CN111139244B - Populus tomentosa MODD1 gene and application thereof - Google Patents

Populus tomentosa MODD1 gene and application thereof Download PDF

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CN111139244B
CN111139244B CN201911404484.5A CN201911404484A CN111139244B CN 111139244 B CN111139244 B CN 111139244B CN 201911404484 A CN201911404484 A CN 201911404484A CN 111139244 B CN111139244 B CN 111139244B
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胡赞民
孙宝成
范成明
苏晓华
丁昌俊
朱一杭
陈宇红
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Research Institute of Forestry of Chinese Academy of Forestry
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Abstract

The invention relates to the field of biotechnology, in particular to a populus tomentosa MODD1 gene and application thereof, wherein the nucleotide sequence of the populus tomentosa MODD1 gene obtained by amplification is shown as SEQ ID NO. 1. According to the invention, the populus tomentosa MODD1 gene is transformed into Arabidopsis thaliana through the agrobacterium GV3101, and the drought resistance of the Arabidopsis thaliana with the populus tomentosa MODD1 gene overexpressed is improved, and the content of abscisic acid is improved. The populus tomentosa MODD1 gene provided by the invention can be used for breeding drought-resistant strains of populus tomentosa and various crops.

Description

Populus tomentosa MODD1 gene and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a populus tomentosa MODD1 gene and application thereof.
Background
Terrestrial plants develop various drought-resistant functions in long-term adaptive evolution, but are still threatened by drought due to climate reasons. Drought affects not only the growth metabolic process of plants, but also the yield and reproduction of commercial crops, and severe drought stress can cause plant death. Therefore, the method has important significance for the research on the drought resistance of plants.
The MODD1 gene of Populus tomentosa belongs to the NINJA (Novel Interactor of JAZ proteins) protein family, which is an interaction protein of JAZ protein found in plants. The activity of the NINJA protein as a transcriptional repressor is mediated by a functional TPL binding domain upstream of this domain. Studies in plants have been shown to be transcriptional repression, e.g. in arabidopsis AFP1 and AFP2 participate in drought regulation by binding to ABI5, negatively regulating ABA signalling, whereas ABF3 is up-regulated by ABA induction (Garcia et al, 2008). In rice, MODD protein, a NINJA family member, inhibits OsbZIP46 activity, and promotes degradation of OsbZIP46 to inhibit drought resistance of rice (Tang et al, 2016). In addition to drought resistance, NINJA is also involved in other signaling pathways, such as in arabidopsis, where it can interact with TOPLESS to negatively regulate the jasmonate signaling pathway (Pauwels et al, 2010).
Disclosure of Invention
The invention aims to improve the drought resistance of plants by using MODD1 gene derived from Chinese white poplar, and provides the Chinese white poplar MODD1 gene and application thereof.
In order to achieve the purpose, according to the annotation of the genome of the populus tomentosa, the cDNA sequence of the populus tomentosa MODD1 gene is screened, oligonucleotide primers are designed and synthesized according to the sequence, cDNA after reverse transcription of mRNA of the populus tomentosa is taken as a template, the full-length cDNA sequence of the populus tomentosa MODD1 gene is cloned and obtained and named PtMODD1, the full-length cDNA sequence is constructed on an entry vector, then the full-length cDNA sequence is constructed on a plant expression vector through recombination reaction, positive clone is screened, and the arabidopsis thaliana is infected after agrobacterium tumefaciens (GV 3101) is transformed.
After the treatment, from experimental results, the PtMODD1 from populus tomentosa is driven by a CaMV35S promoter, the transgenic plants do not have poor agronomic characters, and the drought resistance of PtMODD 1-transformed Arabidopsis is obviously improved. Arabidopsis is a model plant, and genes which can play a role in Arabidopsis have similar effects in various crops, so that PtMODD1 can be used for drought resistance of populus tomentosa and various crops.
Specifically, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a populus tomentosa MODD1 gene, wherein the nucleotide sequence of the populus tomentosa MODD1 gene is:
i) 1, a nucleotide sequence shown in SEQ ID NO; or
ii) the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides to encode the nucleotide sequence of the same functional protein.
The invention further provides a protein coded by the populus tomentosa MODD1 gene, wherein the amino acid sequence of the protein is as follows:
1) An amino acid sequence shown as SEQ ID No. 2; or
2) The amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
The invention further provides a biological material containing the populus tomentosa MODD1 gene, wherein the biological material is one or more of a vector, a transgenic cell line, an engineering bacterium, a host cell or an expression cassette.
In a second aspect, the invention provides an application of the populus tomentosa MODD1 gene, the coding protein thereof or a biological material containing the same in regulating and controlling the drought resistance of plants.
The invention further provides the application of the populus tomentosa MODD1 gene or the coding protein thereof or the biological material containing the populus tomentosa MODD1 gene in plant genetic breeding or transgenic plant preparation.
The application specifically comprises the step of improving the drought resistance of plants by improving the expression level of the populus tomentosa MODD1 gene.
Preferably, the increasing of the expression level of populus tomentosa MODD1 gene is to overexpress the populus tomentosa MODD1 gene in the plant.
The invention further provides the application of the populus tomentosa MODD1 gene or the coding protein thereof or the biological material containing the populus tomentosa MODD1 gene in improving the content of abscisic acid in plants.
In the above application, the plant may be rice, arabidopsis, canola, soybean, cotton, wheat, corn or poplar.
In a third aspect, the present invention provides a method for regulating drought resistance of a plant, comprising: regulating and controlling the expression level of Chinese white poplar MODD1 gene in the plant;
the nucleotide sequence of the populus tomentosa MODD1 gene is as follows:
i) 1, SEQ ID NO; or
ii) the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides to encode the gene of the same functional protein.
Further, the drought resistance of the plant is improved by improving the expression level of the populus tomentosa MODD1 gene in the plant, preferably by improving the content of abscisic acid in the plant; or, a drought-resistant strain is cultivated by crossing the strain which over-expresses the populus tomentosa MODD1 gene with other strains.
Further, the construction method of the line over expressing the populus tomentosa MODD1 gene comprises the following steps:
the populus tomentosa MODD1 gene is connected to a plant expression vector and is obtained by transforming a wild type strain with agrobacterium GV 3101.
The invention provides a populus tomentosa MODD1 gene and application thereof, and has the following beneficial effects:
the invention provides an arabidopsis thaliana strain for over-expressing Chinese white poplar MODD1 gene, and the comparison with a wild type strain shows that the transgenic plant can not have poor agronomic characters under the drive of a CaMV35S promoter by the MODD1 gene from the Chinese white poplar, and the drought resistance of the arabidopsis thaliana transformed with the MODD1 gene of the Chinese white poplar is obviously improved. The arabidopsis thaliana is a model plant, and genes which can play a role in arabidopsis thaliana have similar effects in various crops, so the populus tomentosa MODD1 gene provided by the invention can be used for breeding drought-resistant strains of populus tomentosa and various crops.
Drawings
FIG. 1 is a plasmid map of plasmid pGWC-PtMODD1 obtained by ligating Populus tomentosa MODD1 gene to an entry vector, according to example 1 of the present invention;
FIG. 2 is a plasmid map of plasmid pPtMODD1 obtained by ligating Populus tomentosa MODD1 gene to a plant expression vector, according to example 1 of the present invention;
FIG. 3 is a schematic diagram showing drought resistance comparison between Arabidopsis thaliana (OE-PtMODD 1-1 and OE-PtMODD 1-2) and wild type Arabidopsis thaliana (Col 0) with heterologous expression of the populus tomentosa MODD1 gene, which have the same growth period and are provided in example 3 of the present invention;
FIG. 4 is a schematic diagram showing the comparison of the expression levels of abscisic acid (ABA), malondialdehyde (MDA) and proline (Pro) in Arabidopsis thaliana (OE-PtMODD 1-1 and OE-PtMODD 1-2) and wild type Arabidopsis thaliana (Col 0) for heterologous expression of populus tomentosa MODD1 gene, which are provided in example 4 of the present invention and have consistent growth periods.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
Example 1
Example 1 obtaining of Populus tomentosa MODD1 Gene and construction of expression vector
1. Extraction of Populus tomentosa RNA
(1) Taking the example of the extraction of RNA from leaves as sample treatment, taking fresh leaves to fully grind in liquid nitrogen, wherein the grinding needs to be rapid;
(2) Adding 500 μ L lysate (containing 5 μ L beta-mercaptoethanol), mixing by vortex oscillation, centrifuging at 12,000rpm for 2min, and transferring the supernatant to another centrifuge tube;
(3) Adding 250 μ L of anhydrous ethanol, mixing (precipitate may appear at this time), transferring the obtained solution and precipitate into adsorption column CR3 (the adsorption column is placed in a collecting tube), centrifuging at 12 000rpm for 1min, pouring off waste liquid, and placing the adsorption column CR3 back into the collecting tube;
(4) Adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12 000rpm for 1min, discarding waste liquid, and placing adsorption column CR3 back into the collection tube;
(5) Preparation of Dnase I working solution: putting 10 mu L of DNase I storage solution into a new RNase-free centrifuge tube, adding 70 mu L of RDD solution, and gently and uniformly mixing;
(6) Adding 80 μ L DNase I working solution into the center of the adsorption column CR3, and standing at room temperature for 15min;
(7) Adding 500 μ L deproteinized solution RW1 to the center of adsorption column CR3, centrifuging at 12,000rpm for 1min, discarding waste liquid, and placing the adsorption column back into the collection tube;
(8) Adding 500 μ L of rinsing solution RW (containing ethanol) into adsorption column CR3, standing at room temperature for 2min, centrifuging at 12 000rpm for 1min, pouring off waste liquid, and placing adsorption column CR3 into the collection tube;
(9) Repeating the previous step;
(10) Centrifuging at 12 000rpm for 2min, and pouring off waste liquid. Placing the adsorption column CR3 at room temperature for several minutes to completely dry the eluent remained on the adsorption column;
(11) Transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, and adding 60 μ L ddH 2 O, standing at room temperature for 2min, and centrifuging at 12 000rpm for 2min to obtain an RNA solution.
2. Synthesis of Populus tomentosa cDNA
(1) Adding an RNA template, a primer and RNase-free Water, mixing uniformly, incubating for 5 minutes at 65 ℃, and carrying out ice bath for 2 minutes;
(2) Add 1. Mu.L Random Primer, 10. Mu.L Reaction Mix, 1. Mu.L Transcript Enzyme Mix, 1. Mu.L gDNA Remover;
(3) Lightly mixing uniformly;
(4) After incubation at 25 ℃ for 10 minutes, incubation at 42 ℃ for 15 minutes;
(5) Heat at 85 ℃ for 5 seconds inactivated the Transcript Enzyme and gDNA Remover.
3. Construction of Populus tomentosa MODD1 gene expression vector
Obtaining a Chinese white poplar MODD1 cDNA sequence according to a Chinese white poplar genome database sequence, designing an upstream and a downstream amplification primers according to the sequence:
forward.5’-ATGGGAGAAACAAACGAGAATAG-3’,
reverse.5’-CACAAAGGACGGACCAGAAGAG-3’;
using Populus tomentosa cDNA as template, and pfu enzyme (Trans)
Figure BDA0002348276700000061
Fastpfu DNA Polymerase) amplified the populus tomentosa MODD1 gene, and the obtained gene sequence was designated PtMODD1. The amplification procedure was 35 cycles of pre-denaturation at 2min 98 deg.C, 30s 60 deg.C, 1min 72 deg.C. The PCR product was purified with a kit (Beijing Quanji Biotech Co., ltd.) and used.
The PCR product was ligated to an entry vector (named pGWC-PtMODD1, FIG. 1 is the ligated plasmid map) using an In-fusion system, as follows: mu.L of digested pGWCm (100 ng/. Mu.L, digested with AhdI), 1. Mu.L of LPCR product (80 ng/. Mu.L), 2. Mu.L of In-fusion Mix, 50-60min at 50 ℃; escherichia coli DH 5. Alpha. Was transformed, and positive clones were screened by PCR identification.
After sequencing identification, the gene is constructed on a plant expression vector pHZM 69 through a Gateway system and named as pPtMODD1 (a plasmid map is shown in figure 2), a promoter of the PtMODD1 is CaMV35S, and a screening marker of a transformed plant in the vector is hygromycin and Basta. The recombinant plasmid is transformed into agrobacterium GV3101, and is identified by PCR for later use.
Example 2 genetic transformation of Populus tomentosa MODD1 Gene and screening of Positive transgenic lines
In this example, the genetic transformation of arabidopsis thaliana was performed by the dipping method, and the specific steps were as follows:
agrobacterium harboring the pPtMODD1 plasmid constructed in example 1 was cultured in LB liquid medium (50 mg/L kanamycin, 50mg/L gentamicin, and 200mg/L rifampicin added) to OD =0.8, centrifuged at 10 000rpm for 5min to collect the cells, and an equal volume of suspension (10 mM MgCl. Sub.L. Sub.MgCl.) was used 2 5% sucrose), the cells were collected by centrifugation, suspended in the suspension to OD =1.0, and 0.005% silwet L-77 was added to transform Arabidopsis thaliana at the early stage of flowering once, and dipped into flowers once more at 7-day intervals. After the seeds are mature, collecting T0 generation seeds, planting the seeds in a culture dish, spraying Basta (0.3%) for screening 10 days after seedling emergence, and spraying once again at an interval of 5 days; after PCR identification, the T1 generation positive seedlings are transplanted into a nutrition pot for culture, and seeds are harvested after maturation.
Example 3 evaluation of drought resistance of MODD1 Gene of Populus tomentosa
This example compares arabidopsis thaliana (prepared by the method described in example 2) overexpressing the populus tomentosa MODD1 gene, and Col0 wild-type arabidopsis thaliana, which had consistent growth periods, were planted under the same greenhouse conditions for 14 days of drought treatment (without watering) with a control group, and the comparison results were photographed. As shown in fig. 3, in this example, it is found that the wilting of arabidopsis thaliana (OE-PtMODD 1 and OE-PtMODD 1) over-expressing populus tomentosa MODD1 gene hardly occurs, while the wilting of the control group (Col 0) is more obvious, and the result shows that the populus tomentosa MODD1 gene has a significant effect of promoting the drought resistance improvement of arabidopsis thaliana.
Example 4 analysis of physiological indices of MODD1 Gene of Populus tomentosa
In this embodiment, a kit (enzyme immunoassay) is used to determine the levels of various indexes of plants in a specimen by using a double antibody sandwich method, and the specific steps are as follows:
coating a microporous plate with purified plant abscisic acid (ABA), malondialdehyde (MDA) and proline (Pro) capture antibodies to prepare solid-phase antibodies, sequentially adding the ABA, MDA and Pro into the coated micropores, combining with HRP-labeled detection antibodies to form antibody, antigen and enzyme-labeled antibody complexes, and adding a substrate TMB for color development after thorough washing. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The color shade is positively correlated with the plant ABA, MDA and Pro in the sample. Measuring absorbance (OD value) by using an enzyme-labeling instrument at the wavelength of 450nm, and calculating the content of each index of the plant in the sample through a standard curve, wherein the method comprises the following specific steps:
(1) Sample adding of the standard: setting standard substance holes and sample holes, wherein 50 mu L of standard substances with different concentrations are added into the standard substance holes respectively;
(2) Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. 40 mu L of sample diluent is added into sample holes to be detected on the enzyme-labeled coated plate, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding a sample to the bottom of an enzyme label plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;
(3) Adding an enzyme: adding 100 mu L of enzyme-labeled reagent into each hole except for blank holes;
(4) And (3) incubation: sealing the plate with a sealing plate film, and then incubating at 37 ℃ for 60min;
(5) Preparing liquid: diluting 20 times of the concentrated washing liquid with 20 times of distilled water for later use;
(6) Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30sec, discarding, repeating the steps for 5 times, and patting to dry;
(7) Color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15min;
(8) And (4) terminating: adding 50 mu L of stop solution into each well to stop the reaction (at the moment, the blue color immediately turns to yellow);
(9) And (3) determination: the blank wells were zeroed and the absorbance (OD) of each well was measured sequentially at a wavelength of 450 nm. The measurement should be carried out within 15min after the addition of the stop solution.
The results shown in FIG. 4 (OE-PtMODD 1 represents Arabidopsis thaliana heterologously expressing populus tomentosa MODD1 gene, col0 represents wild type) were obtained by the above experiments, and the Arabidopsis thaliana heterologously expressing populus tomentosa MODD1 gene had increased abscisic acid content and no difference in proline and malondialdehyde content compared with the wild type.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Institute of forestry, Chinese Academy of Forestry Sciences
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Claims (10)

1. The populus tomentosa MODD1 gene is characterized in that the nucleotide sequence of the populus tomentosa MODD1 gene is as follows:
i) 1, and the nucleotide sequence shown in SEQ ID NO.
2. A protein encoded by the Populus tomentosa MODD1 gene of claim 1, wherein the amino acid sequence of the protein is:
1) The amino acid sequence shown as SEQ ID No. 2.
3. A biomaterial comprising one or more of the poplar MODD1 gene as described in claim 1, wherein the biomaterial is a vector, an engineered bacterium or an expression cassette.
4. Use of populus tomentosa MODD1 gene or a protein encoded by the gene or the biomaterial of claim 1 or claim 3 for regulating drought resistance of plants.
5. Use of populus tomentosa MODD1 gene or a protein encoding the same as claimed in claim 1 or a biomaterial as claimed in claim 3 in the genetic breeding of drought-resistant plants or in the preparation of transgenic drought-resistant plants.
6. The use according to claim 4 or 5, wherein the drought resistance of the plant is improved by increasing the expression level of the populus tomentosa MODD1 gene;
the method for improving the expression level of the populus tomentosa MODD1 gene comprises the step of over-expressing the populus tomentosa MODD1 gene in the plant.
7. Use of populus tomentosa MODD1 gene or a protein encoded by the gene or the biomaterial of claim 3 for increasing abscisic acid content in plants.
8. A method of modulating drought resistance in a plant comprising: regulating and controlling the expression level of Chinese white poplar MODD1 gene in plant;
the nucleotide sequence of the populus tomentosa MODD1 gene is as follows:
i) 1, and the nucleotide sequence shown in SEQ ID NO.
9. The method as claimed in claim 8, wherein drought resistance of the plant is increased by increasing the expression level of the populus tomentosa MODD1 gene in the plant, or a drought resistant strain is bred by crossing a strain overexpressing the populus tomentosa MODD1 gene with another strain.
10. The method as claimed in claim 9, wherein the line overexpressing the poplar MODD1 gene is constructed by:
the populus tomentosa MODD1 gene is connected to a plant expression vector and is obtained by transforming a wild type strain with agrobacterium GV 3101.
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