 a and B).
The above results indicate that the transport capacity of zmarmt 9 is dependent on the concentration of cytoplasmic substrates, which is positively correlated with the concentration of substrates in cells.
7. Expression pattern of vacuolar membrane anion channel zmammt 9 response to nitrogen
To investigate the response of maize gene zmammt 9 to nitrogen, wild-type maize was subjected to nitrogen-deficiency treatment for one, two and four days, and quantitative results showed that the expression of zmammt 9 was significantly reduced by the effect of nitrogen deficiency at two and four days of nitrogen-deficiency treatment, to 36% and 27% respectively, and the expression level was reduced with the increase of nitrogen deficiency time (fig. 6 a), indicating that the expression of zmammt 9 was inhibited by nitrogen deficiency. To investigate the effect of restoring nitrogen supply to zmammt 9 expression after nitrogen deficiency, corn plants after nitrogen deficiency treatment were restored to supply ammonium salt and nitrate for 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 72 hours, respectively, and quantitative results showed that the expression level of zmammt 9 exhibited a significant increase with the increase of nitrate supply time, wherein the expression level did not significantly change with time from 6 to 72 hours, and the expression level was about 2 times that during nitrogen deficiency. For the nitrogen deficiency recovery ammonium salt, the expression level of zmarmt 9 did not change significantly with the time of ammonium salt supply (B in fig. 6). The above results demonstrate that zmarmt 9 response to nitrogen is somewhat induced by nitrate, but the degree of induction is relatively low, presumably in a relationship with nitrate utilization of zmarmt 9.
Example 3
1. Plant material
Depending on the functional genome and molecular breeding research center of crops in China agricultural university, the CRISPR/Cas9 gene editing technology is utilized to carry out gene editing on maize inbred ND101, a plurality of transgenic mutant materials of zmalmt9 are successfully obtained, and through screening offspring, stable genetic mutation homozygous lines are obtained. In addition, a plurality of ZmALMT9 overexpression materials are genetically created, stable genetic overexpression homozygous lines are obtained through screening offspring, two independent overexpression transgenic lines are selected for physiological experiment verification through semi-quantitative and quantitative detection, the two independent overexpression transgenic lines are named as ZmALMT9-1 and ZmALMT9-2 respectively, the expression fold of the ZmALMT9-1 is increased by nearly 30 times compared with that of the wild type through normalizing the quantitative results of the overexpression materials, and the expression fold of the ZmALMT9-2 is increased by nearly 60 times (B and C in FIG. 7). The successful acquisition of the mutants and the over-expression transgenic materials can be used for subsequent physiological phenotype experiments.
2. Zmalmt9 mutant phenotype under different nitrogen treatments
To investigate the function of zmammt 9 for nitrogen utilization, the physiological function of zmammt 9 gene was further evaluated by phenotypic identification using low nitrogen (LN, 0.04 mM) and high nitrogen (HN, 4 mM) nutrient treatments set up by hydroponics with ND101, zmammt 9-1 and zmammt 9-2 as materials, and the results showed that the two mutants were not significantly different under low nitrogen conditions compared to wild type ND101, but that the growth of mutants zmammt 9-1 and zmammt 9-2 was inhibited under high nitrogen treatment (a in fig. 8). The physiological indexes of plant height, biomass, SPAD, malic acid concentration, total nitrogen concentration and other anion concentrations are investigated and counted;
biomass and nitrogen content determination: after the corn seedlings are transferred to a water culture solution for nitrogen treatment for 12 days, the overground parts and root systems are separately harvested, and then are deactivated for 30 minutes at 105 ℃, then are dried to constant weight at 70 ℃, the dry weight is weighed, a sample is ground into powder by a grinder, and the carbon and nitrogen content is measured by a carbon and nitrogen analyzer by weighing 100 mg;
malic acid concentration determination: seedlings were watered for 12 days, and the content of transport substrate (malic acid) was measured according to the method described previously (Delhaize E, ryan P R, randall P J. Aluminum tolerance in wheat (Triticum aestivum L.) (II. Aluminum-stimulated excretion of malic acid from root apices) [ J ]. Plant Physiology, 1993, 103 (3): 695-702.). Briefly, samples were ground to a fine powder in liquid nitrogen, 0.1. 0.1 g fresh samples were weighed, added to 1 mL extract and incubated at 75 ℃ for 15 minutes. After centrifugation, the supernatant was taken and mixed with 2.4 mL buffer (0.1M glycine, 0.1M glutamic acid, 2 mM NAD and 2 mL 1KU GOT). The mixture was then pre-incubated for 10 minutes to obtain a stable absorbance reading at 340 nm, then 5 μl of 5 KU malate dehydrogenase was added to activate the reaction for 10 minutes. Absorbance readings before and after malate dehydrogenase addition were recorded for calculation of malate content. Under the condition of low nitrogen, the strain heights of the wild type ND101 and the two mutants are reduced by about 16% and 13% compared with the wild type ND101, the corresponding aboveground biomass is reduced by about 40% and 35%, the SPAD is reduced by 40% and 37%, the malic acid concentration is reduced by about 21% and 14%, the nitrogen concentration is not obviously different, the nitrate ion concentration of the two mutants zmalmt9-1 and zmalmt9-2 is obviously higher than that of the wild type ND101, and the chloride ion, the sulfate ion and the phosphate ion are not obviously different (B-G in figure 8).
The above results indicate that zmammt 9 may be involved in regulating the utilization of nitrogen and malic acid, and considering that the deficiency of zmammt 9 leads to plant growth retardation only under high nitrogen conditions, particularly in plant height, biomass, reduction of SPAD and malic acid concentration and accumulation of nitrate ions, it is presumed that the deficiency of zmammt 9 leads to reduction of plant nitrogen utilization capacity, imbalance of intracellular malic acid steady state, reduction of nitrate ion assimilation capacity, and accumulation of nitrate ions.
3. Phenotype of ZmALMT9 overexpressing plants under different nitrogen treatments
To fully verify the results of the early experiments, the water culture was set up (LN, 0.04 mM) and high nitrogen (HN, 4 mM) according to the previous experimental system, and the obtained overexpressed maize plants were phenotypically observed, and compared with the wild type ND101, the two overexpressers ZmALMT9-1 and ZmALMT9-2 were not significantly different in growth under two nitrogen treatments (FIG. 9A), and physiological indexes of statistical plant height, biomass, SPAD, malic acid concentration, total nitrogen concentration, and other anion concentrations were investigated. The determination of biomass, nitrogen content and malic acid concentration is consistent with the determination of the step 2.
The results showed no significant differences in plant height, biomass, nitrogen concentration, and concentration of the four anions at both treatments (B and C in fig. 9, F and G in fig. 9). There was no significant difference in SPAD values and malic acid concentrations at low nitrogen, and under high nitrogen treatment, the overexpressing bodies zmammt 9-1 and zmammt 9-2 were significantly higher than the wild-type ND101, with 18% and 19% increase in malic acid concentration of the overexpressing bodies zmammt 9-1 and zmammt 9-2, respectively, and 11% and 27% increase in SPAD, respectively (D and E in fig. 9), with the increase in zmammt 9-2 being greater than zmammt 9-1, which may be due to the higher fold of overexpression compared to the overexpressing plant zmammt 9-2 than the overexpressing plant zmammt 9-1 (B and C in fig. 9). Overexpression of zmammt 9 did not affect plant growth, but resulted in increased SPAD and malic acid concentration in plants under high nitrogen conditions. In combination with the mutant phenotype differences, it can be seen that zmarmt 9 plays an important role in regulating malic acid homeostasis in plants, and that changes in malic acid concentration disrupt the process of nitrogen utilization, resulting in a growth-inhibited phenotype.
4. Restoration of zmalmt9 mutant growth phenotype by exogenous malic acid
The above results initially demonstrate that the stunted growth of zmalmt9 mutants is associated with insufficient malate, and in order to further verify that the suppressed growth of the mutants is due to insufficient malate, attempts were made to rescue the growth suppression phenotype of zmalmt9-1 mutants by exogenous supplementation of malate using ND101 and zmalmt9-1 as materials. For this purpose, referring to The previous study (Heng Y, wu C, long Y, et al OsALMT7 maintains panicle size and grain yield In rice by mediating malate transport [ J ]. The Plant Cell, 2018, 30 (4): 889-906; pei Y, deng Y, zhang H, et al EAR APICAL DEGENERATION1 regulates maize ear development by maintaining malate supply for apical inflorescence [ J ]. The Plant Cell, 2022, 34 (6): 2222-41.), three different methods (injection of stems and leaves (In), foliar spray (Sp) and hydroponic exogenous addition (Hy)) were used to apply The 2 mM malic acid solution.
The results showed that only exogenous injection of malic acid completely rescued the phenotype of zmalmt9-1 mutant (a in fig. 10), and that the dry weight on the ground was similar to wild-type ND101 (B in fig. 10). However, foliar spray and hydroponic treatments not only fail to rescue the zmalmt9-1 mutant phenotype, but also pose other hazards to the plant, such as leaf tip cell death due to foliar spray and root growth inhibition due to hydroponic treatment (fig. 10 a). Mutant phenotypes are generated due to reduced malate in the plant body, and exogenous injection of malate can restore the mutant phenotype.
Transcriptome analysis of zmalmt9 mutant transgenic Material
In order to fully understand the molecular mechanism of zmammt 9, the invention performs RNA-seq analysis based on the time and aerial part material of the previously observed wild type and mutant seedling stage growth difference phenotypes, specifically as follows: after 12 days of high and low nitrogen treatment, the upper parts were uniformly taken after 4 hours from the beginning of illumination, and rapidly packed with tinfoil paper and placed in liquid nitrogen to be used as transcriptome sequencing samples. Each treatment was performed with 3 biological replicates. Total RNA was extracted using RNA kit (Ai Kerui). And (5) constructing a library by using NEB Next Ultra RNA Library Prep Kit, and sequencing on an Illumina HiSeq 4000 after the library quality is checked to be qualified. Analysis was performed using a conventional R-packet.
From the obtained transcriptome data, 314 significant up-regulated genes are identified under high nitrogen treatment, 366 down-regulated genes are identified (A in fig. 11), KEGG enrichment analysis is carried out on the differential genes, and results show that a part of differential genes under high nitrogen treatment are obviously enriched in biological processes related to carbon nitrogen metabolism, photosynthesis and organic acid metabolism (B in fig. 11), then heat mapping is carried out on the differential gene expression of the processes, and results show that mutant zmalmt9-1 shows up-regulation to different degrees in terms of carbon nitrogen metabolism compared with wild type ND101, and it is worth noting that key genes involved in malate synthesis and degradation are significantly changed in the mutant, wherein genes coded by malate metabolism related enzymes such as malate dehydrogenase MDH, NAD-malate enzyme NADH, malate synthase ME are expressed up-regulated in the mutant, in addition, nitrate reductase NR and nitrite reductase NIR are expressed up-regulated in the mutant, and the mutant glucose synthase GOGAT, acetamidate, hydrolase NAD-MDH and glutamate synthase are involved in the down-regulation of glutamate metabolism related to different degrees in the mutant, and the mutant also has up-regulation to the corresponding degrees in the mutant, and the mutant has been subjected to the following up-regulation to the following. It is known from the search of related reports that MDH is involved in regulating the light respiratory pathway, IAM is involved in the biosynthesis of auxin and Arabidopsis thaliana in plant development, GDHs may play an important role in the process of nutrient remobilization, and PCK is involved in the process of closed pores of malate metabolism induced by darkness. The above results suggest that the mutant plants involved in carbon and nitrogen metabolism genes were altered, further suggesting that mutant growth inhibition may be due to imbalance in malate homeostasis in cells resulting in carbon and nitrogen metabolism.
Broad target metabolome analysis of zmalmt9 mutant transgenic materials
Earlier studies found that zmarmt 9 participates in regulating and controlling carbon nitrogen and malic acid metabolic processes, so that the step adopts a broad target metabolome to analyze the differential metabolites of wild type ND101 and mutant zmarmt 9-1 under high nitrogen conditions, and the specific steps are as follows: after 12 days of high and low nitrogen treatment, wild ND101 and mutant zmalmt9-1 were uniformly taken from the upper part 4 hours after the beginning of illumination, and rapidly packed with tinfoil paper and placed in liquid nitrogen to be used as a metabolome sequencing sample. Each treatment was repeated for 5 organisms. Sample pretreatment and test conditions depending on the Megaku biological company. Analysis was performed using a conventional R-packet.
The results show that the correlation between the sequencing quality and biological repetition of the experiment is good. The expression mode of the metabolite in each sample and the P value of the metabolite in the VIP and single-dimensional statistics of multivariate statistical analysis are displayed through clustering the heat map and the VIP bar graph, so that the importance and the expression quantity trend change of the differential metabolite are intuitively seen. Among them, the results showed that metabolites of asparagine, glucoside, methyl, fisetin, squalene, benzoic acid, etc. were significantly reduced in the mutants, whereas malonic acid, tartaric semialdehyde, seabuckthorn glycerol, gibberellin a12 aldehyde, trioside, etc. were significantly accumulated in the mutants (a in fig. 12). According to the comparison of metabolites with KEGG pathways, it was found from the secondary classification of KEGG that the differential metabolism was mainly involved in several major metabolic processes such as amino acid metabolism, carbon metabolism, lipid metabolism, etc. (B in fig. 12). Analysis of the enriched pathways revealed that the differential metabolites were mainly involved in the processes of arachidonic acid metabolism, arginine metabolism, flavonoid biosynthesis, lysine degradation, arginine and proline metabolism, alanine, aspartic acid and glutamic acid metabolism, etc. (C in fig. 12). The results show that ZmALMT9 is involved in the metabolism of various amino acids in the carbon-nitrogen metabolic pathway during biological metabolism.
7. Photosynthetic and nitrogen utilization capacity of zmalmt9 mutants
Carbon and nitrogen metabolism are two most basic metabolic processes of plants, carbon metabolism provides a carbon source and energy for nitrogen metabolism, and nitrogen metabolism provides enzymes and photosynthetic pigments for carbon metabolism, which have a common carbon skeleton, ATP and reducing power. Carbon metabolism is based on photosynthesis, and nitrogen metabolism is based on assimilation of nitrogen absorbed by the root system into amino acids. Thus, to demonstrate whether zmarmt 9 affects carbon and nitrogen utilization in plants, determinations of photosynthetic capacity and free amino acids were made;
photosynthetic rate determination: the hydroponic seedlings were selected for 12 d, the newly developed leaves of the plants were selected uniformly after 4 h of the light were turned on, and the leaf photosynthetic rate (Pneoto) was measured using a portable photosynthesis system (Li 6400XT; li-COR, lincoln, NE, USA). Before the machine is used, the quality inspection is required to be qualified. Measurement of the light intensity of the leaf chamber at 500. Mu. Mol.m -2 •s -1 ,CO 2 CO filling into small steel cylinder 2 At a concentration of 400. Mu. Mol -1 Leaf temperature 27 ℃.
Chlorophyll determination: corn leaves (liquid nitrogen ground into powder) or pieces of about 0.2. 0.2 g are weighed, added to a 50 ml centrifuge tube, and 25 ml of a 2:1 acetone ethanol solution is added. Leaching 14 h in the dark (preferably if shaking extraction is possible) until the leaves are completely whitened. The leaf extract was placed in a cuvette of 1 cm, and the extract was used as a blank and subjected to color comparison on a spectrophotometer. (note: dilute under test if absorbance is too great);
The calculation formula is as follows: chlorophyll a (mg/g) = (12.72A663-2.59A645) ×v/1000/W
Chlorophyll b (mg/g) = (22.88 a 645-4.67A663) ×v/1000/W
Total chlorophyll amount (mg/g) = (20.29a645+8.05a663) ×v/1000/W
Determination of free amino acid content: grinding fresh leaves into powder in liquid nitrogen, freeze-drying in a freeze dryer, weighing 20 mg, adding 1 ml of water, ultrasonically extracting for 30 min, centrifuging at 14000 rpm for 10 min, filtering with a 0.1 mu m syringe and a filter, taking 10 mu L of sample, adding 50 mu L of borate buffer solution, mixing uniformly, adding 20 mu L of derivatizing agent while vibrating on an oscillator, immediately vortex mixing uniformly, standing at room temperature for 1 min, vibrating at 55 ℃ for 10 min, cooling to room temperature, centrifuging for 20 min, taking the supernatant, filling into a sample injection plastic chromatographic vial, and detecting by a chromatographic column on the machine: BEH C18.1X100 mm 1.7 μm, column temperature 40℃and flow rate 0.3. 0.3 ml/min.
The results show that under the low nitrogen condition, the photosynthetic efficiency, chlorophyll content and Fv/Fm of the wild ND101 and the mutant zmalmt9 are not obviously different, but under the high nitrogen condition, the photosynthetic efficiency, chlorophyll content and Fv/Fm of the mutant zmalmt9 are obviously smaller than those of the wild ND101. Compared to the wild type, the photosynthetic efficiency of the two mutants was reduced by 19% and 18% (A in FIG. 13), chlorophyll content was reduced by 19% and 12% (B in FIG. 13), fv/Fm was reduced by 2.5% and 4.5% (C in FIG. 13), respectively. Further by targeting changes in metabonomic analysis of amino acids, experimental data showed that the total amino acid concentration of mutant zmalmt9 was significantly less than that of wild type (D in fig. 13), with predominantly glutamine, serine, glutamic acid, asparagine, and aspartic acid being significantly reduced in the mutant (E in fig. 13). Just as the transcriptomic and metabonomic indications are consistent, zmarmt 9 is involved in regulating carbon-nitrogen utilization processes. ZmALMT9 is mainly responsible for transporting excessive malic acid into vacuoles for storage under the condition of high nitrogen, and once the process is lost, the malic acid metabolism in cells is disturbed, photosynthesis and amino acid metabolism are affected, so that the carbon and nitrogen utilization capacity of plants is reduced.
From the above, it can be seen that zmarmt 9 regulates plant carbon-nitrogen metabolism by maintaining malic acid homeostasis to participate in the physiological function of carbon-nitrogen utilization.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. The application of the corn ZmALMT9 gene or the coded protein thereof in regulating and controlling the carbon nitrogen metabolism of plants.
2. The use of claim 1, wherein said modulating plant carbon nitrogen metabolism comprises: regulate photosynthesis and amino acid metabolism of plant.
3. The use of claim 1, wherein said modulating plant carbon nitrogen metabolism comprises: regulate malic acid metabolism of plant.
4. The use according to claim 3, wherein said modulating malic acid metabolism of a plant comprises: and under the condition of 4 mM nitrogen stress, the malic acid metabolism of the plants is regulated and controlled.
5. The use according to claim 4, wherein the nitrogen comprises ammonium salts and/or nitrate salts.
6. The use according to any one of claims 1 to 5, wherein the modulation of plant carbon nitrogen metabolism comprises: upregulating expression of the maize zmarmt 9 gene promotes carbon-nitrogen metabolism of the plant.
7. The use according to claim 6, wherein the plant comprises maize and/or tobacco.
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