Maize monoacylglyceridase gene ZmMAGLL and application thereof

Abstract

The invention is applicable to the field of molecular biology and biotechnology, and provides a corn monoacylglyceridase gene ZmMAGLL and application thereof. The nucleotide sequence of the corn monoacylglyceridase gene ZmMAGLL is as shown in SEQ ID NO: 1, and the amino acid sequence of the corn monoacylglyceridase encoded by the corn monoacylglyceridase gene ZmMAGLL is as shown in SEQ ID NO: 2. The invention provides a monoacylglyceridase gene ZmMAGLL in a cold-resistant corn inbred line W9816 and an amino acid sequence of corn monoacylglyceridase coded by the monoacylglyceridase gene ZmMAGLL. Through 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. The phenotype observation after low-temperature stress treatment shows that the corn monoacylglyceridase gene ZmMAGLL can improve the cold resistance of arabidopsis thaliana.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and biotechnology, and particularly relates to a corn monoacylglycerol esterase gene ZmMAGLL and an application thereof. Background Art

[0002] Corn (Zea may L.), as one of the most widely planted crops in the world, is an important food, feed and cash crop in my country. However, abiotic stress factors such as low temperature and drought pose a serious threat to the growth and yield of corn. Especially in the spring corn production areas of Northeast China and North China, the spring climate in these areas is cold and the accumulated temperature is insufficient, which leads to the harsh conditions of low temperature and high humidity during spring sowing of corn, which in turn causes a series of problems such as seed rot, no seedlings, weak seedlings, and uneven seedlings. Corn is native to tropical latitudes. Low temperatures in the range of 0-15 degrees will affect the fluidity of cell membranes, leading to leakage of intracellular electrolytes, causing energy and metabolic imbalance in plants. At the same time, low temperatures will also promote the accumulation of reactive oxygen species, causing oxidative damage to proteins and DNA, and peroxidation of membrane lipids. In addition, the effect of low temperature on enzyme activity will also directly affect photosynthesis and respiration. The above factors may cause poor germination of corn, slow growth of seedlings, and even plant death in severe cases, reducing crop quality and yield. Especially at sub-zero temperatures, ice crystals will be produced inside and outside cells, which will be more harmful to plant survival. Therefore, identifying low-temperature-related genes and studying their gene functions and molecular mechanisms are of great significance for breeding new cold-tolerant corn varieties.

[0003] Changes in lipid composition and unsaturated lipid content have always been associated with plant cold tolerance. Under low temperature stress, the content of glycolipids, some phospholipids and glycerides in corn leaves will be significantly reduced, while the levels of phosphatidic acid, lysophosphatidic acid and diacylglycerol will be significantly increased. In addition, the levels of polyunsaturated triacylglycerol and phosphatidic acid increased significantly, which also proves that corn will reshape lipids and change their unsaturation under low temperature conditions.

[0004] Monoacylglycerol lipases are involved in catalyzing the hydrolysis of monoacylglycerol into free fatty acids and glycerol, but relatively few studies have been conducted in plants. In Arabidopsis, 16 putative MAGLs (AtMAGLs) have been reported to have a three-dimensional structure similar to that of human MAGL. Through heterologous expression and enzyme assays, it was found that 11 of the encoded proteins did have MAG lipase activity. In addition, some AtMAGLs also showed hydrolase activity in lysophosphatidylcholine and lysophosphatidylethanolamine substrates and were localized in the endoplasmic reticulum, Golgi apparatus, cytoplasm or chloroplasts. Among them, AtMAGL4 was localized in chloroplasts, and its transcription level was upregulated by more than 11 times and 5 times in the aerial parts and roots of 2-3 week-old Arabidopsis after salt, osmotic and cold stress treatments, respectively. In addition, studies have reported the homologous gene of AtMAGL8 in rapeseed, BnaC.MAGL8.a, and it was found that overexpression of BnaC.MAGL8.a in Arabidopsis led to pollen defects. Transcriptome analysis found 398 differentially expressed genes compared with the wild type, which are mainly involved in pollen development and stress response. However, it is still unclear whether the ZmMAGLL gene in corn plays an important role in plant resistance to cold stress and how its specific function is performed. For this reason, the present invention proposes a corn monoacylglycerol esterase gene ZmMAGLL and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a corn monoacylglycerol esterase gene ZmMAGLL and its application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The corn monoacylglycerol esterase gene ZmMAGLL has an open reading frame (ORF) of 1170 bp in full length, an initiation codon of ATG, a termination codon of TGA, and a nucleotide sequence as 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 corn monoacylglycerol esterase gene ZmMAGLL as described above.

[0010] The use of the corn 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 present invention has the following beneficial effects:

[0012] The present invention provides a monoacylglycerol esterase gene ZmMAGLL in a cold-tolerant maize inbred line W9816 and the amino acid sequence of the maize monoacylglycerol esterase encoded by it. The ZmMAGLL plant expression vector was successfully transformed into Arabidopsis thaliana by an Agrobacterium-mediated transformation method, and a homozygous T 3 The present invention provides a new gene resource for analyzing the cold tolerance mechanism of plants, and at the same time provides an important theoretical basis and technical support for the breeding of cold-tolerant crop varieties and the improvement of stress resistance, which has important application value for breeding new cold-tolerant corn varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A multiple sequence alignment.

[0014] Figure 2 Schematic diagram of the phylogenetic tree analysis of the maize ZmMAGLL gene; AtMAGL is Arabidopsis thaliana; ZmMAGLL is maize.

[0015] Figure 3 Schematic diagram of Arabidopsis phenotype under 14℃ treatment conditions.

[0016] Figure 4 Schematic diagram of the fresh weight of the aboveground parts of Arabidopsis thaliana under 14°C treatment conditions.

[0017] Figure 5 Schematic diagram of the determination of photosynthetic fluorescence parameter indicators of Arabidopsis thaliana under 14℃ treatment conditions. DETAILED DESCRIPTION

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

[0019] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

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

[0021] 1. RNA extraction;

[0022] The cold-tolerant maize inbred line W9816 was grown to the three-leaf stage, and the total RNA from the leaves was extracted using the Ultrapure RNA Extraction Kit (CW0581) from Cosmed. The specific steps are as follows:

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

[0024] (2) After adding TRIzon Reagent, gently invert the sample several times to ensure that the sample is fully lysed. Leave it at room temperature for 5 minutes to allow the protein-nucleic acid complex to completely separate.

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

[0026] (4) Centrifuge at 12,000 rpm for 10 min at 4°C, 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 phase solution and mix by inversion.

[0028] (6) Add all the solution obtained in the previous step to the adsorption column (Spin Columns RM) in the collection tube. If the solution cannot be added in one go, add it in multiple times. Centrifuge at 12,000 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 12,000 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 whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 20 s, pour out 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. Leave the adsorption column at room temperature for several minutes to dry.

[0033] (11) Place the adsorption column in a new RNase-free centrifuge tube, add 30-50 μl RNase-Free Water to the middle of the adsorption column, leave at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store at -80°C 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 procedure: 37°C, 2 min; 55°C, 15 min; 85°C, 5 min.

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

[0040] According to the CDS gene sequence of maize ZmMAGLL published by NCBI, the specific cloning primers of the gene were designed using the bioinformatics software Primer 5.0 (following the 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, high-fidelity thermostable DNA polymerase PrimeSTAR GXL DNA polymerase was used to clone ZmMAGLL. The PCR reaction system is shown in Table 2, and the PCR reaction program is shown in Table 3.

[0046] Table 2 PCR reaction system

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

[0048] Table 3 PCR reaction program

[0049]

[0050] 4. Recovery of DNA fragments of ZmMAGLL;

[0051] Use SanPrep column DNA gel recovery kit from Bioengineering to recover the ZmMAGLL target fragment in the PCR product. The specific steps are as follows:

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

[0053] (2) Place the centrifuge tube in a 50°C metal bath for 10 minutes. During this time, invert the tube several times to mix the melted liquid and unmelted gel to accelerate dissolution.

[0054] (3) Place the resulting solution in an adsorption column and centrifuge at 8000 rpm for 30 seconds. If the total volume of the solution is greater than 750 μl, add 750 μl each time and repeat the process 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 seconds, discard the waste liquid, and 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 in it, and let it sit for 10 minutes.

[0058] (7) Add 30 μl TE buffer or ddH 2 O, stand at room temperature for 2 min, centrifuge at 9000 rpm for 60 s. Store the DNA solution obtained in this step in a -20°C refrigerator or use it for subsequent experiments.

[0059] 5. ZmMAGLL was connected to pDONR207 vector;

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

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

[0062] Table 4 BP reaction system

[0063] Reagents Dosage ZmMAGLL glue recovery product 2.5μl pDONR207 Entry Vector 2μl BPEnzyme 0.5μl

[0064] 6.DH5α E. coli competent transformation and PCR detection;

[0065] (1) Thaw 50 μl of DH5α competent E. coli on ice.

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

[0067] (3) After a 30-min ice bath, heat shock was performed at 42°C for 90 s, followed by an ice bath for 5 min. 800 μl of LB liquid culture medium was then added.

[0068] (4) Incubate at 37°C with shaking for 1 hour, and centrifuge at 8000 rpm for 5 minutes. Discard the supernatant, leaving about 50 μl of culture medium in the centrifuge tube. Mix well by pipetting, and then spread on LB solid culture medium containing the corresponding antibiotics. Incubate at 37°C with an inverted incubator for 12-16 hours.

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

[0070] (6) Use Es Taq enzyme to perform PCR molecular detection on the bacterial solution. The bacterial solution with positive PCR results was propagated and 30% glycerol was added to the bacterial solution, and sent to Bio-Gene for sequencing. The original bacterial solution was stored in a -80°C refrigerator.

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

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

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

[0074] To construct the ZmMAGLL plant expression vector, follow these steps:

[0075] 1. According to the sequence of the entry vector pDONR207, add the corresponding sequence when designing primers.

[0076] 2. The genes amplified by high-fidelity heat-resistant DNA polymerase are taken through a series of steps such as recovery, transformation, PCR identification, and sequencing to ensure the correctness of the expressed genes, with complete coding sequences and no mismatches or frameshifts.

[0077] 3. Use Gateway technology to connect the correctly sequenced gene to the plant expression vector pEarleyGate101.

[0078] Extract the pDONR207 entry vector plasmid connected to the target gene with correct sequencing, and use Gateway TM LRClonase TM ∥Enzyme mix replaces the target gene into the plant expression vector. The reaction system is shown in Table 5:

[0079] Table 5LR reaction system

[0080]

[0081]

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

[0083] Example 4: Acquisition and molecular detection of transgenic ZmMAGLL gene Arabidopsis thaliana;

[0084] The floral dip method was used to transform Arabidopsis thaliana. The specific steps are as follows:

[0085] 1. Turn the flowering Arabidopsis thaliana upside down with the flower buds facing downwards and inoculate it with Agrobacterium solution for 1 min 30 s.

[0086] 2. Lay the transformed Arabidopsis plants flat, cover with plastic wrap, grow them under low light intensity for 24 hours, then culture them under normal light conditions, and infect them again one week later.

[0087] 3. After transformation, the plants can bloom and grow normally, and when the siliques are completely yellow and about to crack, the seeds can be harvested. These seeds are T0 generation transgenic seeds.

[0088] 4. Harvest Part T 0 The seeds of the first generation were screened by Basta and identified by PCR to obtain T 1 After two generations of culture, T 3 The Arabidopsis plants can be used for subsequent phenotypic screening.

[0089] Example 5: T 3 Low temperature treatment and survival rate statistics of Arabidopsis thaliana transgenic with ZmMAGLL gene;

[0090] The mutant, wild-type and transgenic Arabidopsis seeds were sown in nutrient soil and grown at 14°C for 30 days to observe the growth of Arabidopsis. Figure 3 and Figure 4As shown, the results showed that under low temperature conditions, the fresh weight of the aboveground part 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: T 3 Analysis of photosynthetic fluorescence parameters of ZmMAGLL gene-transfected Arabidopsis thaliana;

[0092] Photosynthetic fluorescence parameters of Arabidopsis strains cultured at 14°C for 4 weeks were measured. These parameters include 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 (RelativeChlorophyⅠⅠ) and PSⅠ active centers (PSⅠActive Centers). The experimental results show (such as Figure 5 As shown in the figure, after growing 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 than those of the wild type. The Fv / Fm, Phi2 and Relative ChlorophyⅠⅠ of the transgenic Arabidopsis plants (OE-1, OE-2, OE-3) were all improved compared with the mutant, while PhiNPQ and qL were all reduced compared with the mutant. This result shows that the transgenic Arabidopsis plants of the present invention have relatively high cold tolerance, and proves that the heterologous expression of ZmMAGLL in Arabidopsis can restore the weakened photosynthesis caused by low temperature stress, and can improve the cold tolerance of Arabidopsis.

[0093] In summary, the present invention obtained a monoacylglycerol esterase gene ZmMAGLL from the cold-tolerant maize inbred line W9816. The ZmMAGLL plant expression vector was successfully transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, and a homozygous T 3 The results showed that under low temperature stress, the aboveground fresh weight of transgenic Arabidopsis was significantly higher than that of mutants; after low temperature stress treatment, Fv / Fm, Phi2, and RelativeChlorophyⅠⅠ of transgenic Arabidopsis were higher than those of mutants, while PhiNPQ and qL were lower than those of mutants. These results strongly prove that the monoacylglycerol esterase gene ZmMAGLL in the maize inbred line W9816 can improve the cold tolerance of Arabidopsis.

[0094] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Maize monoacylglycerol esterase gene ZmMAGLL, characterized in that: The nucleotide sequence of the corn monoacylglycerol esterase gene ZmMAGLL is shown in SEQ ID NO:

1.

2. The corn monoacylglycerol esterase gene ZmMAGLL according to claim 1, characterized in that: The amino acid sequence of the corn monoacylglycerol esterase encoded by the corn monoacylglycerol esterase gene ZmMAGLL is shown in SEQ ID NO:

2.

3. A recombinant plant expression vector, characterized in that: Contains the corn monoacylglycerol esterase gene ZmMAGLL as claimed in claim 1.

4. Use of the corn monoacylglycerol esterase gene ZmMAGLL according to claim 1 or the recombinant plant expression vector according to claim 3 in improving plant cold tolerance, characterized in that: The plant is Arabidopsis thaliana.

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