CN118147175B - Application of MtCOMT gene in regulation and control of salt tolerance and drought resistance of plants - Google Patents
Application of MtCOMT gene in regulation and control of salt tolerance and drought resistance of plants Download PDFInfo
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- CN118147175B CN118147175B CN202410578380.0A CN202410578380A CN118147175B CN 118147175 B CN118147175 B CN 118147175B CN 202410578380 A CN202410578380 A CN 202410578380A CN 118147175 B CN118147175 B CN 118147175B
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Abstract
The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to application of MtCOMT gene in regulation and control of salt tolerance and drought resistance of plants. The nucleotide sequence of MtCOMT gene is shown as SEQ ID NO.1, and the amino acid sequence of protein expressed by MtCOMT gene is shown as SEQ ID NO. 2. The invention utilizes genetic engineering means to carry out over-expression genetic transformation, and specifically comprises target gene cloning, target gene over-expression vector construction, target gene transformation agrobacterium, target gene transformation arabidopsis thaliana, positive strain identification and phenotype observation, thereby laying a theoretical foundation for research on molecular regulation and control mechanism of salt tolerance and drought resistance of plants.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to application of MtCOMT gene in regulation and control of salt tolerance and drought resistance of plants.
Background
Leguminous plants are important grains and pastures, and provide high-quality nutrients such as proteins, vitamins, trace elements and the like for human beings and animals. The medicago sativa (Medicago truncatula) has the advantages of small genome, short life cycle, strong self-pollination capability, large number of mutants and the like, has close relationship with medicago sativa and high genome homology, and is a model plant for researching biological problems of medicago sativa.
Among the environmental constraints, salinization and drought are the major abiotic stresses affecting plant growth and development and physiological and biochemical metabolism, which ultimately lead to reduced crop yields. Soil salinization has long been recognized as a common environmental phenomenon worldwide and is becoming a global problem of land degradation, especially in arid and semiarid regions. In China, the land area of salinization and drought is increased year by year, the crop yield is seriously affected, and the agricultural development and the grain safety are limited.
Plants accumulate large amounts of reactive oxygen species in cells when subjected to abiotic stresses such as salt and drought, and consequently cause a disturbance in the homeostasis of the cellular redox system. Excess active oxygen inhibits normal physiological metabolic function of cells by destroying nucleic acids, oxidized proteins, etc. to cause destruction of cell structures, thereby limiting plant biomass and yield formation.
Melatonin is an indole hormone commonly existing in higher plants, and plays an important role in regulating plant growth and development such as root organogenesis, flowering, aging and the like. In addition, melatonin has also proven to be an important antioxidant, which can enhance plant tolerance to various abiotic stresses, such as cold, salt, drought, oxidative stress and nutrient deficiency, by either directly scavenging active oxygen or indirectly increasing antioxidant enzyme activity, photosynthetic efficiency, metabolite synthesis, and the like.
COMT (CAFFEIC ACID O-METHYLTRANSFERASE) is one of the functional gene families of plants, involved in plant growth and development regulation and abiotic stress response. At present, the function of regulating and controlling salt tolerance of the COMT gene is confirmed in model plants of arabidopsis thaliana and rice, however, the identification of the COMT gene family of medicago truncatula is not reported, and the function of regulating and controlling salt tolerance and drought resistance of plants is not clear. Namely, no related application of the alfalfa COMT gene family of caltrops in regulating and controlling the salt tolerance and drought resistance of plants exists at present.
Disclosure of Invention
In order to solve the problem that the related application of the alfalfa COMT gene family in regulating and controlling the salt tolerance and drought tolerance of plants does not exist in the prior art, the invention aims to clearly determine the function and specific application of MtCOMT gene in regulating and controlling the salt tolerance and drought tolerance of plants. To achieve the above object, the above technical problems are solved. The invention adopts the following technical scheme:
the applicant subject group of the invention discovers that the alfalfa with different degrees of salt stress and drought stress is treated, and the qRT-PCR is utilized to analyze the change of the expression quantity of part members in the COMT gene family, and the result shows that the expression quantity of MtCOMT under salt stress is 8.96 times of that of a control, and the expression quantity of MtCOMT under drought stress is 3.91 times of that of the control. Therefore, the applicant speculates that MtCOMT gene can participate in the regulation of the salt tolerance and drought resistance of medicago truncatula. In order to verify the conjecture, the MtCOMT gene is subjected to functional research to clarify the function of the MtCOMT gene in regulating and controlling the salt tolerance and drought resistance of plants, and a theoretical basis is laid for genetic improvement of the salt tolerance and drought resistance of plants and creation of new germplasm.
Based on the research, the invention provides application of MtCOMT gene in regulating and controlling salt tolerance and drought resistance of plants, and the coding region sequence of MtCOMT gene is shown as SEQ ID NO. 1.
The MtCOMT gene is a key speed-limiting gene for melatonin biosynthesis, the length of a coding region is 1098bp, the relative molecular weight of the coded protein is 40kDa, the theoretical isoelectric point is 5.67, and the coded protein belongs to relatively stable hydrophilic acidic protein. The invention is obtained from the gene family of the medicago sativa MtCOMT through salt stress and drought stress screening and identification.
The invention also provides a primer pair for amplifying the MtCOMT gene, wherein the primer pair comprises MtCOMT-F and MtCOMT-R;
Wherein, the nucleotide sequence of MtCOMT-F is shown as SEQ ID NO.3; the nucleotide sequence of MtCOMT-R is shown as SEQ ID NO. 4.
The Primer pair is designed by using Primer 5.0 and synthesized by a biological company (Shanghai), and has the function of cloning MtCOMT gene full-length coding region from medicago tribulus genome.
The invention also provides a recombinant expression vector containing the MtCOMT gene, or engineering bacteria or host cells of the recombinant expression vector. And constructing a recombinant expression vector or engineering bacteria or host cells of the recombinant expression vector by utilizing the MtCOMT gene, and transferring the recombinant expression vector or the engineering bacteria or host cells of the recombinant expression vector into plants so as to improve the salt tolerance and drought resistance of the plants.
Wherein the recombinant expression vector comprises the MtCOMT gene and further comprises a plasmid for connecting the MtCOMT gene.
Preferably, the plasmid comprises a pCAMBIA3301 over-expression vector, a pFGC-eYFP vector, PEARLYGATE vector.
Engineering bacteria or host cells containing MtCOMT gene recombinant expression vectors, such as escherichia coli or agrobacterium containing pCAMBIA3301-MtCOMT13, can be obtained by transformation of recombinant expression vectors containing the medicago sativa salt tolerance drought resistance gene MtCOMT.
The engineering bacteria and host cells are understood to be engineering bacteria or host cells used by the person skilled in the art in the transgenic process, such as E.coli competent cells DH 5. Alpha., agrobacterium competent cells EHA105 and Agrobacterium competent cells GV3101. However, with the development of technology, the selection of the engineering bacteria and the host cells may be changed, or the application field of non-transgenic purpose is also related to the utilization of the vector and the engineering bacteria, but only the vector containing the gene or the vector is within the protection scope of the invention.
Transgenic positive plants and seeds which overexpress MtCOMT13 can be created by engineering bacteria or host cells containing the recombinant expression vector of the alfalfa salt-tolerant drought-resistant gene MtCOMT.
The invention also provides application of the MtCOMT gene or the protein coded by the MtCOMT gene or the recombinant vector, engineering bacteria or host cells in improving the salt tolerance and drought resistance of plants.
Wherein the plant comprises Arabidopsis thaliana and alfalfa. In particular, alfalfa is Tribulus terrestris and alfalfa.
Preferably, arabidopsis thaliana is transformed into the MtCOMT gene or a protein encoded thereby as described.
And infecting an arabidopsis inflorescence with an agrobacterium tumefaciens liquid containing the pCAMBIA3301-MtCOMT13 recombinant expression vector by using a flower dipping method, so that a target gene is transformed into the arabidopsis.
Preferably, the MtCOMT gene enhances the salt tolerance and drought resistance of plants by modulating photosynthesis. Specifically, the MtCOMT gene is used for regulating the chlorophyll content of plants to improve the salt tolerance and drought resistance of the plants.
The MtCOMT gene is used for regulating stomatal conductance, transpiration rate and net photosynthetic rate of plants to improve salt tolerance and drought resistance of the plants.
Under the conditions of salt stress and drought stress, compared with wild type arabidopsis, the stomatal conductance, the transpiration rate and the net photosynthetic rate of the transgenic arabidopsis leaves of MtCOMT are obviously improved, which indicates that MtCOMT gene regulates and controls photosynthesis of plants under the salt and drought stress.
The invention also provides a method for cultivating the salt-tolerant drought-resistant transgenic plant, which comprises the step of overexpressing MtCOMT gene in arabidopsis to obtain transgenic arabidopsis with enhanced salt-tolerant drought-resistant property.
The method for cultivating the salt-tolerant drought-resistant transgenic plant provided by the invention specifically comprises the steps of cloning a target gene, constructing a target gene overexpression vector, transforming agrobacterium tumefaciens by the target gene, transforming arabidopsis thaliana by the target gene, identifying positive lines and carrying out phenotypic observation to determine a salt-tolerant drought-resistant material.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers the application of MtCOMT gene in regulating and controlling the salt tolerance and drought resistance of arabidopsis for the first time. The resistance of transgenic arabidopsis to salt stress and drought stress is enhanced by over-expressing the alfalfa MtCOMT gene in arabidopsis. By comparing the gene over-expression arabidopsis plant with a wild type arabidopsis plant, the growth phenotype and photosynthesis of the MtCOMT gene over-expression plant are found to be significantly better than those of the wild type plant. The MtCOMT gene can effectively enhance the salt tolerance and drought resistance of arabidopsis thaliana, can provide theoretical support for the subsequent elucidation of the regulation and control mechanism of plant response to salt stress and drought stress, and also lays a foundation for genetic improvement of the salt tolerance and drought resistance and creation of new germplasm.
Drawings
FIG. 1 is a PCR identification electrophoresis chart of transgenic plants overexpressing MtCOMT in the present invention.
FIG. 2 shows the relative expression levels of the target genes in the transgenic plants overexpressing MtCOMT in the present invention.
FIG. 3 is a phenotypic analysis of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana overexpressed MtCOMT in the present invention; wherein A is the effect of salt and drought stress on the growth phenotype of transgenic Arabidopsis plants; b is the influence of salt and drought stress on the chlorophyll content of transgenic arabidopsis thaliana; c is the influence of salt and drought stress on the stomatal conductance of transgenic Arabidopsis; d is the effect of salt and drought stress on the concentration of CO 2 between cells of transgenic Arabidopsis; e is the effect of salt and drought stress on the transpiration rate of transgenic Arabidopsis; f is the effect of salt and drought stress on the net photosynthetic rate of transgenic arabidopsis.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The invention provides a nucleotide sequence of a salt-tolerant drought-resistant gene MtCOMT of medicago truncatula, which is shown in SEQ ID NO. 1:
ATGGGTTCAACAGGTGAAACTCAAATAACACCAACTCACATATCAGATGAAGAAGCAAACCTCTTCGCCATGCAACTAGCAAGTGCCTCAGTTCTTCCCATGGTTTTAAAATCAGCTCTTGAACTTGATCTCTTAGAAATCATTGCTAAAGCTGGACCTGGTGCTCAAATTTCACCTATTGAAATTGCTTCTCAGCTCCCAACAACTAACCCTGAAGCACCGGTTATGCTGGACCGTATCTTGCGTCTATTGGCTTGTTACAATATCCTCACTTGTTCTGTTCGTACTCAACAAGATGGAAAGGTTCAGAGACTTTATGGTTTGGCTACTGTTGCTAAGTATCTGGTTAAGAATGAAGATGGAGTATCCATTTCTGCTCTTAACCTCATGAATCAGGATAAAGTTCTCATGGAAAGCTGGTACCACCTAAAAGATGCAGTCCTTGATGGGGGCATTCCATTCAACAAGGCTTATGGAATGACAGCCTTTGAATACCATGGAACAGATCCAAGGTTTAACAAGGTTTTCAACAAGGGGATGTCTGATCACTCTACCATCACAATGAAGAAAATTCTTGAGACCTACACAGGTTTTGAAGGCCTTAAATCTCTTGTTGATGTAGGTGGTGGTACTGGAGCTGTAATTAACACGATTGTCTCAAAATATCCCACCATTAAGGGTATTAATTTTGATTTACCCCATGTCATTGAAGATGCTCCATCTTATCCAGGAGTTGAGCATGTTGGTGGAGACATGTTTGTCAGTATTCCAAAGGCTGATGCTGTTTTTATGAAGTGGATTTGTCATGACTGGAGTGATGAGCACTGCTTGAAATTTTTGAAGAACTGCTATGAAGCACTGCCAGACAATGGAAAAGTGATTGTGGCAGAATGCATACTTCCAGTGGCTCCAGATTCAAGCCTGGCCACAAAAGGTGTGGTTCACATTGATGTAATCATGTTGGCTCATAATCCAGGTGGGAAAGAGAGAACACAGAAAGAGTTTGAGGATCTTGCCAAAGGTGCTGGATTCCAAGGTTTCAAAGTTCATTGTAATGCTTTCAACACATACATCATGGAATTTCTTAAGAAGGTTTAA.
The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO. 2:
MGSTGETQITPTHISDEEANLFAMQLASASVLPMVLKSALELDLLEIIAKAGPGAQISPIEIASQLPTTNPEAPVMLDRILRLLACYNILTCSVRTQQDGKVQRLYGLATVAKYLVKNEDGVSISALNLMNQDKVLMESWYHLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGMSDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGINFDLPHVIEDAPSYPGVEHVGGDMFVSIPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECILPVAPDSSLATKGVVHIDVIMLAHNPGGKERTQKEFEDLAKGAGFQGFKVHCNAFNTYIMEFLKKV.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 cloning of the alfalfa MtCOMT Gene
The invention takes diploid medicago sativa R108 as a test material, and the source of the diploid medicago sativa R108 is provided by a laboratory of the university of Qingdao agriculture grass industry.
Primer design
Primer 5.0 software is used for analyzing and designing a Primer for amplifying MtCOMT target fragments, and the designed Primer and the nucleotide sequence are shown as follows:
MtCOMT13-F（SEQ ID NO.3）：5′-ATGGGTTCAACAGGTGAAACTCA-3′；
MtCOMT13-R（SEQ ID NO.4）：5′-TTAAACCTTCTTAAGAAATTCCATGATG-3′。
RNA extraction
Extracting total RNA of a test material by using FastPure Plant Total RNA Isolation Kit (Polysaccharides & polyphenols-rich) RNA extraction kit, performing an extraction process according to the specification of the RNA extraction kit in the whole operation process, and performing reverse transcription by taking the extracted total RNA as a template to obtain cDNA.
Wherein FastPure Plant Total RNA Isolation Kit (Polysaccharides & polyphenols-rich): nuance praise, south Beijing, china.
The reverse transcription reaction procedure was as follows:
In the first step, RNA template was denatured, 1. Mu.L of RNA and 7. Mu.L of ddH 2 O were added to an RNase-free centrifuge tube, heated at 65℃for 5min, rapidly quenched on ice, and allowed to stand on ice for 2min.
And secondly, removing genome DNA, adding 2 mu L of 5X GDNA WIPER Mix into the mixed solution in the previous step, blowing and mixing uniformly by using a pipette, and reacting for 2min at 42 ℃.
Thirdly, a first strand cDNA synthesis reaction solution was prepared, and 2. Mu.L of 10 XRT Mix, 2. Mu. L HISCRIPT III Enzyme Mix, 1. Mu.L of Oligo (dT) 20 VN and 5. Mu.L of ddH 2 O were added to the mixture of the previous step, and then gently mixed by pipetting. Finally, the reaction is carried out in an RT-PCR instrument at 50 ℃ for 45min.
Gene cloning
And (3) taking the obtained cDNA as a template, and carrying out PCR amplification by using primers MtCOMT-F and MtCOMT-R to obtain a 1098bp target fragment.
Wherein, the PCR reaction system: mu.L of cDNA template, 5. Mu.L of 2X PHANTA FLASH MASTER Mix (Dye Plus), 0.5. Mu.L of each of the forward and reverse primers, and finally 10. Mu.L of the mixture was complemented with ddH 2 O;
PCR reaction procedure: pre-denaturation at 98 ℃ for 30sec; denaturation at 98℃for 10sec, annealing at 60℃for 5sec, extension at 72℃for 4sec/kb,34 cycles; thoroughly extend at 72℃for 5min. The product was subjected to agarose gel electrophoresis.
Gel recovery and sequencing
And (3) performing gel recovery and sequencing on the PCR amplification product, wherein the sequencing sequence is shown as SEQ ID NO. 1.
The coding region sequences of the MtCOMT genes are identical through comparison, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2.
EXAMPLE 2 construction of recombinant plant overexpression vector pCAMBIA3301-MtCOMT13
1. Double enzyme cutting over-expression vector
After the gel is recovered, the recovered PCR amplified product and the plasmid of the pCAMBIA3301 over-expression vector are respectively subjected to double enzyme digestion by using Nco I and Pml I restriction enzymes, and specific steps are referred to NEB specifications.
Detecting the enzyme digestion sequence by using a gel electrophoresis method, purifying and recovering the target strip by using a product purification kit, wherein the specific operation is referred to the specification of the product purification kit. The purified PCR amplified product was stored in a-20℃refrigerator.
Wherein, the product purification kit: fastPure @ Gel DNA Extraction Mini Kit product purification kit: nuance praise, south Beijing, china.
2. Homologous recombination for connecting target gene and over-expression vector
And (3) connecting the target gene fragment with the double-enzyme-digested overexpression vector by using a homologous recombination kit, wherein specific steps refer to the specification of the homologous recombination kit.
Wherein, homologous recombination kit: clonExpress @ Ultra One Step Cloning Kit homologous recombination kit: nuance praise, south Beijing, china.
PCR reaction system: taking 2 mu L of target fragment, 3 mu L of linear vector and 5 mu L of 2 x ClonExpress Mix;
PCR reaction procedure: after 30min of connection at 50℃immediately 4 ℃.
3. Recombinant plasmid transformed E.coli
After the target band is connected to the digested pCAMBIA3301 over-expression vector, the connection product is transferred into an escherichia coli competent cell DH5 alpha, and specific steps are referred to the specification of the escherichia coli competent cell DH5 alpha.
Wherein, the competent cell DH5 alpha of the escherichia coli: shanghai, uygur biotechnology Co Ltd.
After the transformation was completed, the E.coli bacterial liquid was spread on LB solid medium containing 50mg/L kanamycin, and cultured in an incubator at 37℃for 36 hours in an inverted state. Monoclonal was picked with a sterile gun head, and bacterial liquid PCR was identified and sequenced with pCAMBIA3301-F/R universal primers.
Wherein, the pCAMBIA3301-F/R universal primer and the nucleotide sequence thereof are as follows:
pCAMBIA3301-F（SEQ ID NO.5）：5'-GAAACCTCCTCGGATTCCATTG-3'；
pCAMBIA3301-R（SEQ ID NO.6）：5'-ATTGCGGGACTCTAATCATAAAAAC-3'。
And (3) activating, preserving and extracting plasmids from the monoclonal bacterial liquid with correct sequencing, wherein specific steps refer to a plasmid extraction instruction.
Wherein, the plasmid: fastPure PLASMID MINI KIT plasmid extraction kit: nuance praise, south Beijing, china.
LB medium: LB broth, model HB0128, soo, qingdao, china.
4. Recombinant plasmid transformed agrobacterium
MtCOMT13 the over-expression vector plasmid was transferred into Agrobacterium competent cell EHA105, for specific procedures in accordance with the instructions for Agrobacterium competent cell EHA 105. The transformed Agrobacterium solution was spread on LB solid medium containing 50mg/L kanamycin and 50mg/L rifampicin, and was placed in an incubator at 28℃for 36 hours. The single clone is picked up by a sterile gun head, and bacterial liquid PCR identification is carried out by using pCAMBIA3301-F/R universal primer. Activating and preserving the identified correct monoclonal colony to obtain the agrobacterium containing pCAMBIA3301-MtCOMT recombinant vector.
Among them, agrobacterium competent cell EHA105: shanghai, uygur biotechnology Co Ltd.
pCAMBIA3301-F（SEQ ID NO.5）：5'-GAAACCTCCTCGGATTCCATTG-3'；
pCAMBIA3301-R（SEQ ID NO.6）：5'-ATTGCGGGACTCTAATCATAAAAAC-3'。
Example 3 transgenic functional verification-MtCOMT 13 transgenic Arabidopsis plants selection and phenotypic analysis
1. Arabidopsis plant preparation
200 Arabidopsis seeds were placed in a 1.5mL centrifuge tube in an ultra clean bench, 1mL ddH 2 O was added thereto, and after shaking and shaking, the mixture was placed in a small centrifuge for centrifugation. Subsequently, 1mL of 75% alcohol was added to sterilize for 30s, and the solution was washed once again with ddH 2 O. NaClO 10% by mass was added and sterilized for 6min, during which the tube was shaken by hand. After 3 washes of ddH 2 O, 1mL ddH 2 O was added for soaking, and swelling was performed for 45min. The arabidopsis seeds are sucked by a 1mL sterile gun head and evenly spread on a 1/2MS solid culture medium, after germination for 3d at 4 ℃, the culture dish is transferred to an illumination incubator at 24 ℃ for 16h illumination/8 h darkness for cultivation, and after 3 true leaves grow out, the arabidopsis seeds can be transplanted into soil. The arabidopsis is continuously grown in soil, and is normally watered and managed until bolting is carried out, so that the arabidopsis is ready for genetic transformation.
Wherein, 1/2MS solid culture medium; agar powder Agar: bootto, beijing, china; MS powder: model M519, phytoTech, usa.
2. Preparation of infectious microbe liquid
Agrobacterium cells containing the recombinant vector pCAMBIA3301-MtCOMT were picked up and cultured overnight on a 28℃constant temperature shaking table with 200rpm in LB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin until OD 600 was about 0.8. 200mL of the shaken bacterial solution was placed in a centrifuge tube, and centrifuged at 8000rpm at 24℃for 10min to collect the bacterial cells. And pouring out the supernatant after centrifugation, adding a sucrose solution with the mass fraction of 5% prepared in advance, uniformly mixing and re-suspending the supernatant until the OD 600 is about 1.0, and preparing a re-suspension bacterial liquid. Then adding surfactant sliwet-77 with mass fraction of 0.03% to obtain infection liquid for standby.
Wherein, 5% sucrose solution is prepared by 1/2MS solution. The formulation registry of the 1/2MS solution was that after adding 2.22g of MS powder and 8g of sucrose to a beaker, ultrapure water was added to a constant volume of 1L. Stirring with magnetic stirrer to dissolve thoroughly, and adjusting pH to 5.85.
Surfactant sliwet-77: model S9430, soribao, beijing, china.
3. Pattern dipping method for infecting arabidopsis thaliana
The arabidopsis inflorescence is immersed in the dye liquor for 60s, immediately taken out, cultured for 24h under dark condition, and then transferred to normal condition for continuous growth. After 7d, the arabidopsis inflorescences are smeared with a cotton swab dip-on solution for secondary infection.
After infection, arabidopsis thaliana is normally managed until seeds are mature, and transgenic Arabidopsis thaliana mature seeds are harvested.
Wherein, the arabidopsis inflorescence refers to the removal of the pod and the flowers which are fully bloomed, and only the buds which are not bloomed are remained.
The method/condition of the normal management of the arabidopsis is that the arabidopsis plants are cultivated under the conditions of 24 ℃ and 16h illumination/8 h darkness, and the water is supplemented by Hoagland nutrient solution. Hoagland nutrient solution formulations are shown in Table 1:
Table 1 Hoagland nutrient solution formulation
4. Identification of transgenic plants
After the transgenic Arabidopsis mature seeds were sterilized, they were spread evenly on 1/2MS solid medium containing 7.5. Mu.g/mL PPT (glufosinate). The specific steps are the same as the steps of the arabidopsis planting method. After the arabidopsis seedling grows to 6 leaves, taking the leaves with good growth, extracting DNA by using a CTAB method, taking a wild arabidopsis plant as a negative control, and identifying a positive strain by using MtCOMT-F/pCAMBIA 3301-R primers through PCR amplification and agarose gel electrophoresis.
Wherein, the DNA extraction step by CTAB method is as follows:
(1) 40mL of CTAB after preheating at 65℃was mixed with 20. Mu.L of RNase according to 1:2000 volume ratio.
(2) 0.2G of blades are put into a 2mL centrifuge tube, homogenized, centrifuged simply, the homogenized at the mouth of the centrifuge tube is thrown to the bottom of the centrifuge tube, and 700 mu L of CTAB is added.
(3) Preserving for 30min in a water bath kettle at 65 ℃ with the centrifuge tube reversed for 3 times.
(4) 700. Mu.L of the DNA extract was added thereto, and the mixture was thoroughly mixed by shaking. This step requires protection from light, preventing DNA from degradation.
(5) The mixture was centrifuged at 13000rpm for 15min at room temperature using a high-speed refrigerated centrifuge (Legend Micro 21R,Thermo Fisher, USA).
(6) 600. Mu.L of the supernatant was transferred to a new centrifuge tube, 400. Mu.L of isopropanol was added, and the mixture was left to stand at-20℃for 15 minutes.
(7) The reaction mixture was centrifuged at 13000rpm at 4℃for 15min, and the supernatant was removed, and then 400. Mu.L of 75% ethanol was added thereto and washed three times.
(8) Centrifuge 13000 rpm min at room temperature, remove the upper isopropanol layer and aspirate the excess liquid. Air-dried in a super clean bench, and 100. Mu.L of ddH 2 O was added after air-drying.
(9) The DNA was obtained by standing overnight at-20℃and stored for later use.
The PCR amplification conditions were RT-PCR identification using Arabidopsis DNA as a template and a 2X PHANTA FLASH MASTER Mix (Dye Plus) kit (Noruzan, nanjing, china).
PCR reaction system: mu.L of the DNA template, 5. Mu.L of 2X PHANTA FLASH MASTER Mix (Dye Plus), 0.5. Mu.L of each of the MtCOMT-F, pCAMBIA3301-R primers, and finally 10. Mu.L of the primer were used as the primer, and the primer was made up with ddH 2 O.
PCR reaction procedure: first step, pre-denaturation at 98℃for 30sec; the second step of denaturation at 98℃for 10sec, annealing at 60℃for 5sec and extension at 72℃for 5sec/kb was performed for 34 cycles; finally, the mixture is thoroughly extended for 5min at 72 ℃.
The source of wild type Arabidopsis plants was provided by the university of Qingdao agricultural grass laboratory.
MtCOMT13-F（SEQ ID NO.3）：5′-ATGGGTTCAACAGGTGAAACTCA-3′；
pCAMBIA3301-R（SEQ ID NO.6）：5′-ATTGCGGGACTCTAATCATAAAAAC-3′。
As shown in FIG. 1, the 1098bp fragment appeared in the transgenic line, whereas the wild-type line did not, indicating that MtCOMT over-expression vector had been successfully introduced into Arabidopsis.
When transgenic positive Arabidopsis grows to 6 leaves, tender leaves of Arabidopsis are cut into a sterilized 2mL centrifuge tube (strip steel beads), and the 2mL centrifuge tube is placed into liquid nitrogen for quick freezing, and is ground by using a freeze grinder (JXFSTPRP-CLN, jing Xin, china). Reagents were added according to FastPure Plant Total RNA Isolation Kit (Polysaccharides & polyphenols-rich) RNA extraction kit (Northenzan, nanjing, china) instructions to extract positive plant young leaf RNA, and RNA concentration was determined using an ultra-micro spectrophotometer (Nano Drop One, thermo Fisher, USA). The extracted RNA is stored in an ultralow temperature refrigerator at the temperature of-80 ℃ for standby. After RNA integrity testing, the RNA was reverse transcribed into cDNA using HISCRIPT III a 1st Strand cDNA Synthesis Kit (+ GDNA WIPER) reverse transcription kit (nuozhen, south kyi, china). The cDNA was diluted to 200 ng/. Mu.L and qRT-PCR was performed to detect the relative expression level of MtCOMT gene using qMtCOMT-F/qMtCOMT-R as primer.
Wherein qMtCOMT-F (SEQ ID NO. 7): 5'-CCAACAACTAACCCTGAA-3';
qMtCOMT13-R（SEQ ID NO.8）：5'-CACCTACATCAACAAGAGA-3'。
the qRT-PCR condition is that the alfalfa action gene of tribulus is used as an internal reference gene.
QRT-PCR reaction system: the total volume was 20. Mu.L, as specified in Table 2 below.
QRT-PCR reaction procedure: qRT-PCR amplification was performed using a BioRad instrument. The specific procedure is as follows: the first step of pre-denaturation is carried out at 95 ℃ for 30sec; in the second step, the reaction is carried out for 10sec at 95 ℃ and 30sec at 60 ℃ for 40 circulation reactions; and thirdly, generating a dissolution curve at 85 ℃ after the cycle is finished.
TABLE 2 real-time fluorescent quantitative PCR reaction system
As a result, as shown in FIG. 2, the relative expression amount of MtCOMT in the transgenic plant was significantly higher than that in the wild-type plant, indicating that the MtCOMT gene-overexpressing plant was successfully obtained. 3 representative strains were propagated and T3 generation seeds were harvested for subsequent testing.
The specific propagation steps are as follows: 200 Arabidopsis seeds were placed in a 1.5mL centrifuge tube in an ultra clean bench, 1mL ddH 2 O was added thereto, and after shaking and shaking, the mixture was placed in a small centrifuge for centrifugation. Subsequently, 1mL of 75% alcohol was added to sterilize for 30s, and the solution was washed once again with ddH 2 O. NaClO 10% by mass was added and sterilized for 6min, during which the tube was shaken by hand. After 3 washes of ddH 2 O, 1mL ddH 2 O was added for soaking, and swelling was performed for 45min. The arabidopsis seeds are sucked by a 1mL sterile gun head and evenly spread on a 1/2MS solid culture medium, after germination for 3d at 4 ℃, the culture dish is transferred to an illumination incubator at 24 ℃ for 16h illumination/8 h darkness for cultivation, and after 3 true leaves grow out, the arabidopsis seeds can be transplanted into soil. The arabidopsis is continuously grown in soil, and is normally watered and managed until the seeds are mature, and transgenic arabidopsis seeds are harvested.
5. Transgenic Arabidopsis phenotype observations
Transplanting wild type MtCOMT over-expressed Arabidopsis seedlings with consistent growth vigor after germination for 7d into a flowerpot filled with vermiculite, and respectively carrying out salt stress treatment and drought stress treatment on the plants after 21d growth. Three biological replicates were set for each treatment group. Following stress treatment, plants continued to grow for 3 weeks, immediately following which the plant phenotype was noted by observation. And photosynthetic parameters such as chlorophyll content, stomatal conductivity, intercellular CO 2 concentration, transpiration rate, net photosynthetic rate and the like are measured by using a photosynthetic instrument.
Wherein, photosynthetic apparatus: model LI-6800, china Ecotek.
Salt stress treatment: salt stress treatment is carried out by adopting 300mM NaCl, namely, the arabidopsis plants grown in the flowerpots filled with vermiculite for 21d are directly irrigated with nutrient solution containing 300mM NaCl, and the nutrient solution is supplemented every 3 days until the salt treatment is carried out for 21d. Three biological replicates were set.
Drought stress: and (3) performing drought stress treatment by adopting a natural drought method for stopping watering, namely stopping watering the arabidopsis plants grown for 21d in the flowerpot filled with vermiculite until the drought treatment is performed for 21d. Three biological replicates were set.
As a result, as shown in FIG. 3, leaves of the transgenic Arabidopsis overexpressing MtCOMT a remain dark green under salt stress, compared to severe wilting and partial chlorosis of rosette leaves of wild-type plants. After natural drought stress, leaves of wild type arabidopsis completely withered, while leaves of transgenic arabidopsis overexpressing MtCOMT were still pale green.
In addition, the stomatal conductance, transpiration rate, and net photosynthetic rate of overexpressed MtCOMT Arabidopsis were significantly higher than that of wild-type Arabidopsis, while the intercellular CO 2 concentration was significantly lower than that of wild-type Arabidopsis. The over-expression MtCOMT gene improves the salt tolerance and drought resistance of the arabidopsis.
The result shows that MtCOMT is an important gene involved in regulating and controlling the salt tolerance and drought resistance of arabidopsis thaliana, and can be used for regulating and controlling the salt tolerance and drought resistance of plants.
Example 4 application of MtCOMT13 Gene in genetic improvement of salt tolerance and drought resistance of alfalfa and Tribulus terrestris
In production practice, the gene can be used for transforming the alfalfa and alfalfa explants, and then the transformed calli can be cultivated into plants. The plant expression vector is used to transform the explant cells to cultivate the salt-tolerant drought-resistant medicago sativa and medicago sativa by a transgenic method, so that the salt-tolerant drought-resistant property of the medicago sativa and the medicago sativa can be improved.
In production practice, the efficiency and accuracy of breeding targets can be enhanced by using the genes through a molecular marker assisted selection breeding method. If the molecular marker is used to correlate the target gene with the salt-tolerant drought-resistant characters of the medicago truncatula and medicago sativa, the target characters can be selected by detecting the existence of the target gene. The invention discovers the function and specific application of MtCOMT gene in regulating and controlling the salt tolerance and drought resistance of plants, provides new gene resources for breeding the salt tolerance and drought resistance of the medicago truncatula and medicago sativa, and lays a foundation for further analyzing the molecular mechanism of MtCOMT for regulating and controlling the salt tolerance and drought resistance of plants.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without undue burden by those of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the present invention by a person skilled in the art shall be within the scope of protection defined by the claims.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (5)
- The application of the MtCOMT13 gene or the protein encoded by the MtCOMT13 gene in improving the salt tolerance and drought resistance of arabidopsis thaliana or alfalfa is characterized in that the nucleotide sequence of the MtCOMT gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein expressed by the MtCOMT gene is shown as SEQ ID NO. 2;Constructing a recombinant expression vector by utilizing the MtCOMT gene, and transferring the recombinant expression vector into arabidopsis thaliana or alfalfa so as to improve the salt tolerance and drought resistance of the arabidopsis thaliana or alfalfa;The MtCOMT gene improves the salt tolerance and drought resistance of arabidopsis thaliana or alfalfa by regulating photosynthesis; the MtCOMT gene is used for regulating the chlorophyll content of the arabidopsis thaliana or the alfalfa to improve the salt tolerance and drought resistance of the arabidopsis thaliana or the alfalfa; the MtCOMT gene is used for regulating the stomatal conductance, the transpiration rate and the net photosynthetic rate of the arabidopsis thaliana or the alfalfa to improve the salt tolerance and drought resistance of the arabidopsis thaliana or the alfalfa.
- 2. Use of MtCOMT gene or protein encoded thereby according to claim 1to improve salt and drought tolerance in arabidopsis or alfalfa, wherein the primer pair for amplifying MtCOMT gene according to claim 1 comprises MtCOMT-F and MtCOMT-R;wherein, the nucleotide sequence of MtCOMT-F is shown as SEQ ID NO. 3; the nucleotide sequence of MtCOMT-R is shown as SEQ ID NO. 4.
- 3. Use of MtCOMT gene or a protein encoded by it according to claim 1 to improve salt and drought tolerance in arabidopsis thaliana or alfalfa, wherein the recombinant expression vector comprises the MtCOMT gene and further comprises a plasmid for ligation of the MtCOMT gene.
- 4. Use of MtCOMT gene or protein encoded thereby according to claim 3 to increase salt and drought tolerance in arabidopsis or alfalfa, wherein the plasmid comprises a pCAMBIA3301 overexpression vector.
- 5. Use of MtCOMT gene or its encoded protein according to claim 1 to improve salt tolerance and drought resistance of arabidopsis thaliana or alfalfa, wherein transgenic arabidopsis thaliana with enhanced salt tolerance and drought resistance is obtained by over-expression of MtCOMT gene according to claim 1 in arabidopsis thaliana.
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| WO2018198049A1 (en) * | 2017-04-25 | 2018-11-01 | Cellectis | Alfalfa with reduced lignin composition |
| CN109536509B (en) * | 2018-11-23 | 2021-11-23 | 中国农业科学院油料作物研究所 | Sesame drought-resistant, moisture-resistant and salt-tolerant gene SiNAC56, protein coded by same and application of sesame drought-resistant, moisture-resistant and salt-tolerant gene SiNAC56 |
| CN111440780B (en) * | 2020-06-01 | 2021-04-06 | 扬州大学 | Paeonia ostii PoCCoAOMT gene cDNA full-length sequence and application thereof in plant drought resistance |
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Non-Patent Citations (3)
| Title |
|---|
| Evolution and Analysis of Caffeic Acid Transferase (COMT) in Seed Plants;Yinghui Gao等;Biochemical Genetics;20231006;第4-5页及摘要 * |
| Regulation of Na+/H+ exchangers, Na+/K+ transporters, and lignin biosynthesis genes, along with lignin accumulation, sodium extrusion, and antioxidant defense, confers salt tolerance in alfalfa;Md Atikur Rahman等;Frontiers in Plant Science;20221107;摘要及图6，第13页右栏 * |
| S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase [ Medicago sativa ]，GenBank: ACY06328.1.Genbank.2009,1. * |
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