CN113621625B - Application of sesame SiERF103 gene in enhancing plant resistance - Google Patents

Abstract

The invention discloses application of a sesame SiERF103 gene in enhancing plant resistance, belonging to the technical field of plant genetic engineering, wherein the nucleotide sequence of the sesame SiERF103 gene is shown as SEQ ID NO.1, and the resistance comprises resistance to waterlogging and/or drought. The invention reports the application of the sesame SiERF103 gene in enhancing plant waterlogging resistance and/or drought resistance for the first time. Through constructing SiERF103 gene over-expression vector, it is over-expressed in Arabidopsis thaliana, and is subjected to drought or waterlogging stress treatment, researches find that after drought or waterlogging stress treatment, normal growth condition is recovered, and transgenic Arabidopsis thaliana can be recovered to normal growth in large quantity. The invention proves that the sesame SiERF103 gene has the function of improving plant waterlogging and drought resistance. The gene source is provided for drought resistance and stain resistance genetic improvement of crops, and has important significance for promoting stable yield of crops.

Description

Application of sesame SiERF103 gene in enhancing plant resistance

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of sesame SiERF103 genes in enhancing plant resistance.

Background

Sesame (Sesamum indicum l.) is a specialty oil crop in our country, cultivated in a long history, cultivated worldwide, and china is a major production and trade country, but is self-sufficient. In recent years, the yield of sesame in China is seriously reduced, and the sesame has a great relationship with the influence of various abiotic stresses such as drought, waterlogging and the like on the sesame in the growing and developing process. The sesame industry technology system researches and analyzes most of sesame main production areas in China, and discovers that sesame in China is influenced by waterlogging and drought stress throughout the year, so that the growth and development of the sesame are seriously influenced, and the sesame is an important cause for reducing the sesame yield. Therefore, the drought resistance and the stain resistance of sesame are improved, the high and stable yield is promoted, and the sesame is particularly important to promote the sesame production and development in China and solve the problem of insufficient total quantity. At present, the researches on the functions of the sesame drought resistance and stain resistance related genes are deficient, and the researches on molecular correlation are relatively few, so that the development of the sesame stress resistance functional genes has important significance in developing sesame stress resistance molecular breeding and improving sesame stress resistance.

The plant is often influenced by a plurality of environmental factors in the growth and development, when the stress occurs, the plant can rapidly activate a large number of stress response genes and synthesize various functional proteins, and a series of complex signal networks are formed to resist adverse environments, maintain the normal physiological functions of the plant and achieve the aim of avoiding the stress injury. The transcription factors are protein molecules which can be specifically combined with cis-acting elements in a promoter region of a eukaryotic gene, so that the target gene is ensured to be expressed in specific strength, specific time and space, and important function is played in plant stress resistance. For example, arabidopsis RAP2.2 resists hypoxia stress by regulating the expression of hypoxia stress related genes such as ADH1, PDC1 and the like, and genes related to carbohydrate metabolism and ethylene synthesis. Rice Sub1A-1 inhibits ethylene production and transcription of genes related to growth and metabolism under flooding conditions, reduces decomposition of starch and sugar, and enables plant bodies to keep a low metabolism state and survive. Sesame is used as an important oil crop, and the separation and identification of the stress resistance gene of the sesame has very important significance for cultivating new stress resistance sesame varieties.

Disclosure of Invention

Aiming at the blank of the prior art, the invention provides the application of the sesame SiERF103 gene in enhancing plant resistance, and through the over-expression experiment of the SiERF103 gene in model plant transgenic arabidopsis, the invention verifies that the sesame SiERF103 gene has the functions of enhancing plant waterlogging resistance and drought resistance for the first time, and provides gene resources for plant drought resistance and stain resistance breeding.

One of the purposes of the invention is to provide an application of a sesame SiERF103 gene in enhancing plant resistance, wherein the nucleotide sequence of the sesame SiERF103 gene is shown in a sequence table SEQ ID NO.1, and the resistance comprises: anti-waterlogging and/or drought.

Further, the amino acid sequence of the protein coded by the sesame SiERF103 gene is shown in a sequence table SEQ ID NO. 2.

Further, the primer sequence for amplifying the sesame SiERF103 gene is shown in a sequence table SEQ ID No. 3-4.

The second aim of the invention is to provide an application of the vector and/or the cell containing the sesame SiERF103 gene in enhancing plant waterlogging resistance and/or drought resistance.

Further, the vector containing the sesame SiERF103 gene is a cloning vector or an over-expression vector.

Further, the over-expression vector is a vector pCAMBIA1301S-SiERF103 constructed by connecting a sesame SiERF103 gene with a plant expression vector pCAMBIA 1301S.

Further, the construction method of the sesame SiERF103 gene-containing cell comprises the following steps: amplifying to obtain sesame SiERF103 gene shown in SEQ ID NO.1, carrying out double enzyme digestion to obtain a linearization expression vector, connecting the sesame SiERF103 gene with the linearization vector, transferring the linearization vector into receptor cells, culturing, screening and identifying to obtain the sesame SiERF103 gene.

The invention also aims at providing the sesame SiERF103 gene, or the protein encoded by the sesame SiERF103 gene, or the application of the vector and/or the cell containing the sesame SiERF103 gene in enhancing the plant waterlogging resistance and/or drought resistance.

It is a fourth object of the present invention to provide a method for improving plant waterlogging and/or drought resistance comprising: and transferring an over-expression vector containing the sesame SiERF103 gene into an agrobacterium competent cell, and infecting plants to obtain plants with improved waterlogging and drought resistance.

Further, the plant with increased waterlogging and drought resistance may resume normal growth after drought stress and/or waterlogging stress treatment.

Compared with the prior art, the invention has the beneficial effects that: the invention reports the application of the sesame SiERF103 gene in enhancing plant resistance for the first time, in particular enhancing plant waterlogging resistance and/or drought resistance. Through constructing SiERF103 gene over-expression vector, it is over-expressed in model plant Arabidopsis thaliana, and drought or waterlogging stress treatment is carried out, and researches find that after drought or waterlogging stress treatment, normal growth condition is recovered, and transgenic Arabidopsis thaliana can be recovered to normal growth in large quantity. The invention proves that the sesame SiERF103 gene has the function of improving plant waterlogging and drought resistance. The gene source is provided for drought resistance and stain resistance genetic improvement of crops, and has important significance for promoting stable yield of crops.

Drawings

FIG. 1 shows the construction of a plant expression vector pCAMBIA1301S-SiERF103 comprising the SiERF103 gene of example 1 of the present invention;

FIG. 2 shows the result of fluorescent quantitative PCR detection of T1 generation plants of Arabidopsis thaliana transformed with SiERF103 gene in example 2 of the present invention;

FIG. 3 is a result of determining drought resistance of SiERF103 transgenic Arabidopsis thaliana in example 2 of the present invention, wherein FIG. 3-A is a phenotype of SiERF103 transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana that resumes growth after normal growth and drought stress; FIG. 3-B shows the survival rates of transgenic SiERF103 Arabidopsis thaliana and wild type Arabidopsis thaliana after drought stress;

FIG. 4 is a graph showing the results of a stain resistance assay of transgenic SiERF103 Arabidopsis thaliana in example 2 of the present invention, wherein FIG. 4-A shows the phenotype of transgenic SiERF103 Arabidopsis thaliana and wild type Arabidopsis thaliana that resumes growth after normal growth and a detrimental stress; FIG. 4-B shows the survival rate of transgenic SiERF103 Arabidopsis thaliana and wild type Arabidopsis thaliana after waterlogging stress.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

The method of cloning the drought-resistance-related gene SiERF103 described in the present invention is a method commonly used in the art. There are also a number of well-established techniques for extracting mRNA, and kits (EASY spin Plus Plant RNA kit) are available commercially (available from Aidlab Biotechnologies co., ltd). Methods of constructing the vectors described in the present invention and methods of cutting, ligating, inflorescence infection, etc., used to transfer the vectors into plants are also common techniques in the art. The plasmids (pCAMBIA 1301) involved therein, the medium for transfection (Agrobacterium tumefaciens LBA4404 and the reagent components used, such as sucrose, kan, hygromycin, etc.) are commercially available.

Example 1 acquisition of sesame SiERF103 Gene and construction of overexpression vector

1. Obtaining of sesame SiERF103 Gene

Using the protein sequence of Arabidopsis ERF gene, 114 sesame ERF genes including SiERF103 gene (accession number: LOC 105175593) were identified in NCBI database by homology sequence comparison analysis.

In order to analyze the function of the SiERF103 gene in stress resistance, primers capable of amplifying the complete CDS sequence of the SiERF103 gene are designed according to the CDS sequence of the SiERF103 gene. Root tissues of sesame subjected to 75 waterlogged stress for 7 days in the early-flowering stress-resistant sesame variety are taken, total RNA is extracted by using an EASYspin Plus plant RNA extraction kit (purchased from Aidlab company), and the total RNA is subjected to reverse transcription into cDNA (the method is operated according to the reagent specifications of reverse transcriptase of Vazyme company) by using reverse transcriptase (purchased from Vazyme company). Conventional PCR amplification was performed using the reverse transcribed cDNA as a template and designed primers. Wherein the names and sequences (including modified bases) of the primers designed are as follows:

SiERF103FL-F:

AGCTTTCGCGAGCTCGGTACCATGAGAATGATTCTCAAGAAAT (shown as SEQ ID NO. 3)

SiERF103FL-R:

CAGGTCGACTCTAGAGGATCCTCAAACCATCTCACTTGACACACTAC (shown as SEQ ID NO. 4)

The primer is used for amplifying a cDNA fragment containing the complete coding region of the SiERF103 gene, and the amplified fragment is sequenced to obtain the nucleotide sequence of the SiERF103 gene, which is shown as SEQ ID NO. 1. The amino acid sequence of the protein coded by the polypeptide is shown as SEQ ID NO. 2.

2. Construction of overexpression vector containing sesame SiERF103 gene

The sesame SiERF103 gene obtained by amplification is connected with pCAMBIA1301S (provided by the laboratory) plasmid by utilizing a homologous recombination method to construct a plant expression vector, which is named pCAMBIA1301S-SiERF103 (the map is shown in figure 1), and the specific operation is as follows:

(1) the linearized vector was first obtained by a double cleavage (KpnI and BamHI) (Takara) method, and purified by agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical Co., ltd.) to obtain a high purity linearized vector.

(2) Adding target fragment DNA and linearization vector into a 1.5ml centrifuge tube in a molar ratio of 3:1 for recombination reaction, placing in a 37 ℃ water bath for about 30min after uniform mixing, adding 10 mu L of reaction solution into 50 mu L of DH5a competent cells which are melted for 10min on ice from a-80 ℃ refrigerator, slightly mixing by a pipette, incubating for 20min on ice, and rapidly placing on ice for cooling for 2min after heat shock in a 42 ℃ water bath for 45 seconds.

(3) 300. Mu.L of LB liquid medium was added to the clean bench, and incubated at 37℃for 60min. The cells were collected by centrifugation at 13,000rpm for 1min, a part of the supernatant was discarded, the cells were resuspended in the remaining medium, gently smeared with a sterile smear bar on LB solid medium containing Kan resistance, and cultured in an incubator at 37℃for 16-24h in an inverted manner.

(4) And (3) selecting a plurality of clones transformed by the recombination reaction for colony PCR identification, then respectively selecting each single colony which is identified as positive, culturing in a liquid LB culture medium containing Kan antibiotics at 37 ℃ and 200rpm for overnight, extracting plasmids or directly sequencing bacterial liquid and identifying the accuracy of the vector by enzyme digestion electrophoresis.

Example 2 application of sesame SiERF103 Gene in enhancing plant waterlogging and drought resistance

1. Acquisition of SiERF 103-transformed Arabidopsis thaliana Material

(1) Transfer of recombinant vector into Agrobacterium LBA4404

(1) 2 mug of plasmid DNA is added into each 100 mug of LBA4404 agrobacterium competent cells, the mixture is gently stirred at the bottom of the tube by hand and then mixed, and the mixture is sequentially placed on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min.

(2) 700. Mu.L of LB liquid medium without antibiotics was added thereto, and the culture was continued at 28℃for 5 hours with shaking. The cells were collected by centrifugation at 13,000rpm for 1min, about 100. Mu.L of the supernatant was left, resuspended cells were gently blown, uniformly spread on LB solid medium containing Kan and Rif, cultured in an incubator at 28℃for 2 days in an inverted state, several positive clones were picked up, colony PCR was performed using the primers SiERF103FL-F/R in example 1, and it was confirmed that the vector pCAMBIA1301S-SiERF103 had been transferred into Agrobacterium tumefaciens LBA 4404.

(2) Arabidopsis thaliana culture

(1) A number of arabidopsis seeds were counted as required for the experiment and placed in a sterile 1.5mL centrifuge tube.

(2) 1mL of 75% ethanol was added, mixed upside down, and the supernatant was discarded and repeated 1 time. Placing into a shaking table at 37deg.C and 200rpm, shaking for 10min, and sterilizing.

(3) Discard 75% ethanol, add 1ml of 95% ethanol, mix upside down, discard supernatant, and repeat 1 time. In an ultra clean bench, 300-500. Mu.L of absolute ethanol was added to each tube of seeds, and the seeds were sprayed onto sterile filter paper with absolute ethanol using a 1mL pipette.

(4) After the ethanol had evaporated, the dried Arabidopsis seeds were spot-sown with toothpicks on plates of the prepared MS medium.

(5) Sealing the flat plate, placing the flat plate in a dark condition for vernalization at 4 ℃ for 48 hours, placing the flat plate in an illumination incubator for vertical culture after vernalization, and transplanting after seedling emergence for one week.

(6) The seedlings are planted into the soil of the small basin by forceps, firstly, the seedlings are moisturized for 48 hours by using a preservative film, and the seedlings are placed in a plant growth room for cultivation until the arabidopsis grows and is bolting (about one month) for transformation experiments.

(3) Genetic transformation

(1) Agrobacterium activation: 20 mu L of Rif and Kan are respectively added into 20mL of LB liquid medium, the mixture is shaken uniformly and inoculated, and the mixture is subjected to shaking activation at 220rpm at 28 ℃ for 8-10h.

(2) And (3) culturing agrobacterium tumefaciens in an enlarged manner: 200 mu L of Rif and Kan are respectively added into 200mL of LB liquid culture medium, 5-10mL of activated bacterial liquid is added, shake culture is carried out at 28 ℃ and 220rpm for 14-16h until the OD value is 1.6-2.0, centrifugation is carried out at 4500rpm for 10min, supernatant is discarded from the precipitate bacterial body, and natural drying is carried out.

(3) 100mL of 5% sucrose solution is added to the precipitated thalli to resuspend the thalli, and a pipettor blows the thalli evenly to resuspend the thalli.

(4) The bacterial solution in the centrifuge flask was added to a dish, 100mL of 5% sucrose solution was added, 40. Mu.L Silwet-L-77 (0.02%) was added before conversion, and the dish was shaken and mixed well.

(5) The arabidopsis inflorescences are closed, immersed in a plate and gently rocked for 15s.

(6) The plants are covered by a black bag and kept for 24 hours in a dark place.

(7) The transformation was repeated once more after one week.

(4) T1 generation positive plant screening and fluorescent quantitative PCR detection

Planting the seeds harvested in the T0 generation of arabidopsis thaliana, sterilizing the seeds, inoculating an MS screening culture medium containing 30mg/L hygromycin (25 mg/L cephalosporin is added for bacteriostasis), carrying out illumination culture at 22 ℃ for 7-10 days, screening to obtain positive plants (plants with seedlings and root systems which normally grow), transplanting the positive seedlings into soil, covering the positive plants with a preservative film for 2-3 days, removing the film, and then normally growing.

Taking young leaves of positive transgenic plants and wild arabidopsis plants, extracting total RNA of the leaves by using an RNA extraction kit (Beijing Aidelai biotechnology Co., ltd.), then obtaining cDNA by using a reverse transcription kit (Nanjinouzan biotechnology Co., ltd.), taking the respective cDNA as a template and arabidopsis beta-actin as an internal reference, wherein the primers of the beta-actin gene comprise:

actin-F:5'-CCCGCTATGTATGTCGCCA-3' (SEQ ID NO. 7);

actin-R:5'-AACCCTCGTAGATTGGCACAG-3' (SEQ ID NO. 8);

SiERF103QRT-F/R with SiERF103 specific primer:

SiERF103QRT-F:5'-GAACTACAGAGGCGTGAG-3' (SEQ ID NO. 5);

SiERF103QRT-R:5'-TTATCGTAAGCCAAAGC-3' (SEQ ID NO. 6).

qRT-PCR expression verification of target Gene (qRT-PCR Mix: nanjinouzan Biotechnology Co., ltd., instrument: roche)

480)。

The qRT-PCR detection result is shown in figure 2, and the result shows that the expression quantity of SiERF103 genes in the detected 3 transgenic Arabidopsis thaliana strains (SiERF 103-OE#1, -OE#2, -OE#3) is obviously improved by taking a wild plant as a control.

And (3) carrying out single plant seed collection on transgenic T1 generation plants with SiERF103 over-expression, and continuously carrying out hygromycin screening on the seeds harvested from the T1 generation positive plants to obtain T2 generation positive plants and carrying out single plant seed collection. Propagation is continued until homozygous SiERF103 over-expressed transgenic material is obtained.

2. Determination of drought resistance of transgenic Arabidopsis thaliana

And (3) respectively carrying out drought stress treatment (i.e. no watering) on the arabidopsis transgenic plant and the wild arabidopsis plant for 14 days when the T3 generation homozygous transgenic arabidopsis grows to 3 pairs of leaves, then carrying out rehydration and recovery growth for 7 days, observing and counting the number of plants recovered to normal growth of each group, and calculating the survival rate. Wherein the transgenic line and wild type material were each subjected to 3 replicates, each replicate subjected to stress treatment on 27 plants.

The drought resistance measurement results are shown in fig. 3, and the results show that only 5-7 plants of the wild type recover to be normal after rehydration, and 23-26 plants of the transgenic material recover, and about 90% of arabidopsis thaliana strains transformed with SiERF103 genes are calculated to recover to be normal growth, and only about 22% of plants of the wild type arabidopsis thaliana survive. The result shows that the over-expression of the sesame SiERF103 gene can obviously improve the drought resistance of plants.

3. Transgenic arabidopsis stain resistance assay

When the T3 generation homozygous transgenic arabidopsis grows to 3 pairs of leaf periods, carrying out the waterlogging stress treatment on the arabidopsis transgenic plant and the wild arabidopsis plant for 18 days, wherein the waterlogging stress treatment specifically comprises the following steps: placing the box for planting plants in water, wherein the water is 0.5cm higher than the soil surface, so as to ensure that the roots of the plants are immersed in the water; normal growth was then resumed for 7 days. And observing and counting the number of plants in each group which recover normal growth, and calculating the survival rate. Wherein the transgenic line and wild type material were each subjected to 3 replicates, each replicate subjected to stress treatment on 27 plants.

The results of the stain resistance measurement are shown in FIG. 4, and the results show that after normal treatment for 7 days, 6-9 strains of wild type are recovered, and 18-24 strains of transgenic materials are recovered. Through calculation, over 75% of SiERF103 over-expression transgenic lines gradually recover to normal growth, and only about 25% of wild arabidopsis thaliana recovers to normal, which indicates that over-expression of sesame SiERF103 genes can improve the stain resistance of plants.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Sequence listing

<110> institute of oil crop and oil crop at national academy of agricultural sciences

Application of <120> sesame SiERF103 gene in enhancing plant resistance

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acaaagaaga actacagagg cgtgaggcgg cggccctggg ggaagttcgc agccgagatt 180

cgggactcca accggcaggg ggcaagaata tggctgggga cattcgaaac agccgaagaa 240

gctgctttgg cttacgataa ggcggcattt agaatgcgag gcacaaaggc tctcctcaat 300

ttccctcctg aaatcgtggc tgctgctgct tcttcatcga gcactccaac acttgatcgc 360

gatcaaaaac atttggataa ttctagaaac tgtttgtctg atgatgcaag tagtgtgtca 420

agtgagatgg tttga 435

<210> 2

<211> 144

<212> PRT

<213> sesame (Sesamum indicum l.)

<400> 2

Met Arg Met Ile Leu Lys Lys Ser Tyr Tyr Thr Asn Ser Ser Thr Arg

1 5 10 15

Pro Asp Ala Leu Asn Thr Ser Ser His Gln Ile Arg Ser Gln Ser Phe

20 25 30

Asn Leu Arg Pro Ser His Ser Val Thr Lys Lys Asn Tyr Arg Gly Val

35 40 45

Arg Arg Arg Pro Trp Gly Lys Phe Ala Ala Glu Ile Arg Asp Ser Asn

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65 70 75 80

Ala Ala Leu Ala Tyr Asp Lys Ala Ala Phe Arg Met Arg Gly Thr Lys

85 90 95

Ala Leu Leu Asn Phe Pro Pro Glu Ile Val Ala Ala Ala Ala Ser Ser

100 105 110

Ser Ser Thr Pro Thr Leu Asp Arg Asp Gln Lys His Leu Asp Asn Ser

115 120 125

Arg Asn Cys Leu Ser Asp Asp Ala Ser Ser Val Ser Ser Glu Met Val

130 135 140

<210> 3

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cccgctatgt atgtcgcca 19

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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aaccctcgta gattggcaca g 21

Claims (7)

1. The application of the sesame SiERF103 gene in enhancing plant resistance is characterized in that the nucleotide sequence of the sesame SiERF103 gene is shown in a sequence table SEQ ID NO.1, and the resistance is anti-waterlogging and/or anti-drought.

2. The application of claim 1, wherein the amino acid sequence of the protein encoded by the sesame SiERF103 gene is shown in a sequence table SEQ ID NO. 2.

3. The use according to claim 1, wherein the primer sequence for amplifying the sesame SiERF103 gene is as shown in sequence table seq id No. 3-4.

4. Use of an overexpression vector comprising the sesame SiERF103 gene as defined in claim 1 for enhancing the resistance to plant waterlogging and/or drought.

5. The use according to claim 4, wherein the over-expression vector is a vector pCAMBIA1301S-SiERF103 constructed by connecting the sesame SiERF103 gene with a plant expression vector pCAMBIA 1301S.

6. A method of increasing plant vigour and/or drought resistance, the method comprising: transferring an over-expression vector containing the sesame SiERF103 gene as claimed in claim 1 into agrobacterium competent cells, and infecting plants to obtain plants with improved waterlogging and drought resistance.

7. The method as claimed in claim 6, characterized in that the plant with increased resistance to waterlogging and drought is able to resume normal growth after treatment with drought stress and/or waterlogging stress.

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