Corn monoglyceride lipase gene ZmMAGLL and application

Abstract

The application belongs to the field of molecular biology and biotechnology, and provides a maize monoacylglycerol lipase gene ZmMAGLL and application. The maize monoacylglycerol lipase gene ZmMAGLL has a nucleotide sequence shown as SEQ ID NO:1, and the maize monoacylglycerol lipase gene ZmMAGLL encodes a maize monoacylglycerol lipase with an amino acid sequence shown as SEQ ID NO:2. The application provides a cold-resistant maize inbred line W9816, a monoacylglycerol lipase gene ZmMAGLL and an amino acid sequence of a maize monoacylglycerol lipase encoded by the maize monoacylglycerol lipase gene ZmMAGLL. By using an agrobacterium-mediated transformation method, the ZmMAGLL plant expression vector is successfully transformed into Arabidopsis thaliana, and a homozygous T3 generation transgenic Arabidopsis thaliana plant is obtained. After low-temperature stress treatment, the phenotype observation shows that the maize monoacylglycerol lipase gene ZmMAGLL can improve the cold resistance of Arabidopsis thaliana.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biotechnology, and particularly relates to the corn monoacylglycerol esterase gene ZmMAGLL and its applications. Background Technology

[0002] Maize (Zea mays L.), one of the most widely cultivated crops in the world, is an important food, feed, and cash crop in my country. However, abiotic stresses such as low temperature and drought pose a serious threat to maize growth and yield. This is particularly true in the spring maize production areas of Northeast and North China, where cold spring weather and insufficient accumulated temperature lead to harsh conditions of low temperature and high humidity during spring sowing, resulting in a series of problems such as seed rot, failure to germinate, weak seedlings, and uneven emergence. Maize originated in tropical latitudes; low temperatures between 0-15 degrees Celsius affect cell membrane fluidity, causing electrolyte leakage and energy and metabolic imbalances within the plant. Simultaneously, low temperatures promote the accumulation of reactive oxygen species, causing oxidative damage to proteins and DNA, and peroxidation of membrane lipids. Furthermore, the impact of low temperatures on enzyme activity directly affects photosynthesis and respiration. All these factors can lead to poor germination, slow seedling growth, and in severe cases, even plant death, reducing crop quality and yield. Especially at temperatures below zero degrees Celsius, ice crystals form inside and outside cells, posing an even greater threat to plant survival. Therefore, identifying low-temperature-related genes and studying their gene functions and molecular mechanisms is of great significance for breeding new cold-resistant maize varieties.

[0003] Changes in lipid composition and unsaturation have long been associated with plant cold tolerance. Under low-temperature stress, the contents of glycolipids, some phospholipids, and glycerides in maize leaves decrease significantly, while the levels of phosphatidic acid, lysophosphatidic acid, and diglycerides increase significantly. Furthermore, the significantly elevated levels of polyunsaturated triacylglycerols and phosphatidic acid also demonstrate that maize remodels its lipids and alters their unsaturation under low-temperature conditions.

[0004] Monoacylglycerol lipases are involved in catalyzing the hydrolysis of monoacylglycerol into free fatty acids and glycerol, but research on them in plants is relatively limited. In Arabidopsis, 16 hypothesized MAGLs (AtMAGLs) have been reported to have three-dimensional structures similar to human MAGLs. Heterologous expression and enzyme assays revealed that 11 of these encoded proteins indeed possess MAG lipase activity. Furthermore, some AtMAGLs also exhibit hydrolytic activity on lysophosphatidylcholine and lysophosphatidylethanolamine substrates, and are located in the endoplasmic reticulum, Golgi apparatus, cytoplasm, or chloroplasts. AtMAGL4, located in chloroplasts, showed that its transcriptional level was upregulated by more than 11-fold and 5-fold in the aboveground parts and roots of 2-3 week old Arabidopsis after salt, osmotic, and cold stress treatments, respectively. In addition, a study reported the homolog of AtMAGL8 in rapeseed, BnaC.MAGL8.a, and found that overexpression of BnaC.MAGL8.a in Arabidopsis led to pollen defects. Transcriptome analysis revealed 398 differentially expressed genes compared to the wild type, primarily involved in pollen development and stress response. However, whether the ZmMAGLL gene in maize plays a crucial role in plant resistance to cold stress, and how it specifically functions, remains unclear. Therefore, this invention proposes the maize monoacylglycerol esterase gene ZmMAGLL and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide the maize monoacylglycerol esterase gene ZmMAGLL and its applications, aiming to solve the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The maize monoacylglycerol esterase gene ZmMAGLL has an open reading frame (ORF) of 1170 bp, with the start codon ATG and the stop codon TGA. Its nucleotide sequence is shown in SEQ ID NO:1.

[0008] Furthermore, the amino acid sequence of the corn monoacylglycerol esterase encoded by the corn monoacylglycerol esterase gene ZmMAGLL is shown in SEQ ID NO:2.

[0009] The recombinant plant expression vector contains the maize monoacylglycerol esterase gene ZmMAGLL as described above.

[0010] The application of the maize monoacylglycerol esterase gene ZmMAGLL or the recombinant plant expression vector described above in improving plant cold tolerance, wherein the plant is Arabidopsis thaliana.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] This invention provides the monoacylglycerol esterase gene ZmMAGLL from the cold-tolerant maize inbred line W9816 and the amino acid sequence of the encoded maize monoacylglycerol esterase. Using Agrobacterium-mediated transformation, the ZmMAGLL plant expression vector was successfully transformed into Arabidopsis thaliana, yielding homozygous T3 generation transgenic Arabidopsis plants. Phenotypic observation after low-temperature stress treatment showed that the maize monoacylglycerol esterase gene ZmMAGLL can improve the cold tolerance of Arabidopsis thaliana. This invention provides a new gene resource for elucidating the mechanisms of plant cold tolerance, and also provides important theoretical basis and technical support for the breeding of cold-tolerant crop varieties and the improvement of stress resistance, possessing significant application value for the breeding of new cold-tolerant maize varieties. Attached Figure Description

[0013] Figure 1 This is a multiple sequence alignment diagram.

[0014] Figure 2 This is a schematic diagram of the phylogenetic analysis of the ZmMAGLL gene in maize; where AtMAGL represents Arabidopsis thaliana and ZmMAGLL represents maize.

[0015] Figure 3 This is a schematic diagram of Arabidopsis thaliana phenotypes under 14℃ treatment conditions.

[0016] Figure 4 A schematic diagram showing the fresh weight of the aboveground parts of Arabidopsis thaliana under 14℃ treatment.

[0017] Figure 5 This is a schematic diagram showing the determination of photosynthetic fluorescence parameters in Arabidopsis thaliana under 14℃ treatment conditions. Detailed Implementation

[0018] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0019] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0020] Example 1: Cloning of the maize monoacylglycerol esterase gene ZmMAGLL;

[0021] 1. RNA extraction;

[0022] Total RNA was extracted from the leaves of cold-tolerant maize inbred line W9816 at the three-leaf stage using a Kangwei Century ultrapure RNA extraction kit (CW0581). The specific steps are as follows:

[0023] (1) Take fresh corn leaves, grind them thoroughly in liquid nitrogen, add 1 ml of TRIzonReagent for every 30-50 mg of tissue, and mix well.

[0024] (2) After adding TRIzon Reagent, gently invert the sample several times to ensure complete lysis. Incubate at room temperature for 5 minutes to allow the protein-nucleic acid complex to be completely separated.

[0025] (3) Add 200 μl of chloroform, cover the centrifuge tube, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes.

[0026] (4) Centrifuge at 12000 rpm for 10 min at 4℃, aspirate 550 μl of the upper aqueous phase and transfer it to a new RNase-Free centrifuge tube.

[0027] (5) Add 550 μl of 70% ethanol (prepared with RNase-free water) to the aqueous solution and mix by inverting.

[0028] (6) Add all the solution obtained in the previous step to the adsorption column (Spin Columns RM) that has been loaded into the collection tube. If the solution cannot be added all at once, it can be added in multiple batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0029] (7) Add 700 μl of Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0030] (8) Add 500 μl of Buffer RW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0031] (9) Repeat step (8).

[0032] (10) Centrifuge at 12000 rpm for 2 min and discard the waste liquid in the collection tube. Place the adsorption column at room temperature for a few minutes to dry.

[0033] (11) Place the adsorption column in a new RNase-free centrifuge tube, add 30-50 μl of RNase-Free Water to the middle of the adsorption column, incubate at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, collect the RNA solution, and store at -80℃ to prevent degradation.

[0034] 2. Reverse transcription;

[0035] The extracted RNA was reverse transcribed using US EVERBRIGHT RT mix with DNase (All-in One). The reaction system is shown in Table 1.

[0036] Table 1 Reverse transcription reaction system

[0037] reagents volume 5×RTAll-in-OneMix 4μl DNase 1μl Template RNA 1μl RNaseFreeWater 14μl 20μl

[0038] Reaction program: 37℃, 2 min; 55℃, 15 min; 85℃, 5 min.

[0039] 3. Amplification of the full-length CDS of the ZmMAGLL gene;

[0040] Based on the CDS gene sequence of maize ZmMAGLL published by NCBI, specific cloning primers for this gene were designed using the bioinformatics software Primer 5.0 (following primer design principles):

[0041] ZmMAGLL-F:

[0042] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGGGAGCATGC-3' (as shown in SEQ ID NO: 3);

[0043] ZmMAGLL-R:

[0044] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACAACCCTGCGGCTCA-3' (shown in SEQ ID NO: 4).

[0045] Using the cDNA obtained by reverse transcription as a template, ZmMAGLL was cloned using the high-fidelity thermostable DNA polymerase PrimeSTAR GXL. The PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0046] Table 2 PCR reaction system

[0047] Components volume 5×PrimeSTARGXLBuffer 10μl dNTPMixture 4μl ZmMAGLL-F 1μl ZmMAGLL-R 1μl cDNA 2μl PrimeSTARGXL DNA Polymerase 1μl Sterile distilled water 31μl

[0048] Table 3 PCR reaction procedure

[0049]

[0050] 4. Recovery of ZmMAGLL DNA fragments;

[0051] The ZmMAGLL target fragment in the PCR product was recovered using SanGong's SanPrep column-based DNA gel recovery kit. The specific steps are as follows:

[0052] (1) After electrophoresis, cut the gel block containing the ZmMAGLL target fragment, weigh it, and place it in a 1.5ml centrifuge tube. Add the corresponding amount of Buffer B2 according to the weight of the gel block (300μl of Buffer B2 for every 100mg of gel).

[0053] (2) Place the centrifuge tube in a 50°C metal bath for 10 minutes. During this period, the centrifuge tube can be inverted several times to mix the melted liquid and unmelted gel, thus accelerating the melting process.

[0054] (3) Place the obtained solution in an adsorption column and centrifuge at 8000 rpm for 30 s. If the total volume of the solution is greater than 750 μl, add 750 μl each time and repeat the operation multiple times.

[0055] (4) Add 300 μl of Buffer B2 to the adsorption column, centrifuge at 9000 rpm for 30 s, and then pour out the waste liquid.

[0056] (5) Add 500 μl of Buffer B2 to the adsorption column, centrifuge at 9000 rpm for 30 s, and discard the waste liquid. Repeat once more.

[0057] (6) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9000 rpm for 60 seconds. Take a new 1.5 ml centrifuge tube, place the adsorption column into it, and let it air dry for 10 minutes.

[0058] (7) Add 30 μl of TE buffer or ddH2O to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge at 9000 rpm for 60 s. Store the DNA solution obtained in this step in a refrigerator at -20℃ or use it for subsequent experiments.

[0059] 5. ZmMAGLL ligation to pDONR207 vector;

[0060] Using Gateway TM BP Clonase TM ∥Enzyme mix ligates the target fragment with the pDONR207 vector to obtain a recombinant vector for gene sequencing.

[0061] The connection reaction system is shown in Table 4 below:

[0062] Table 4 BP Reaction System

[0063] reagents Dosage ZmMAGLL recycled products 2.5μl pDONR207 entry-level carrier 2μl BPEnzyme 0.5μl

[0064] 6. Transformation of DH5α Escherichia coli competent cells and PCR detection;

[0065] (1) Place 50 μl of DH5α Escherichia coli competent cells on ice to thaw.

[0066] (2) Use a pipette to draw 5 μl of the ligation product or recombinant plasmid and add it to 50 μl of DH5α Escherichia coli competent cells.

[0067] (3) After an ice bath for 30 min, heat shock at 42℃ for 90 s, followed by an ice bath for 5 min. Then add 800 μl of LB liquid culture medium.

[0068] (4) Incubate at 37℃ with shaking for 1 hour, then centrifuge at 8000 rpm for 5 minutes. Discard the supernatant, leaving approximately 50 μl of culture medium in the centrifuge tube. Mix well with a pipette, then spread it onto LB solid medium containing the corresponding antibiotic, and incubate at 37℃ inverted for 12-16 hours.

[0069] (5) Pick a single colony and put it into 800 μl of LB liquid medium containing the corresponding antibiotic. Place the centrifuge tube in a shaker and incubate at 37°C and 180 rpm for about 10 hours.

[0070] (6) PCR molecular detection of bacterial culture was performed using Es Taq enzyme. The bacterial culture with positive PCR results was amplified and shaken, and 30% glycerol was added to the bacterial culture. The culture was then sent to Sangon Biotech for sequencing. The original bacterial culture was stored at -80℃.

[0071] Example 2: Bioinformatics analysis of the maize ZmMAGLL gene;

[0072] The ZmMAGLL gene encodes a maize monoacylglycerol esterase with an open reading frame (ORF) of 1170 bp (as shown in SEQ ID NO:1) encoding 389 amino acids (as shown in SEQ ID NO:2). To gain a deeper understanding of the characteristics of this gene, we performed multiple sequence alignment and phylogenetic analysis at the amino acid level. Figure 1 and Figure 2 As shown, the results indicate that ZmMAGLL has the highest homology with AtMAGL4 in Arabidopsis thaliana.

[0073] Example 3: Construction of ZmMAGLL plant expression vector;

[0074] To construct the ZmMAGLL plant expression vector, the following steps were performed:

[0075] 1. Based on the sequence of the introductory vector pDONR207, the corresponding sequence was incorporated into the primer design.

[0076] 2. The gene amplified by high-fidelity thermostable DNA polymerase is then subjected to a series of steps, including recovery, transformation, PCR identification, and sequencing, to ensure the correctness of the expressed gene, that it has a complete coding sequence, and that there are no mismatches or frameshifts.

[0077] 3. The correctly sequenced gene was ligated into the plant expression vector pEarleyGate101 using Gateway technology.

[0078] Extract the pDONR207 introductory vector plasmid containing the correctly sequenced target gene and use Gateway... TM LRClonase TM The target gene was replaced into the plant expression vector using Enzyme mix, and the reaction system is shown in Table 5.

[0079] Table 5 LR reaction system

[0080]

[0081]

[0082] The reaction was carried out overnight at 22°C in a connector for E. coli transformation.

[0083] Example 4: Obtaining and molecularly detecting Arabidopsis thaliana transgenic with the ZmMAGLL gene;

[0084] The specific steps for gene transformation in Arabidopsis thaliana using the flower-dipping method are as follows:

[0085] 1. Invert the flowering Arabidopsis thaliana so that the flower buds face down and immerse it in Agrobacterium tumefaciens solution for 1 minute and 30 seconds.

[0086] 2. Place the transformed Arabidopsis plants flat, cover them with plastic wrap, and grow them under low light intensity for 24 hours. Then, place them under normal light conditions for cultivation. After one week, infect them again as above.

[0087] 3. After transformation, the plants can flower and grow normally. When the siliques are completely withered and about to split open, the seeds can be harvested. These seeds are the T0 generation transgenic seeds.

[0088] 4. The harvested T0 generation seeds were screened by Basta and identified by PCR to obtain T1 generation transgenic plants. After two generations of culture, T3 generation Arabidopsis plants were obtained, which can be used for subsequent phenotypic screening.

[0089] Example 5: Low-temperature treatment and survival rate statistics of T3 generation Arabidopsis thaliana transgenic with ZmMAGLL gene;

[0090] Seeds of Arabidopsis thaliana from mutant, wild-type, and transgenic plants were sown in nutrient soil and grown at 14℃ for 30 days. The growth of the Arabidopsis thaliana was then observed. Figure 3 and Figure 4As shown, the results indicate that under low temperature conditions, the fresh weight of the aboveground parts of transgenic Arabidopsis plants (OE-1, OE-2, OE-3) was significantly higher than that of the mutant (atmagl4), indicating that heterologous expression of ZmMAGLL can improve the cold tolerance of Arabidopsis.

[0091] Example 6: Analysis of photosynthetic fluorescence parameters in T3 generation Arabidopsis thaliana transgenic with the ZmMAGLL gene;

[0092] The photosynthetic fluorescence parameters of various Arabidopsis thaliana lines cultured at 14℃ to 4 weeks of age were measured. These parameters included maximum photochemical quantum efficiency (Fv / Fm), actual photochemical quantum efficiency (Phi2), quantum yield of photochemical quenching (qL), quantum yield of non-photochemical quenching (PhiNPQ), relative chlorophyll content (Relative ChlorophyllⅠⅠ), and PSI active centers (PSⅠActive Centers). The experimental results showed (e.g.) Figure 5 As shown in the figure, after growth under low temperature conditions, the Fv / Fm, Phi2, and Relative ChlorophyⅠⅠ of the mutant (atmagl4) were significantly lower than those of the wild type, while PhiNPQ and qL were significantly higher. The Fv / Fm, Phi2, and Relative ChlorophyⅠⅠ of the transgenic Arabidopsis plants (OE-1, OE-2, OE-3) were all higher than those of the mutant, while PhiNPQ and qL were lower. These results indicate that the transgenic Arabidopsis plants of this invention have relatively high cold tolerance, and demonstrate that heterologous expression of ZmMAGLL in Arabidopsis can restore the reduced photosynthesis caused by low temperature stress and improve the cold tolerance of Arabidopsis.

[0093] In summary, this invention obtained a monoacylglycerol esterase gene, ZmMAGLL, from the cold-tolerant maize inbred line W9816. Using Agrobacterium-mediated transformation, the ZmMAGLL plant expression vector was successfully transformed into Arabidopsis thaliana, yielding homozygous T3 generation transgenic Arabidopsis plants. Experimental results showed that under low-temperature stress, the aboveground fresh weight of the transgenic Arabidopsis was significantly higher than that of the mutant. After low-temperature stress treatment, the Fv / Fm, Phi2, and RelativeChlorophyⅠⅠ values ​​of the transgenic Arabidopsis were all increased compared to the mutant, while PhiNPQ and qL were decreased. These results strongly demonstrate that the monoacylglycerol esterase gene ZmMAGLL from the maize inbred line W9816 can improve the cold tolerance of Arabidopsis thaliana.

[0094] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. Maize monoacylglycerol esterase gene ZmMAGLL Its application in improving plant cold tolerance is characterized by, The corn monoacylglycerol esterase gene ZmMAGLL The nucleotide sequence of the corn monoacylglycerol esterase gene is shown in SEQ ID NO:

1. ZmMAGLL The amino acid sequence of the encoded maize monoacylglycerol esterase is shown in SEQ ID NO:2; the plant is Arabidopsis thaliana.

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