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

Abstract

The invention discloses the application of the sesame SiERF103 gene in enhancing plant resistance, and belongs to the technical field of plant genetic engineering, wherein the nucleotide sequence of the sesame SiERF103 gene is shown in SEQ ID NO.1, and the resistance includes stain resistance damage and/or drought resistance. The present invention reports for the first time the application of the sesame SiERF103 gene in enhancing plant waterlogging resistance and/or drought resistance. By constructing the overexpression vector of SiERF103 gene, it was overexpressed in Arabidopsis, and treated with drought or waterlogging stress, the study found that after drought or waterlogging stress treatment, returning to normal growth conditions can make a large amount of transgenic Arabidopsis recover Normal growth. That is, the present invention confirms that the sesame SiERF103 gene has the function of improving plant waterlogging and drought resistance. This provides genetic resources for genetic improvement of crop drought resistance and waterlogging tolerance, and is of great significance for promoting stable crop yield.

Description

Application of Sesame SiERF103 Gene in Improving Plant Resistance

technical field

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

Background technique

Sesamum (Sesamum indicum L.) is a special oil crop in my country. It has a long history of cultivation and is planted all over the country and all over the world. China is a major producer and trading country, but its self-sufficiency is insufficient. In recent years, the yield of sesame in my country has declined seriously, which has a lot to do with the impact of various abiotic stresses such as drought and waterlogging during the growth and development of sesame. The Sesame Industry Technology System has investigated and analyzed most of the main sesame producing areas in my country and found that the sesame in our country has been affected by waterlogging and drought stress all year round, which seriously affects the growth and development of sesame and is an important reason for the reduction of sesame production. Therefore, improving the drought resistance and waterlogging tolerance of sesame and promoting high and stable yield are particularly important to promote the development of sesame production in my country and solve the problem of insufficient self-sufficiency in total amount. At present, the research on the functions of sesame drought resistance and waterlogging tolerance related genes is still relatively scarce, and there are relatively few molecular related research reports. Therefore, the discovery of sesame stress resistance functional genes is of great significance for the development of sesame stress resistance molecular breeding and the improvement of sesame stress resistance. .

Plants are often affected by many environmental factors during their growth and development. When stress occurs, plants can quickly activate a large number of stress response genes and synthesize various functional proteins, forming a series of complex signaling networks to resist adverse environments. Maintain its own normal physiological functions to achieve the purpose of avoiding coercion damage. Transcription factors are a class of protein molecules that can specifically bind to cis-acting elements in the promoter region of eukaryotic genes, thereby ensuring the expression of the target gene at a specific intensity, at a specific time and space, and in plant stress resistance. It played an important role. For example, Arabidopsis RAP2.2 resists hypoxic stress by regulating the expression of ADH1, PDC1 and other hypoxic stress-related genes, sugar metabolism and ethylene synthesis-related genes. Rice Sub1A-1 inhibits the production of ethylene and the transcription of growth and metabolism-related genes under flooding conditions, reduces the decomposition of starch and sugar, and keeps the plant in a low metabolic state to survive. Sesame is an important oil crop, and the isolation and identification of its stress-resistant genes is of great significance for the breeding of new stress-resistant sesame varieties.

Contents of the invention

Aiming at the gap in the prior art, the present invention provides the application of the sesame SiERF103 gene in enhancing plant resistance. Through the overexpression experiment of the SiERF103 gene in the model plant transgenic Arabidopsis, it is confirmed for the first time that the sesame SiERF103 gene has the function of enhancing plant waterlogging. The functions of drought resistance and drought resistance provide genetic resources for plant drought resistance and waterlogging tolerance breeding.

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

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

Further, the primer sequences for amplifying the sesame SiERF103 gene are shown in SEQ ID NO.3-4 in the sequence table.

The second object of the present invention is to provide an application of a vector and/or cell comprising the above-mentioned 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 overexpression vector.

Further, the overexpression vector is the vector pCAMBIA1301S-SiERF103 constructed by connecting the sesame SiERF103 gene with the plant expression vector pCAMBIA1301S.

Further, the method for constructing the cell containing the sesame SiERF103 gene is as follows: amplify the sesame SiERF103 gene shown in SEQ ID NO. Connected and transferred into recipient cells, obtained after culture, screening and identification.

The third object of the present invention is to provide the sesame SiERF103 gene, or the protein encoded by the sesame SiERF103 gene, or the vector and/or cells comprising the sesame SiERF103 gene in enhancing plant waterlogging and/or drought tolerance Applications.

The fourth object of the present invention is to provide a method for improving waterlogging and/or drought resistance of plants, comprising: transferring the overexpression vector comprising the sesame SiERF103 gene into Agrobacterium competent cells, and infecting the plants to obtain Plants with increased resistance to waterlogging and drought.

Further, the plants with improved waterlogging and drought resistance can recover to normal growth after being treated with drought stress and/or waterlogging stress.

Compared with the prior art, the beneficial effects of the present invention are: the present invention reports for the first time the application of the sesame SiERF103 gene in enhancing plant resistance, specifically enhancing plant waterlogging resistance and/or drought resistance. By constructing the SiERF103 gene overexpression vector, it was overexpressed in the model plant Arabidopsis thaliana, and subjected to drought or waterlogging stress treatment. It was found that the normal growth conditions could be restored after drought or waterlogging stress treatment, and the transgenic Arabidopsis thaliana Many returned to normal growth. That is, the present invention confirms that the sesame SiERF103 gene has the function of improving plant waterlogging and drought resistance. This provides genetic resources for the genetic improvement of crop drought resistance and waterlogging tolerance, which is of great significance for promoting stable crop yield.

Description of drawings

Fig. 1 is the construction of the plant expression vector pCAMBIA1301S-SiERF103 comprising SiERF103 gene in Example 1 of the present invention;

Fig. 2 is the detection result of fluorescent quantitative PCR of T1 generation plant of Arabidopsis thaliana transgenic SiERF103 gene in Example 2 of the present invention;

Fig. 3 is the test result of the drought resistance of the SiERF103 gene Arabidopsis in Example 2 of the present invention, wherein Fig. 3-A is the phenotype of the SiERF103 gene Arabidopsis and wild-type Arabidopsis that recover after normal growth and drought stress; Fig. 3-B is the survival rate of SiERF103 transgenic Arabidopsis and wild-type Arabidopsis after drought stress;

Fig. 4 is the test result of the waterlogging resistance of the SiERF103 gene transgenic Arabidopsis in Example 2 of the present invention, wherein Fig. 4-A shows the phenotypes of the SiERF103 gene transgenic Arabidopsis and wild type Arabidopsis after normal growth and recovery of growth after waterlogging stress ; Figure 4-B is the survival rate of transgenic Arabidopsis thaliana and wild-type Arabidopsis after waterlogging stress.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The method of cloning the drought-resistance-related gene SiERF103 described in the present invention is a commonly used method in the art. There are also various mature technologies for the method of extracting mRNA, and a kit (EASY spin Plus Plant RNA kit) can be used, which can be obtained from commercial sources (purchased from Aidlab Biotechnologies Co., Ltd). Methods such as enzyme cutting, ligation, and inflorescence infection used to construct the vector described in the present invention and transfer the vector into plants are also common techniques in the art. The plasmid (pCAMBIA1301) involved therein, the media for transfection (Agrobacterium tumefaciens LBA4404 and reagent components used such as sucrose, Kan, hygromycin, etc.) can be obtained from commercial sources.

Embodiment 1 Sesame SiERF103 gene acquisition and overexpression vector construction

1. Acquisition of Sesame SiERF103 Gene

Using the protein sequence of Arabidopsis ERF gene, 114 sesame ERF genes were identified in the NCBI database through homologous sequence analysis, including SiERF103 gene (gene accession number: LOC105175593).

In order to analyze the function of SiERF103 gene in stress resistance, according to the CDS sequence of SiERF103 gene, design primers that can amplify its complete CDS sequence. Take the root tissues of the stress-resistant sesame variety Zhongzhi 75 at the initial flowering stage after 7 days of waterlogging stress, use the EASYspin Plus Plant RNA Extraction Kit (purchased from Aidlab Company) to extract total RNA, and use reverse transcriptase (purchased from Vazyme Company) to extract the total RNA. It was reverse-transcribed to synthesize cDNA (the method was operated according to the instructions of Vazyme reverse transcriptase reagent). Using the reverse-transcribed cDNA as a template, conventional PCR amplification was performed with the designed primers. The primer names and sequences (including modified bases) designed are as follows:

SiERF103FL-F:

AGCTTTCGCGAGCTCGGTACCATGAGAATGATTCTCAAGAAAT (as shown in SEQ ID NO.3)

SiERF103FL-R:

CAGGTCGACTCTAGAGGATCCTCAAACCATCTCACTTGACACACTAC (as shown in SEQ ID NO.4)

A cDNA fragment containing the complete coding region of the SiERF103 gene was amplified with the above primers, and the amplified fragment was sequenced to obtain the nucleotide sequence of the SiERF103 gene, as shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

2. Construction of an overexpression vector comprising the sesame SiERF103 gene

The sesame SiERF103 gene that above-mentioned amplification obtains utilizes the method for homologous recombination and pCAMBIA1301S (provided by this laboratory) plasmid connection construction plant expression vector, named pCAMBIA1301S-SiERF103 (illustration as shown in Figure 1), concrete operation is as follows:

① Firstly, the linearized vector was obtained by double enzyme digestion (KpnI and BamHI) (Takara), and purified by agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical Technology Co., Ltd.) to obtain a high-purity linearized vector.

②Add the DNA of the target fragment and the linearized carrier to a 1.5ml centrifuge tube at a molar ratio of 3:1 for recombination reaction, mix well and place in a water bath at 37°C for about 30min, add 10μL of the reaction solution to temperature from -80°C Take out 50 μL of DH5a competent cells that have been thawed on ice for 10 minutes in the refrigerator, mix gently with a pipette, and incubate on ice for 20 minutes, heat shock in a water bath at 42°C for 45 seconds, and then quickly cool on ice for 2 minutes.

③Add 300 μL LB liquid medium to the clean bench and incubate at 37°C for 60 minutes. Centrifuge at 13,000rpm for 1min to collect the bacteria, discard part of the supernatant, resuspend the bacteria with the remaining medium, and spread evenly on the LB solid medium containing Kan resistance with a sterile spreader, place in a 37°C incubator Inverted for 16-24h.

④Pick several clones transformed by the recombination reaction for colony PCR identification, and then pick each single colony that is identified as positive, and culture them overnight in a liquid LB medium containing Kan antibiotics at 37°C and 200rpm in an incubator to extract plasmids Or directly sequence the bacterial liquid and identify the accuracy of the carrier by enzyme digestion and electrophoresis.

Example 2 Application of Sesame SiERF103 Gene in Enhancing Waterlogging and Drought Resistance of Plants

1. Acquisition of SiERF103 transgenic Arabidopsis materials

(1) The recombinant vector was transformed into Agrobacterium LBA4404

① Add 2 μg of plasmid DNA to each 100 μL of LBA4404 Agrobacterium competent cells, gently tap the bottom of the tube to mix, and then put it on ice for 5 minutes, liquid nitrogen for 5 minutes, 37 ° C water bath for 5 minutes, and ice bath for 5 minutes.

②Add 700 μL of LB liquid medium without antibiotics, and shake and culture at 28°C for 5h. Centrifuge at 13,000 rpm for 1 min to collect the bacteria, take about 100 μL of the supernatant, gently blow and blow the resuspended bacteria, spread it evenly on the LB solid medium containing Kan and Rif, and culture it upside down in a 28°C incubator for 2 days. Pick several positive clones, and perform colony PCR with the primer SiERF103FL-F/R in Example 1 to verify that the vector pCAMBIA1301S-SiERF103 has been transformed into Agrobacterium tumefaciens LBA4404.

(2) Arabidopsis culture

①According to the experimental needs, count out a certain number of Arabidopsis seeds and put them in a sterile 1.5mL centrifuge tube.

② Add 1mL 75% ethanol, mix up and down, discard the supernatant, and repeat once. Put it into a 37°C 200rpm shaker and shake for 10min to sterilize the surface.

③ Discard 75% ethanol, add 1mL 95% ethanol, mix up and down, discard the supernatant, and repeat once. In an ultra-clean bench, add 300-500 μL of absolute ethanol to each tube of seeds, and spray the seeds and absolute ethanol onto sterile filter paper with a 1 mL pipette.

④ After the ethanol has evaporated, sow the dried Arabidopsis seeds on the prepared MS medium plate with a toothpick.

⑤Seal the plate and place it upside down at 4°C for vernalization for 48 hours in the dark. After vernalization, place the plate in a light incubator for vertical cultivation, and transplant the seedlings one week after emergence.

⑥Use tweezers to plant the seedlings into the soil in a small pot, first use plastic wrap to keep moisture for 48 hours, place them in the plant growth room and cultivate them until Arabidopsis thaliana grows and bolts (about one month) for transformation experiments.

(3) Genetic transformation

① Agrobacterium activation: Add 20 μL of Rif and Kan to 20 mL of LB liquid medium, shake well and inoculate the bacteria, shake and activate at 28°C and 220 rpm for 8-10 hours.

② Agrobacterium expansion culture: add 200μL Rif and Kan to 200mL LB liquid medium respectively, then add 5-10mL activated bacteria solution, shake and culture at 28℃220rpm for 14-16h, until the OD value is 1.6-2.0, centrifuge at 4500rpm After 10 minutes, the precipitated cells were discarded and the supernatant was discarded, and dried naturally.

③Add 100mL of 5% sucrose solution to the precipitated cells to resuspend the cells, pipette evenly to resuspend the cells.

④ Add the bacterial liquid in the centrifuge bottle to the plate, then add 100 mL of 5% sucrose solution, add 40 μL Silwet-L-77 (0.02%) before transformation, and shake the plate to mix.

⑤ Close the inflorescences of Arabidopsis thaliana, immerse in the plate, and shake gently for 15 seconds.

⑥ Cover the plants with a black bag and keep them protected from light for 24 hours.

⑦Repeat the transformation one week later.

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

Plant the harvested seeds of Arabidopsis thaliana T0 generation, sterilize them, inoculate MS screening medium containing 30 mg/L hygromycin (adding 25 mg/L cephalosporin for antibacterial) and cultivate under light at 22°C for 7-10 days, and select Obtain positive plants (plants with normal growth of seedlings and root systems), transplant the positive seedlings into the soil, cover them with plastic wrap for 2-3 days, then remove the film, and then grow normally.

Take the young leaves of positive transgenic plants and wild-type Arabidopsis plants, use the RNA extraction kit (Beijing Aidelai Biotechnology Co., Ltd.) to extract total RNA from the leaves, and then use the reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain cDNA, with the respective cDNA as a template and Arabidopsis β-actin as an internal reference, wherein the primers for the β-actin gene include:

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

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

With SiERF103 specific primer SiERF103QRT-F/R:

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

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

480)。qRT-PCR expression verification of the target gene (qRT-PCR Mix: Nanjing Nuoweizan Biotechnology Co., Ltd.; instrument: Roche

480).

The results of qRT-PCR detection are shown in Figure 2. The results showed that, using wild-type plants as controls, the SiERF103 gene was detected in three transgenic Arabidopsis lines (SiERF103-OE#1, -OE#2, -OE#3 ) were significantly increased.

The transgenic T1 generation plants overexpressing SiERF103 were harvested as a single plant, and the seeds harvested from the positive plants of the T1 generation were further screened with hygromycin to obtain positive plants of the T2 generation and harvested as a single plant. Continue to breed until a homozygous SiERF103 overexpression transgenic material is obtained.

2. Determination of drought resistance of transgenic Arabidopsis

After the homozygous transgenic Arabidopsis thaliana of the T3 generation grew to the stage of three pairs of leaves, the transgenic Arabidopsis plants and the wild-type Arabidopsis plants were subjected to drought stress (i.e. no watering) treatment for 14 days, and then rewatered to resume growth for 7 days , observe and count the number of plants that returned to normal growth in each group, and calculate the survival rate. The transgenic family and the wild-type material were repeated 3 times, and 27 plants were subjected to stress treatment in each repetition.

The results of the drought resistance test are shown in Figure 3. The results showed that only 5-7 plants of the wild type returned to normal after rehydration, while 23-26 plants of the transgenic materials recovered. It was calculated that about 90% of the plants of the Arabidopsis transgenic SiERF103 gene The thaliana lines returned to normal growth, while only about 22% of wild-type Arabidopsis plants survived. The results indicated that the overexpression of SiERF103 gene in sesame can significantly improve the drought resistance of plants.

3. Determination of water resistance of transgenic Arabidopsis

After the homozygous transgenic Arabidopsis thaliana of the T3 generation grew to the stage of three pairs of leaves, the transgenic Arabidopsis plants and the wild-type Arabidopsis plants were subjected to waterlogging stress treatment for 18 days. Place in water, the water is 0.5cm higher than the soil surface, to ensure that the plant roots are submerged in water; then resume normal growth for 7 days. The number of plants returning to normal growth in each group was observed and counted, and the survival rate was calculated. The transgenic family and the wild-type material were repeated 3 times, and 27 plants were subjected to stress treatment in each repetition.

The results of the stain resistance test are shown in Figure 4. The results showed that after 7 days of normal treatment, 6-9 strains of the wild type recovered, and 18-24 strains of the transgenic material recovered. According to calculations, more than 75% of SiERF103 overexpressed transgenic lines gradually returned to normal growth, while only about 25% of wild-type Arabidopsis returned to normal, indicating that the overexpression of SiERF103 gene in sesame can improve the water resistance of plants.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention.

sequence listing

<110> Institute of Oil Crops, Chinese Academy of Agricultural Sciences

<120> Application of Sesame SiERF103 Gene in Enhancing Plant Resistance

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 435

<212>DNA

<213> Sesamum (Sesamum indicum L.)

<400> 1

atgagaatga ttctcaagaa atcgtattac accaactcct caacgaggcc tgatgcctta 60

aacacttctt cgcatcagat caggagtcag agttttaacc tccgcccgtc tcattccgtc 120

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> Sesamum (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

50 55 60

Arg Gln Gly Ala Arg Ile Trp Leu Gly Thr Phe Glu Thr Ala Glu Glu

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

<211> 43

<212>DNA

<213> Artificial Sequence

<400> 3

agctttcgcg agctcggtac catgagaatg attctcaaga aat 43

<210> 4

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 4

caggtcgact ctagaggatc ctcaaaccat ctcacttgac acactac 47

<210> 5

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 5

gaactacaga ggcgtgag 18

<210> 6

<211> 17

<212>DNA

<213> Artificial Sequence

<400> 6

ttatcgtaag ccaaagc 17

<210> 7

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 7

cccgctatgt atgtcgcca 19

<210> 8

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 8

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.

Images