CN104651331A - Key enzyme protein MzASMT9 for synthesizing Malus zumi melatonin as well as encoding gene and application of key enzyme protein MzASMT9 - Google Patents

Abstract

The invention relates to the key enzyme protein MzASMT9 for melatonin synthesis of Begonia melatonin and its coding gene and application. The protein is the protein shown in the following (a) or (b): (a) a protein composed of the amino acid sequence shown in sequence 2 in the sequence listing; (b) the amino acid sequence of sequence 2 in the sequence listing, after a Or the substitution and/or deletion and/or addition of several amino acid residues, the protein derived from (a) obtained and related to plant stress tolerance. Experiments have proved that the protein of the present invention and its coding gene have the function of improving the stress resistance of plants, and the stress resistance of plants transferred to the gene of the present invention is significantly higher than that of plants that are not transferred to the gene of the present invention, such as transgenic Arabidopsis thaliana, after salt After treatment and drought treatment, the growth of transgenic plants was significantly better than that of non-transgenic Arabidopsis, the photosynthetic rate was higher than that of wild-type Arabidopsis, and the root length and biomass were significantly improved compared with the control group. Therefore, the protein of the present invention and its coding gene will have broad application prospects in the fields of cultivating stress-resistant plants, crop breeding and the like.

Description

The Key Enzyme Protein MzASMT9 of Melatonin Synthesis in Begonia Zhumei and Its Encoding Gene and Application

technical field

The present invention relates to the cloning and gene application of the key enzyme ASMT gene for the synthesis of melatonin related to adversity stress. Application in drought stress. Belongs to the field of biotechnology.

Background technique

Plants are subject to a variety of biotic and abiotic stresses in nature, mainly environmental stresses, such as salt stress, high temperature stress, low temperature stress, drought stress, etc. These factors will affect the growth and development of plants, and affect the quality and quality of agricultural production. Yield.

Improving the stress tolerance of plants at the molecular level is a better research method for achieving breeding goals. Genetic engineering is a complex technology for manipulating genes at the molecular level. Genetic engineering breeding enables genes to be "assembled" into new gene combinations according to people's needs.

Melatonin is a broad-spectrum antioxidant that protects DNA, membrane lipids, and proteins from oxidative damage caused by reactive oxygen species and reactive nitrogen species, and can significantly improve the damage caused by reactive oxygen species and reactive nitrogen species. The research on melatonin in plants was carried out later than in animals, but recent studies have shown that it also has the effect of scavenging active oxygen free radicals in plants, and plays an important role in abiotic stress (abiotic stress) . Therefore, the study of melatonin and its synthesis is of great significance to improve the stress tolerance of plants.

The role of melatonin in adversity stress has been reported, but the rate-limiting enzyme of melatonin synthesis in plants is still controversial. There have been studies to transfer human and animal HIOMT (ie ASMT) gene and AANAT gene into plants.

The invention is to transfer the ASMT gene in the plant into the plant: the plant expression vector of the melatonin synthase gene that has been successfully constructed is used to introduce the key enzyme MzASMT9 gene of melatonin synthesis into the model plant Arabidopsis mediated by Agrobacterium Mustard (Arabidopsis thaliana) to obtain transgenic plants expressing melatonin biosynthetic enzyme genes, and then analyze the phenotype of transgenic plants caused by the increase of melatonin content, infer the role of melatonin synthase in plant abiotic stress Function.

Contents of the invention

The object of the present invention is to provide a key enzyme protein MzASMT9 of Begonia melatonin synthesis and its coding gene, and another object of the present invention is to provide the application of the Begonia protein MzASMT9.

The present invention provides a protein related to plant stress tolerance and its coding gene. The protein provided by the present invention is named MzASMT9, which is derived from Begonia Zhumei.

The Zhumei crabapple protein MzASMT9 provided by the present invention is specifically the protein shown in (a) or (b) below:

(a) a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing;

(b) A protein derived from (a) obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of Sequence 2 in the sequence listing and related to plant stress tolerance.

Sequence 2 in the sequence listing consists of 341 amino acids, wherein, from the 8th to the 64th, it is a protein dimer related to the N-terminal of the methyltransferase, and from the 75th to the 317th, there is a nitrogen acetyl serotonin methyltransferase Functional Area.

The substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and /or deletion and/or addition; the protein dimer related to the methyltransferases is the 8th-64th amino residue from the amino terminal of sequence 2, and the sequence of the nitrogen acetylserotonin methyltransferase is from Amino residues 75-317 at the amino terminal of Sequence 2.

In order to facilitate the purification of the protein in (a) or (b), tags shown in Table 1 can be attached to the N-terminus (amino-terminus) or C-terminus (carboxyl-terminus) of the protein.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in (a) or (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the protein in (b) above can be deleted by deleting one or several amino acid residue codons from the DNA sequence shown in Sequence 1 in the Sequence Listing, and/or performing missense mutations of one or several base pairs , and/or connect the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

Some of the substitutions, substitutions and/or additions of one or more amino acid residues in the amino acid sequences of the above proteins are caused by naturally occurring variations, and some are caused by artificial mutagenesis.

The gene encoding the protein also belongs to the protection scope of the present invention.

The coding gene provided by the present invention may specifically be the gene shown in 1) or 2) or 3) as follows:

1) The DNA molecule shown in sequence 1 in the sequence listing;

2) a DNA molecule that hybridizes with the DNA molecule defined in 1) under stringent conditions and encodes the plant stress tolerance-related protein;

3) A DNA molecule having at least 70% homology to the DNA sequence defined in 1) and encoding the plant stress tolerance-related protein.

The above-mentioned stringent conditions can be hybridization at 65° C. in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

The gene encoding the protein MzASMT9 is denoted as MzASMT9, and the sequence 1 in the sequence listing consists of 1026 deoxyribonucleotides. The deoxyribonucleotides starting from the 5' end are the start codon ATG of MzASMT9, and the deoxyribonucleotides at positions 1023 to 1026 from the 5' end are the stop codon TGA of MzASMT9.

Recombinant vectors, recombinant bacteria, transgenic cell lines or expression cassettes containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.

The recombinant expression vector can specifically be MzASMT9-pGWB405 obtained by inserting the deoxynucleotides from the start codon ATG to the stop codon TGA of sequence 1 in the sequence listing.

The recombinant expression vector containing MzASMT9 gene can be constructed by existing plant expression vector.

The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions.

When using MzASMT9 to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, maize ubiquitin Promoters (Ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, these enhancers The region can be the start codon of ATG or the start codon of the adjacent region, etc., but it must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.), or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

A pair of primers for amplifying the full length of any of the above-mentioned coding genes or any fragment thereof also falls within the protection scope of the present invention.

The primer pair can specifically be as follows:

1) The sequence of one primer is shown in sequence 3 in the sequence listing, and the sequence of the second primer is shown in sequence 4 in the sequence listing. This pair of primers is the primer for amplifying the full length of the gene; As shown in Table 5, the primer pair of sequence 3 and sequence 5 is to express the GFP tag carried by the target vector after the downstream primer sequence 5 removes the stop codon TGA when constructing the MzASMT9-pGWB405 plant expression vector.

A final object of the present invention is to provide a method for cultivating stress-tolerant plants.

The method for cultivating stress-tolerant plants provided by the present invention is to introduce any one of the above-mentioned coding genes into plants, and obtain stress-tolerant plants after cultivation.

In the above process, the stress tolerance may specifically be salt tolerance and drought tolerance.

Using any vector that can guide the expression of exogenous genes in plants, the gene encoding MzASMT9 provided by the present invention can be introduced into plant cells, and transgenic cell lines and transgenic cells with enhanced tolerance to salt stress and drought stress can be obtained. plants. The expression vector carrying the coding gene can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and transform the transformed The plant tissue is grown into a plant.

The plants can be monocotyledonous plants or dicotyledonous plants, such as: Arabidopsis thaliana, soybean, rice, crabapple, corn, cucumber, tomato, poplar, lawn grass, alfalfa and the like.

The application of any of the above-mentioned proteins or any of the above-mentioned coding genes in the cultivation of stress-tolerant plants also falls within the protection scope of the present invention. In the above-mentioned applications, the stress tolerance can specifically be salt tolerance and drought tolerance.

Experiments have proved that the protein of the present invention and its coding gene have the function of improving the stress resistance of plants, and the stress resistance of plants transferred to the gene of the present invention is significantly higher than that of plants that are not transferred to the gene of the present invention, such as transgenic Arabidopsis thaliana, after salt or drought treatment, the growth of transgenic plants was significantly better than that of non-transgenic Arabidopsis. Under salt stress, the photosynthetic rate was higher than that of wild-type Arabidopsis, and the root length and biomass were significantly increased compared with the control group. Therefore, the protein of the present invention and its coding gene will have broad application prospects in the fields of cultivating stress-resistant plants, crop breeding and the like.

Detailed ways

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The tissue-cultured seedlings of Begonia Zhumei and the seeds of wild-type Arabidopsis thaliana (Col-0) were preserved by the Laboratory of Fruit Tree Stress Physiology and Molecular Biology, China Agricultural University.

Embodiment 1, the cloning of gene

In the previous work of this experiment, the homology analysis was carried out with the OsASMT1 gene in rice, and the gene MzASMT9 with higher homology was screened from apple. Total RNA was extracted from Zhumei crabapple seedlings, and cDNA was synthesized from the RNA with reverse transcriptase, which was used as a template to amplify the full length of the MzASMT9 gene. The primers are: MzASMT9 F: CACATGTTTGGTTTTGCTG (SEQ ID NO: 3), MzASMT9 R: TTAGTTCACATAGGGTAGGC (SEQ ID NO: 4). The PCR program is as follows: 94°C for 3 minutes; 28 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 90s; 72°C for 10 minutes.

The PCR product was detected by 1.0% agarose gel electrophoresis, and a band with a molecular weight of about 1 kb was obtained, which was consistent with the expected result. This fragment was recovered using an agarose gel recovery kit (CWBIO). Combine this recycled fragment with the (Invitrogen) connection, using the heat shock method, the connection product was transformed into Escherichia coli Trans10 competent cells, according to The positive clones were screened by the spectinomycin resistance marker on the vector, and the recombinant plasmid containing the target fragment was obtained. Using the M13 Primers on the recombinant plasmid vector as primers to determine its nucleotide sequence, the sequencing results show that the nucleotide sequence of the amplified MzASMT9 gene is shown in sequence 1 in the sequence table, consisting of 1026 deoxyribonucleosides Its open reading frame (ORF) is the deoxyribonucleotide starting from the 5' end of sequence 1 in the sequence listing, and the encoded amino acid sequence is the protein of sequence 2 in the sequence listing (referred to as protein MzASMT9). The recombinant vector containing the MzASMT9 gene was named as MzASMT9-

The expression characteristic of MzASMT9 gene under the treatment of embodiment 2, salt, drought stress

Zhumei crabapple seedlings grown for 2 months were treated with 0mM, 50mM, 100mM, 200mM NaCl saline for stress treatment under the conditions of light 16/8h and temperature 22°C, and the leaves were collected at 0h and 12h. After quick-freezing in liquid nitrogen, store in a -80°C refrigerator for later use. Cultivate Zhumei Begonia seedlings with a size of 5-6cm and consistent growth in complete nutrient solution, apply 300mM mannitol, collect leaves at 0h, 4h, 8h, and 12h after treatment, and store them in a -80°C refrigerator after quick-freezing in liquid nitrogen spare. Collect 1 g of the above-mentioned leaves and grind them in liquid nitrogen, extract plant RNA by CTAB method, and remove DNA by DNase I digestion. Take 0.5 μL RNA, and check the integrity of RNA by 1% agarose gel electrophoresis. RT-PCR analysis was performed using Promega reverse transcription kit.

The results showed that the expression of MzASMT9 gene was induced by salt and drought treatments.

Embodiment 3, the application of MzASMT9 gene in cultivating salt-tolerant and drought-tolerant plants

1) Construction of MzASMT9 plant expression vector MzASMT9-pGWB405

The cDNA obtained by reverse transcription of the total RNA of the leaves of Zhumei Begonia was used as a template for PCR amplification; the obtained recombinant vector MzASMT9- The recombinant expression vector MzASMT9-pGWB405 was obtained by connecting the recombinase LR clonase II (invitrogen) to the Gateway system vector pGWB405; the recombinant vector was subjected to single-clonal sequencing, and the results showed that the structure of the recombinant vector was correct, and the sequence of the gene inserted into the vector was correct. The gene MzASMT9 was positively inserted behind the CaMV35S promoter of the plant binary expression vector pGWB405.

The primer sequences are as follows:

5'-CACATGTTTGGTTTTGCTG-3' (SEQ ID NO: 3)

5'-TTAGTTCACATAGGGTAGGC-3' (SEQ ID NO: 4)

5'-CATAGGGTAGGCCTCAAC-3' (SEQ ID NO: 5).

2) Acquisition and identification of transgenic MzASMT9 gene plants

The recombinant plasmid MzASMT9-pGWB405 was transformed into competent GV3101 Agrobacterium by freeze-thaw method. The wild-type Arabidopsis Col-0 was infected by flower dipping method.

Wild-type Arabidopsis Col-0, which had grown for 6 weeks and had bolted and flowered, was used for Agrobacterium transformation. The recombinant Agrobacterium MzASMT9-pGWB405 was inoculated in 5ml YEP+50μg/ml rifampicin+50μg/ml spectinomycin+5μg/ml tetracycline medium, and the bacteria were shaken overnight. The next day, the bottle was transferred to 50 ml of the above-mentioned liquid medium, and cultured at 28° C. until the OD value was 0.6-0.8. The bacterial cells were collected by centrifugation at 6000 rpm, and Agrobacterium was suspended with 50 ml of 5% sucrose solution. The inflorescence part above the rosette leaves of the plant 4-6 days after pruning is completely immersed in the suspension for 10-12s. Place the plant pot on its side in the dark for 24 hours. Then cultivate under normal conditions, cultivate the plants until fruiting, and harvest mature seeds (T ₀ generation).

After drying for about two weeks, sterilize the T ₀ generation seeds, sow them on 1/2 MS medium containing 25mg/L kanamycin and culture them at 4°C for 2-3 days. Transfer to a culture room with 16/8 hours of light and a temperature of 25°C for cultivation. Screen for resistant transformed plants. After 7-10 days, the normally growing Arabidopsis thaliana was transferred to culture. After maturity, the seeds were harvested from individual plants (T ₁ generation).

The seeds of the _T1 generation were sterilized, screened according to the method for screening the first generation seeds, and Arabidopsis thaliana strains with a separation ratio of 3:1 were transplanted into the soil, and the _T2 generation seeds were harvested per plant. The _T2 generation seeds were still screened with 25 mg/L kanamycin after sowing, and the homozygous transgenic plants were no longer segregated, and the _T3 generation seeds produced by these plants were the homozygous lines of the transgenic plants.

Extract the total RNA of the untransformed wild-type Arabidopsis plants and the T ₃ generation plants of the MzASMT9 gene for RT-PCR identification and analysis, and the primers are as follows:

5'-CACATGTTTGGTTTTGCTG-3' (SEQ ID NO: 3)

5'-CATAGGGTAGGCCTCAAC-3' (SEQ ID NO: 5)

The results showed that 28 of the 28 MzASMT9 gene-transferred T ₁ plants could detect the expression of MzASMT9 gene, while the wild-type control plants could not detect the expression of the target gene. Harvest the seeds of the identified positive plants, sow the seeds of each individual plant respectively, and screen with 25 mg/L kanamycin to observe the separation of the _T1 generation, and so on until the _T3 generation obtains a genetically stable transgenic line. A total of 10 stable genetic transgenic lines were obtained.

3) Salt tolerance identification of transgenic Arabidopsis plants with MzASMT9 gene

After the three homozygous lines with high expression level of the transgenic MzASMT9 gene were sterilized, they were cultured vertically in 1/2 MS culture dish on demand. After 7 days, Arabidopsis seedlings with roots of about 1 cm were transferred to MS medium containing 100 mM L ^-1 NaCl. Cultivate at room temperature 25°C and light cycle 8/16h.

Dish statistics: root length. Under salt stress, the Arabidopsis thaliana grown in a petri dish for one week were subjected to 100mM L ^-1 NaCl stress for 7 days, and statistics were made, and each strain system counted 48 plants. Results Compared with the wild-type control group, the root length of Arabidopsis lines transfected with MzASMT9 gene increased significantly. Among them, the average root length of the wild-type control group was 4.1 cm, and the average root length of the transgenic Arabidopsis thaliana with elevated melatonin was 5.4 cm.

Biomass. Weighed the Arabidopsis thaliana transgenic MzASMT9 gene line and the wild-type control group after stressing in 100mM L ^-1 NaCl dish for 7 days, and weighed 48 plants for each line. Average 0.015g.

Salt tolerance phenotype in soil. The 12-day-old Arabidopsis grown in the dish was moved to the soil, and after 10 days of growth under normal conditions (room temperature 24°C, light 8/16h), the MzASMT9 gene-transferred Arabidopsis and the wild-type control group were treated with salt, and a concentration of It was 200mM saline, and after one week of treatment, the photosynthetic rates of transgenic MzASMT9 gene and wild-type Arabidopsis were measured. The photosynthetic rate of transgenic Arabidopsis was 1.8μmol CO ₂ m ^-2 s ^-1 . The wild-type control group was 1.1 μmol CO ₂ m ^-2 s ^-1 . After 15 days, the growth of the MzASMT9 gene-transferred line was hindered but showed a salt-tolerant phenotype. The wild-type Arabidopsis in the control group partly died, and the growth status was worse than that of the MzASMT9-transferred Arabidopsis.

4) Identification of drought tolerance of transgenic Arabidopsis plants with MzASMT9 gene

Drought tolerance phenotype in soil. The 12-day-old Arabidopsis thaliana grown in the dish was moved to the soil, and after 10 days under the growth condition of room temperature 24°C and light 8/16h, the MzASMT9 gene-transferred Arabidopsis and the wild-type control group were subjected to drought treatment, and watering was stopped. Two weeks later, the survival rate and biomass of transgenic MzASMT9 gene and wild-type Arabidopsis were compared. The mortality rate of transgenic Arabidopsis was 35%, compared with 70% in the wild control group. The biomass was significantly larger than that of the wild-type control Arabidopsis thaliana. The weight of the MzASMT9 gene transfected strain was 0.12g per plant, and that of the wild-type plant was 0.05g. The growth of the transgenic Arabidopsis was stunted but showed a drought-tolerant phenotype, and the growth status of the wild-type Arabidopsis in the control group was worse than that of the transgenic Arabidopsis with MzASMT9 gene.

The analysis results showed that the MzASMT9 gene transgenic Arabidopsis had longer root length and higher biomass than the wild type under salt stress, and the growth in soil also showed a strong salt-tolerant phenotype, and the photosynthetic efficiency was higher than that of the wild type. Under drought stress, the survival rate and biomass of Arabidopsis transgenic for MzASMT9 were significantly better than those of the wild control group. Therefore, MzASMT9 is a key enzyme in increasing melatonin synthesis. Its overexpressed plants showed an increase in melatonin content, and also showed an increase in salt tolerance and drought tolerance, indicating that melatonin can play a role in adversity. important role in coercion.

Claims (10)

1. A protein, which is the protein shown in (a) or (b) below: (a) a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing; (b) A protein derived from (a) obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of Sequence 2 in the sequence listing and related to plant stress tolerance. 2. The protein according to claim 1, characterized in that: the substitution and/or deletion and/or addition of said one or several amino acid residues refers to the protein two related to methyltransferases in sequence 2 Polymer, nitrogen acetyl serotonin methyltransferase sequence is substituted and/or deleted and/or added; the protein dimer related to the methyltransferase is the 8th-64th amino residue from the amino terminal of sequence 2 The sequence of the nitrogen acetylserotonin methyltransferase is amino residues 75-317 from the amino terminal of sequence 2. 3. The coding gene of the protein described in claim 1 or 2. 4. The coding gene as claimed in claim 3, characterized in that: the coding gene can specifically be the gene shown in 1) or 2) or 3) as follows: 1) The DNA molecule shown in sequence 1 in the sequence listing; 2) a DNA molecule that hybridizes with the DNA molecule defined in 1) under stringent conditions and encodes the plant stress tolerance-related protein; 3) A DNA molecule having at least 70% homology to the DNA sequence defined in 1) and encoding the plant stress tolerance-related protein. 5. A recombinant vector, a recombinant bacterium, a transgenic cell line or an expression cassette containing the coding gene of claim 3 or 4. 6. A pair of primers for amplifying the full length of the coding gene of claim 3 or 4 or any fragment thereof. 7. The pair of primers according to claim 6, characterized in that: the pair of primers can specifically be as shown in 1) below: 1) The sequence of one primer is shown in sequence 3 in the sequence listing, and the sequence of the second primer is shown in sequence 4 in the sequence listing. This pair of primers is the primer for amplifying the full length of the gene; As shown in Table 5, the primer pair of sequence 3 and sequence 5 is to express the GFP tag carried by the target vector after the downstream primer sequence 5 removes the stop codon TGA when constructing the MzASMT9-pGWB405 plant expression vector. 8. A method for cultivating stress-tolerant plants, which is to introduce the coding gene described in claim 3 or 4 into the plants, and obtain stress-tolerant plants after cultivation. 9. The method according to claim 8, characterized in that: said stress tolerance is salt tolerance and drought tolerance. 10. The method according to claim 8 or 9, wherein the plant is a monocot or a dicot.