CN111394368A - Hevea brasiliensis EPSPS gene with 182 th site mutation and application thereof - Google Patents
Hevea brasiliensis EPSPS gene with 182 th site mutation and application thereof Download PDFInfo
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Abstract
The invention discloses a new mutant gene form of rubber tree 5-enolpyruvylshikimate-3-phosphate synthase, the 544 th nucleotide of an open reading frame is mutated from A to G, the generated mutation is that 182 th amino acid is mutated from T to A, and the verification of transgenic arabidopsis thaliana gene proves that point mutation can improve the resistance to glyphosate, therefore, the EPSPS mutant gene provided by the invention provides a basis for disclosing a phytotoxicity mechanism of plant glyphosate and transgenic detection.
Description
Technical Field
The invention relates to the field of agricultural pharmacy and molecular biology, in particular to a hevea brasiliensis EPSPS gene with a 182 th site mutated and application thereof.
Background
Glyphosate is widely used due to its broad spectrum, high efficiency, environmental friendliness, etc., but the negative effects of glyphosate on non-target plants are not insignificant. Under the restriction of various factors, glyphosate frequently drifts off target to non-target plants or pollutes soil and water bodies in the pesticide application process, so that the glyphosate causes damage to the plants. The glyphosate enters the plant body and is seeped out from the roots or the stubble to return to the soil; after the plant dies, glyphosate is released into the soil again. The action site of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase, namely EPSPS.
The brazilian rubber tree (Hevea brasiliensis muell. arg.) is native to amazon forest in south america, and is the main planting area of the brazilian rubber tree in china, the southern hai, Yunnan, Guangdong, and the like. The Brazilian rubber tree is a main source of natural rubber, and makes great contribution to the economic construction and social development of China. Along with the development of rubber tree seedling culture work and the scale expansion of rubber planting industry in China, the use of herbicides such as glyphosate and the like is increased due to the harm brought by field weeds. In production, glyphosate is found to be harmful to both mature rubber trees and rubber seedlings. When the nursery garden and the forest are used for removing weeds, glyphosate drifting liquid drops are absorbed by the rubber tree seedling leaves. Glyphosate and its metabolites remain in the soil or flow into the water, resulting in the production of phytotoxicity. The glyphosate is sprayed on rubber bud seedlings in the earlier stage, and the application of the glyphosate can cause yellowing, deformity, withering, falling off and deformity of newly grown first fluffy leaves of rubber trees. The change of the leaf structure affects the chloroplast structure, the deformed leaves are in different periods, and the physiological indexes such as chlorophyll, sugar and the like in the leaves are obviously changed compared with the untreated leaves: the activity of superoxide dismutase (SOD) is increased, the activity of Peroxidase (POD) is increased, the content of soluble sugar is reduced, and the content of chlorophyll is reduced. With the development and maturation of molecular biology technology, one or a section of protein of EPSPS gene can be subjected to site-directed mutagenesis, and the resistance to glyphosate is generated on the premise of not influencing the activity of EPSPS. However, no report has been found on the research of EPSPS point mutation of rubber tree glyphosate target gene.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a mutant gene of rubber tree 5-enolpyruvylshikimate-3-phosphate synthase (HbEPSPS), wherein the mutant site is positioned in a core region of the HbEPSPS, namely a 182 th site of the rubber tree EPSPS gene with mutation, so that the transgenic arabidopsis thaliana has the function of improving the resistance to glyphosate;
the second purpose of the invention is to provide a point mutation amplification primer of the EPSPS gene;
the third purpose of the invention is to provide a construction method of EPSPS point mutation transgenic plants.
The purpose of the invention is realized by the following technical scheme: an EPSPS mutant gene, wherein the amino acid of the EPSPS gene at the 182 th site is mutated from T to A, and the nucleotide sequence of the EPSPS gene is shown as SEQ ID NO: 1 is shown.
Application of EPSPS mutant gene in preparation of a kit for detecting glyphosate resistance in plant samples.
Further, the plant is rubber tree or arabidopsis thaliana.
A point mutation amplification primer of an EPSPS gene is HbEPSPS-T182A-F and HbEPSPS-T182A-R, and the sequence of HbEPSPS-T182A-F is shown as SEQ ID NO: 6, the sequence of HbEPSPS-T182A-R is shown as SEQ ID NO: 7, specifically:
HbEPSPS-T182A-F：5′-TTTTCCTCGGAAATGCAGGAGCAGCAATGCGTCC-3′，
HbEPSPS-T182A-R：5′-CTCCTGCATTTCCGAGGAAAAGTTCAACATCA-3′。
a method for constructing an EPSPS point mutation transgenic plant comprises the following steps:
s1, extracting total RNA in a rubber tree leaf, and performing reverse transcription to obtain a cDNA first chain;
s2, obtaining a cDNA sequence of the rubber tree HbEPSPS gene through PCR amplification reaction by taking the cDNA first chain as a template and the HbEPSPS-F and the HbEPSPS-R as primers;
wherein the sequence of the primer HbEPSPS-F is shown as SEQ ID NO: 2, the sequence of the primer HbEPSPS-R is shown as SEQ ID NO: 3, specifically:
HbEPSPS-F：5′-TCAGCAGACAAGTTGGTTGG-3′，
HbEPSPS-R：5′-ATGGTTGCAGACTTGCAGTG-3′；
s3, connecting the cDNA fragment of the HbEPSPS gene obtained by PCR amplification reaction to a pMD18-T vector, converting the cDNA fragment into an Escherichia coli DH5 α strain, and cloning and sequencing verification, wherein a plasmid with correct sequencing verification is named as HbEPSPS-18T;
s4, taking HbEPSPS-18T as a template, adopting point mutation primers HbEPSPS-T182A-F and HbEPSPS-T182A-R as primers for PCR amplification reaction to obtain a cDNA sequence of the HbEPSPS gene, connecting a cDNA fragment of the cDNA sequence to a pMD18-T vector, converting the cDNA fragment into an Escherichia coli DH5 α strain for cloning and sequencing verification, and naming a plasmid with correct sequencing verification as HbEPSPST 182A-18T;
s5, with HbEPSPST182A-18T as a template, and primers HbEPSPS-HindIII-F and HbEPSPS-BamHI-R with enzyme cutting sites to amplify a cDNA sequence, wherein the sequence of the primer HbEPSPS-HindIII-F is shown as SEQ ID NO: 4, the sequence of the primer HbEPSPS-BamHI-R is shown as SEQ ID NO: 5, specifically:
HbEPSPS-HindIII-F：5′-CCCAAGCTTATGGCTGTTACT-3' (the restriction sites are underlined),
HbEPSPS-BamHI-R：5′-CGGGATCCCTCAATCTTACT-3' (the restriction sites are underlined);
and transforming the plasmid with the HbEPSPS gene cDNA sequence into agrobacterium and transforming a target plant Arabidopsis thaliana wild type Col-0 by an electric shock method to obtain a transgenic stably-inherited Arabidopsis thaliana plant.
The transgenic arabidopsis thaliana is treated by glyphosate with different concentrations, and the glyphosate resistance is obviously improved, mainly expressed in that the content of shikimic acid is improved and the weight of seeds is improved.
Further, the reaction procedure of the amplification PCR instrument in step S2 is as follows: pre-denaturation at 94 ℃ for 3min, then denaturation at 94 ℃ for 30s, and annealing at 50 ℃ for 50 s; extending for 2min and 30s at 72 ℃; circulating for 35 times, finally extending for 10min at 72 ℃, and storing at 4 ℃.
The invention has the following advantages: the invention discloses a new mutation gene form of rubber tree 5-enol pyruvylshikimate-3-phosphate synthase (HbEPSPS), the 544 th nucleotide of an Open Reading Frame (ORF) is mutated from A to G, the generated mutation is that 182 th amino acid is mutated from T to A, and the verification of transgenic arabidopsis thaliana gene proves that point mutation can improve the resistance to glyphosate, therefore, the EPSPS mutation gene provided by the invention provides a basis for disclosing a phytotoxicity mechanism and transgenic detection of plant glyphosate.
Drawings
FIG. 1 shows the HbEPSPS point mutation sequence information in example 1; a, a T182APCR product gel electrophoresis picture; b, the nucleotide sequence of the coding region of the HbEPSPS gene T182A; c, HbEPSPS gene coding region 182 amino acid from T mutation to A sequence.
FIG. 2 shows the HbEPSPS point mutation sequence information in example 1; a, the 544 th nucleotide is mutated from A to G; b, mutation of amino acid 182 from T to A; and C, the mutation site is located in the core region of HbEPSPS.
FIG. 3 shows that the HbEPSPS point mutation gene in example 2 transforms Arabidopsis thaliana to improve glyphosate resistance, A, the content of shikimic acid in leaves after 0-20. mu. mol L-1 concentration of glyphosate is sprayed on wild type (Col-0) and over-expression point mutation (T182A) Arabidopsis thaliana, and B, the seed weight after 0-20. mu. mol L-1 concentration of glyphosate is sprayed on wild type (Col-0) and over-expression point mutation (T182A) Arabidopsis thaliana.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
example 1: acquisition of HbEPSPS182 amino acid point mutant gene
(1) Extracting total RNA in the leaves of the rubber tree, and performing reverse transcription to obtain a cDNA first chain;
(2) obtaining a cDNA sequence of the rubber tree HbEPSPS gene through PCR amplification reaction by taking the first chain of the cDNA as a template and the HbEPSPS-F and the HbEPSPS-R as primers; wherein the sequences of the primers HbEPSPS-F and HbEPSPS-R in the step (2) are as follows:
HbEPSPS-F：5′-TCAGCAGACAAGTTGGTTGG-3′，
HbEPSPS-R：5′-ATGGTTGCAGACTTGCAGTG-3′。
the PCR instrument has the following reaction program that pre-denaturation is carried out at 94 ℃ for 3min, then denaturation is carried out at 94 ℃ for 30s, annealing is carried out at 50 ℃ for 50s, extension is carried out at 72 ℃ for 2min for 30s, circulation is carried out for 35 times, finally extension is carried out at 72 ℃ for 10min, preservation is carried out at 4 ℃, a cDNA sequence of the HbEPSPS gene is obtained through PCR amplification reaction, a cDNA fragment is connected into a pMD18-T vector and is transformed into an escherichia coli DH5 α strain for cloning and sequencing verification, and a plasmid with correct sequencing verification is named as HbEPSPS-18T;
(3) HbEPSPS-18T is taken as a template, and a point mutation primer is taken as
HbEPSPS-T182A-F：5′-TTTTCCTCGGAAATGCAGGAGCAGCAATGCGTCC-3′，
HbEPSPS-T182A-R：5′-CTCCTGCATTTCCGAGGAAAAGTTCAACATCA-3′
The PCR amplification PCR instrument is carried out by the following reaction procedures of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 50s, extension at 72 ℃ for 2min for 30s, circulation for 35 times, final extension at 72 ℃ for 10min, storage at 4 ℃, obtaining the cDNA sequence of the HbEPSPS gene through PCR amplification reaction, connecting the cDNA fragment to pMD18-T vector, transforming into Escherichia coli DH5 α strain for cloning and sequencing verification, wherein the plasmid with correct sequencing verification is named as HbEPSPST182A-18T, and the experimental results are shown in figures 1 and 2.
Example 2: HbEPSPST182A amino acid point mutation transgenic arabidopsis thaliana for improving glyphosate resistance
(1) HbEPSPST182A-18T is used as a template, and a primer with an enzyme cutting site is used
HbEPSPS-HindIII-F：5′-CCCAAGCTTATGGCTGTTACT-3′，
HbEPSPS-BamHI-R: 5'-CGGGATCCCTCAATCTTACT-3' amplifying the cDNA sequence;
(2) transforming the plasmid with the cDNA sequence of the HbEPSPS gene into agrobacterium and transforming a target plant Arabidopsis thaliana wild type Col-0 by an electric shock method to obtain a transgenic stable genetic point mutation overexpression Arabidopsis thaliana plant (T182A);
(3) with 0, 0.1, 1, 10, 15 and 20. mu. mol L-1Treating wild Col-0 and T182A mutant transgenic Arabidopsis by glyphosate;
(4) mixing 0, 0.1, 1, 10, 15 and 20 mu mol L-1The leaves of wild Col-0 and T182A mutant transgenic Arabidopsis thaliana treated by glyphosate are ground by liquid nitrogen, and about 0.1g of leaves are put into a 2.0m L centrifugal tubeAdding 0.25mol L of 1.0m L-1HC L, centrifuging at 12000rpm at 4 deg.C for 30min, collecting 200 μ L supernatant, centrifuging in 10m L centrifuge tube, adding 2.0m L oxidant solution (1% periodic acid and 1% sodium periodate solution), standing at room temperature for 3 hr, and adding 2.0m L1.0.0 mol L-1NaOH solution, 1.2m L0.1.0.1 mol L-1Mixing glycine, standing for 5min, comparing color at 380nm with ultraviolet spectrophotometer at 0.25 mol/L-1HC L is a blank control, the experimental results are shown in FIG. 3, and the results show that the resistance of T182A mutant transgenic Arabidopsis thaliana to glyphosate is remarkably improved, and the tolerance concentration reaches 15 mol. L-1The seed number after the seeds of the Arabidopsis were subjected to seed setting analysis, and the T182A mutant transgenic Arabidopsis seeds were found to have a weight of 15 mol. L-1Still 0.2g can be reached.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Sequence listing
<110> university of Hainan
<120> hevea brasiliensis EPSPS gene with 182 th site mutation and use thereof
<130>1
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>1572
<212>DNA
<213> rubber Tree (unknown)
<400>1
atggcgcaag cgagcaaaat ctgcaatggg gctcaaaata attgtacttt cctcaatctc 60
tcgaaacccc aaagacccaa atatctatct tcaatttcat ttagatcaca gcttcagggg 120
tcttcacttt catggggttc aaaacagtgt caaagaaggg ctgattctac agattctaca 180
gttggtacag ttaagatgag tcctgttaga gtttcggctt cagtcgccac agcagagaag 240
tcggcaccag agatagtctt gcaacccatt aaagaaatct ccggtaccgt ctacttgccg 300
ggttccaagt ctctgtccaa tcggattctc cttcttgctg ctctttctga gggtacaact 360
gttgttgaca acttgctgaa tagtgatgat gttcgttaca tgcttggtgc actgagaacg 420
cttggattgc gtgtggaaga caatagtgaa ctcaaacaag ccattgtaga aggttgtgga 480
ggtcattttc cggtgggtaa agaatcaaag aatgatgttg aacttttcct cggaaatgca 540
ggaacagcaa tgcgtccatt gactgctgct gttactgcag cgggtggaaa ttcaagctac 600
atacttgatg gggttccacg aatgcgagag agaccaattg gagatttggt tgctggtctt 660
aagcagcttg gtgcagatgt tcaatgttct gatactaact gtccccctgt tcgtgtaaat 720
ggaaaaggag gacttcctgg gggaaaggtt aagctctctg gatcaattag tagtcaatac 780
ttgactgctt tgctcatggc agctcctttg gctcttggag atgtagaaat tgagattatt 840
gataaactga tatccattcc ttatgttgag atgactttga agttaatgga gcgatatgga 900
gtcaccatag aacacagtgg tagctgggat cgtttcttcg ttcgtggtgg tcaaaagtac 960
aagtctcctg gaaattcttt tgttgaaggt gatgcttcaa gtgccagtta tttcctggct 1020
ggtgcagcaa tcaccggtgg aaccatcact gttgaaggtt gtgggacgag cagtttgcag 1080
ggggatgtaa aatttgctga ggttcttgat aaaatgggag ctaaagttac ctggacagag 1140
aacagtgtta ctgtcactgg accaccacgc aattctccta gtaaaaaaca cttgcgcgct 1200
attgatgtca acatgaacaa aatgccagat gttgctatga cgctggccgt ggttgcactt 1260
tttgctgatg gccctactgc cataagagac gtggccagtt ggagagtgaa agaaacagaa 1320
cggatgatcg caatttgtac agagctcagg aagttaggag caaatgttga agaggggcaa 1380
gattactgtg ttattactcc acctgagaaa ctaaaagtcg cagagatcga cacttatgat 1440
gatcacagaa tggccgtggc attctccctt gctgcctgtg gggatgttcc agtcacaatc 1500
aaggaccctg gttgcacaag aaaaactttt cccgactact ttgaagtcct tgagaggttc 1560
actaagcatt ga 1572
<210>2
<211>20
<212>DNA
<213> Artificial sequence (unknown)
<400>2
<210>3
<211>20
<212>DNA
<213> Artificial sequence (unknown)
<400>3
<210>4
<211>21
<212>DNA
<213> Artificial sequence (unknown)
<400>4
cccaagctta tggctgttac t 21
<210>5
<211>20
<212>DNA
<213> Artificial sequence (unknown)
<400>5
<210>6
<211>34
<212>DNA
<213> Artificial sequence (unknown)
<400>6
ttttcctcgg aaatgcagga gcagcaatgc gtcc 34
<210>7
<211>32
<212>DNA
<213> Artificial sequence (unknown)
<400>7
ctcctgcatt tccgaggaaa agttcaacat ca 32
Claims (6)
1. An EPSPS mutant gene, which is characterized in that the amino acid of the EPSPS gene at the 182 th site is mutated from T to A, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
2. Use of the EPSPS mutant gene of claim 1 in the preparation of a kit for detecting glyphosate resistance in a plant sample.
3. Use according to claim 2, wherein the plant is rubber tree or Arabidopsis thaliana.
4. A point mutation amplification primer of an EPSPS gene is characterized by comprising HbEPSPS-T182A-F and HbEPSPS-T182A-R, wherein the sequence of HbEPSPS-T182A-F is shown as SEQ ID NO: 6, the sequence of HbEPSPS-T182A-R is shown as SEQ ID NO: shown at 7.
5. A method for constructing an EPSPS point mutation transgenic plant is characterized by comprising the following steps:
s1, extracting total RNA in a rubber tree leaf, and performing reverse transcription to obtain a cDNA first chain;
s2, obtaining a cDNA sequence of the rubber tree HbEPSPS gene through PCR amplification reaction by taking the cDNA first chain as a template and the HbEPSPS-F and the HbEPSPS-R as primers;
wherein the sequence of the primer HbEPSPS-F is shown as SEQ ID NO: 2, the sequence of the primer HbEPSPS-R is shown as SEQ ID NO: 3 is shown in the specification;
s3, connecting the cDNA fragment of the HbEPSPS gene obtained by PCR amplification reaction to a pMD18-T vector, converting the cDNA fragment into an Escherichia coli DH5 α strain, and cloning and sequencing verification, wherein a plasmid with correct sequencing verification is named as HbEPSPS-18T;
s4, taking HbEPSPS-18T as a template, adopting point mutation primers HbEPSPS-T182A-F and HbEPSPS-T182A-R as primers for PCR amplification reaction to obtain a cDNA sequence of the HbEPSPS gene, connecting a cDNA fragment of the cDNA sequence to a pMD18-T vector, converting the cDNA fragment into an Escherichia coli DH5 α strain for cloning and sequencing verification, and naming a plasmid with correct sequencing verification as HbEPSPST 182A-18T;
s5, with HbEPSPST182A-18T as a template, and primers HbEPSPS-HindIII-F and HbEPSPS-BamHI-R with enzyme cutting sites to amplify a cDNA sequence, wherein the sequence of the primer HbEPSPS-HindIII-F is shown as SEQ ID NO: 4, the sequence of the primer HbEPSPS-BamHI-R is shown as SEQ ID NO: and 5, transforming the plasmid with the HbEPSPS gene cDNA sequence into agrobacterium and transforming a target plant Arabidopsis thaliana wild type Col-0 by an electric shock method to obtain a transgenic stably-inherited Arabidopsis thaliana plant.
6. The method for constructing the EPSPS point mutation transgenic plant according to claim 5, wherein the reaction program of the amplification PCR instrument in the step S2 is as follows: pre-denaturation at 94 ℃ for 3min, then denaturation at 94 ℃ for 30s, and annealing at 50 ℃ for 50 s; extending for 2min and 30s at 72 ℃; circulating for 35 times, finally extending for 10min at 72 ℃, and storing at 4 ℃.
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Citations (5)
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