CN110734915B - Plant gene and application - Google Patents

Abstract

The invention discloses a plant gene and application thereof. Wherein the nucleotide sequence of the gene TeLTF4 is shown as SEQ ID NO.1, and the amino acid sequence thereof is shown as SEQ ID NO. 2. The invention clones the TelTF4 gene from marigold. The gene expression level analysis result shows that the expression level of the TeLTF4 is higher in marigold leaves, and the expression level is further obviously increased in leaves inoculated with pathogenic microorganisms. When the TeLTF4 gene is expressed in tobacco leaf, it can trigger cell death hypersensitivity and increase the expression level of disease-resistant gene. The TeLTF4 gene cloned from marigold plays a role in disease resistance regulation and control and can be used for enhancing the disease resistance of plants.

Description

Plant gene and application

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a gene obtained by cloning marigold and application of the gene in the aspect of regulating and controlling plant disease resistance.

Background

Tagetes erecta (Tagetes erecta L.) belongs to the family of Compositae and annual herbaceous plants, and is native to Mexico and America. As the flowers are yellow to orange red, the color is bright, the florescence is long, and the ornamental plants become garden ornamental plants. In addition, because marigold flowers are rich in carotenoid, the marigold flowers can be used as a main plant resource for extracting the carotenoid, especially lutein, by cultivating and planting varieties with high pigment content. Therefore, marigold has recently been grown in large scale in Yunnan, Shandong, inner Mongolia, etc. of China. Besides carotenoid, marigold is rich in secondary metabolites such as phenols and terpenoids, so that the marigold can adapt to various stresses, is easy to cultivate and has high edible and medicinal values.

The mechanism and control of plant diseases caused by pathogenic microorganisms are always important fields in agricultural research and production. A complex interaction relationship exists between plants and pathogenic invaders, and whether diseases occur or not is also the result of the attack and defense game of the plants and pathogenic microorganisms. In attack and defense war between pathogenic bacteria and host plants invaded by the pathogenic bacteria, pathogenic microorganisms firstly utilize pores on the surfaces of plants such as wounds and stomata, and then inject toxic effect proteins (effectors) into the plants to reopen the closed stomata, and break through the physical barriers on the surfaces of the plants; breaking through the protective barrier of plant enzyme system by inhibiting the destructive action of cell wall degrading enzyme, protease, etc.; by blocking the signal conduction of plants, the synthesis of plant antibacterial compounds and the remote transmission of infection information are prevented, the immune defense response defense line of the plants is broken through, the life activities of the plants are finally destroyed and interfered, and the nutrition is obtained from the plants to realize the colonization of pathogenic microorganisms. The plant needle front relatively forms a PTI immune mechanism (PAMP-triggered immunity) for recognizing the conservative components of the microorganism, and senses the existence of the microorganism in the environment to activate defense reaction by finding the common and necessary structure and molecules of the microorganism; then, aiming at the blocking and destruction of pathogenic bacteria effector protein to PTI pathway, an ETI (effector-induced immunity) immune mechanism for recognizing effector protein is evolved, a rapid immune response similar to that of animals appears, programmed cell death is rapidly induced in an infected part to generate a hypersensitivity reaction, and pathogenic microorganisms are prevented from spreading from the infected part. Meanwhile, signals are transmitted to uninfected parts of plants through systemic acquired resistance to carry out defense mobilization. Therefore, the plants and pathogenic microorganisms in the living environment form an attack and defense co-evolution mode.

As described above, plants produce a disease-resistant defense response against pathogenic bacteria by finding and sensing pathogenic microorganisms, activating signaling pathways, and resetting gene transcription. An important link in the defense response is to rapidly regulate the expression of plant immune response genes through the anti-disease transcription factor protein. Through research on model plants, a basic path of a plant immune defense mechanism is discovered and some disease-resistant transcription genes are identified in recent years, but corresponding gene resources are still to be separated from non-model plants with stronger disease resistance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a plant disease-resistant regulatory gene.

The invention also provides an application of the gene in regulating and controlling plant disease resistance.

For the plant disease-resistant regulatory gene, the technical scheme provided by the invention is that the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention also comprises the application of the gene in enhancing the disease resistance of plants.

The invention clones disease-resistant related regulatory genes from marigold, which are named as Tagetes erecta Leaf transformation Factor 4 (hereinafter referred to as TeLTF 4).

The invention clones the TelTF4 gene from marigold: performing data assembly on the TelTF4 gene according to the high-throughput transcriptome deep sequencing of marigold; then, Primer design software Primer Premier is used for designing nested PCR specific primers, total RNA is extracted from marigold leaves, and reverse transcription-polymerase chain reaction (RT-PCR) cloning is carried out to obtain the TELTF4 gene.

The quantitative analysis result of gene expression shows that the expression level of the TeLTF4 in the leaves of marigold plants is higher, and the expression level is obviously increased after the inoculation of pathogenic microorganisms. When the TelTF4 gene is expressed in tobacco leaves, it can trigger the hypersensitivity reaction of cell death and induce the expression of disease-resistant related gene. The TelTF4 gene plays a role in immune defense reaction and can be used for regulating and controlling the disease resistance of plants.

The invention has the beneficial effects that:

the marigold gene TelTF4 provides a new regulatory gene resource for improving the disease resistance of plants, and can be used for cultivating and improving disease-resistant plant materials.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 shows the results of analysis of the expression level of the TelTF4 gene in marigold roots, leaves, flowers, and leaves inoculated with a pathogenic microorganism according to an example of the present invention.

FIG. 2 shows the Western blot Western blot detection result (A) of the expression of the TeLTF4 gene in tobacco leaf, and the detection result (B) of the hypersensitivity mediated by the expression of TeLTF4 and the detection result (C) of the expression level of the disease resistance marker gene PRI in tobacco leaf.

Detailed Description

In the following examples, the specific experimental conditions, where not indicated, are according to conventional conditions well known to those skilled in the art, such as those described in the molecular cloning Laboratory Manual of Sambrook J. and Russell, D.W. (New York: Cold Spring Harbor Laboratory Press,2001), or according to the manufacturer's recommendations.

Example 1: high throughput transcriptome sequencing of marigold

As the genome of marigold is not determined, in order to obtain a functional gene transcript sequence, a tissue sample of a marigold cultivated variety is utilized, and high-throughput transcriptome sequence determination and assembly annotation are carried out on 9 samples in total by respectively extracting leaf, immature flower and mature flower RNA of three individual plants.

1. Reagent

The plant RNA extraction reagent Trizol is purchased from Invitrogen company, DNase I (dnase I) is purchased from TaKaRa company, the RNA Library preparation Kit (RNA Library Prep Kit) is from Beijing Baimaike biotechnology limited, and other reagents are imported subpackaged or domestic analysis pure products.

2. Plant material

Marigold (Tagetes erecta L.) cultivar juwang is purchased from kaki-sp.

3. Method of producing a composite material

3.1RNA extraction

1) Crushing 100mg of plant tissues by using a liquid nitrogen grinding method, transferring the plant tissues into a 1.5mL centrifuge tube, adding 1mL Trizol, violently shaking, and standing at room temperature for 5 min;

2) adding 200 μ L chloroform into the centrifuge tube, shaking for 30s, mixing, and standing at room temperature for 5 min;

3) centrifuging at 12000rpm for 15min at 4 deg.C;

4) transferring 700 mu L of the supernatant into a 1.5mL centrifuge tube, wherein the lower organic phase and the middle layer have protein and other impurities to avoid touching and absorbing;

5) adding equal volume of isopropanol into the supernatant, mixing, and standing at room temperature for 10 min;

6) centrifuging at 4 deg.C and 12000rpm for 15min, and removing supernatant;

7) adding 1mL of 70% ethanol, gently oscillating the centrifugal tube, and suspending and precipitating;

8) centrifuging at 4 deg.C and 12000rpm for 5min, and removing supernatant;

9) drying at room temperature for 5-10 min;

10) adding 50. mu.L RNase-free H water₂O), dissolving RNA;

11) mu.g of RNA was taken according to the concentration of the RNA solution, and 5. mu.L of 10 Xbuffer (400mM Tris-HCl, pH 7.5,80mM MgCl)₂50mM DTT), 5. mu.L of Dnase I and 2. mu.L of RNase inhibitor, and reacting at 37 ℃ for 30 min;

12) adding 2.5 μ L0.5M EDTA, inactivating Dnase I at 80 deg.C for 2 min;

13) adding 10 μ L of 3M sodium acetate and 250 μ L of precooled ethanol, standing at-80 deg.C for 20 min;

14) centrifuging at 4 deg.C and 12000rpm for 10min, and removing supernatant;

15) adding 1mL of 70% ethanol to clean RNA;

16) centrifuging at 4 deg.C and 12000rpm for 5min, and removing supernatant;

17) drying at room temperature for 5-10 min;

18) adding 50 mu L of RNase-free water to dissolve RNA;

19) and detecting the purity and concentration of the RNA sample.

3.2 transcriptome sequencing Assembly and Annotation

Transcriptome sequencing was performed using the RNA Library Prep Kit via Illumina HiSeq high throughput sequencing platform, according to the following steps:

1) enriching eukaryotic RNA by magnetic beads with oligo (dt), and randomly breaking mRNA;

2) taking mRNA as a template, synthesizing a first cDNA chain by using a hexabasic random primer, then adding dNTPs, RNase H and DNA polymerase I to synthesize a second cDNA chain, and purifying the cDNA by using beads (beads);

3) carrying out end repair on the purified double-stranded cDNA, connecting a sequencing joint, then carrying out fragment size selection by using microbeads, and obtaining a cDNA library through PCR enrichment;

4) detecting the concentration of the library and the size of the insert;

5) sequencing the cDNA library by using an Illumina HiSeq high-throughput sequencing platform, wherein the sequencing read length is PE 125;

6) cutting sequencing joints and primer sequences of sequencing fragments (reads), and filtering low-quality value data to obtain high-quality sequencing data;

7) extending the high-quality sequencing read into longer fragments (contigs) by using Trinity assembly software, obtaining fragment sets (components) by using the overlapping of the fragments, and finally obtaining a transcript sequence (unigene) by using a method of a De Bruijn graph;

8) aligning transcript sequences to the NR (NCBI non-redundant database), Swiss-Prot (database maintained by the European bioinformatics institute), GO (Gene ontology), COG (Clusters of organizations), KOG (eKaryotic organizations), KEGG (Kyoto Encyclopedia of Genes and genomes) databases using BLAST software;

9) the coding region sequence of unigene and the corresponding amino acid sequence are predicted by using TransDecoder software, and the annotation information of unigene is obtained by comparing HMMER software with Pfam (protein family) database.

4. Results

RNA extraction, library construction and high-throughput transcriptome deep sequencing are carried out on marigold tissue materials, and then assembly and gene function prediction are carried out on a sequencing sequence. The results of the annotation and analysis of the assembly of the transcription factor gene show that 9 genes with transcription regulatory domains in marigold have higher expression level in leaves and complete protein coding sequences. Wherein the expression of Tagetes erecta Leaf transformation Factor 4(TeLTF4) in the Leaf blade is 61.4 times that of the immature flower tissue.

Example 2: cloning of marigold TelTF4 Gene

According to the data assembly of the marigold TelTF4 gene in example 1, PCR specific primers were designed using Primer design software Primer Premier 5.0, total RNA was extracted from mature marigold flowers, and the TelTF4 gene was cloned by reverse transcription-PCR (RT-PCR).

1. Reagent

The plant RNA extraction reagent Trizol is purchased from Invitrogen company, DNase I (dnase I) is purchased from TaKaRa company, reverse transcriptase, dNTP and high-fidelity DNA polymerase are purchased from Beijing Quanjin biotechnology limited company, primers are synthesized by the company Limited in the biological engineering (Shanghai), and other reagents are imported subpackaged or domestic analysis pure products.

2. Vectors and strains

Cloning Vector pEASY-Blunt Simple Cloning Vector was purchased from Beijing Quanyujin Biotechnology Co., Ltd, and E.coli (Escherichia coli) strain DH 5. alpha. was purchased from Beijing Quanyujin Biotechnology Co., Ltd.

3. Culture medium and reagent

LB culture medium, tryptone 10g/L, yeast powder 5g/L, NaCl 10 g/L. Adjusting pH to 7.0 with NaOH, and autoclaving.

100×Mg²⁺Solution 20.33g MgCl₂.6H₂O、24.65g MgSO₄.7H₂O constant volume in 100mL H₂And O, autoclaving.

SOC culture medium including tryptone 20g/L, yeast powder 5g/L, NaCl 0.58g/L, KCl 0.19g/L, 100 XMg ²⁺10 mL. Adjusting pH to 7.0 with NaOH, and autoclaving. Then 2mL of filter sterilized 1mol/L glucose was added.

1000 Xampicillin: 100mg/mL, dissolved in sterile deionized water and stored at-20 ℃.

4. Method of producing a composite material

4.1 extraction of RNA from leaf tissue of Tagetes erecta

The procedure was as described in 3.1 of example 1.

4.2 RT-PCR

4.2.1 RT

1) Taking 1. mu.g of total RNA and 1. mu.L of polyT₁₈(10. mu.M) primer mix with RNase-free ddH₂Complementing the amount of O to 12.75 mu L, and gently mixing;

2) preserving heat at 65 ℃ for 5min, immediately transferring to an ice bath, and standing for 2 min;

3) adding 5 × reaction buffer 4 μ L, 10mM dNTP 2 μ L, RNA inhibitor 0.25 μ L (40U/μ L), reverse transcriptase 1 μ L (100U/μ L), 42 deg.C for 1h, and synthesizing first chain cDNA;

4) heating at 95 deg.C for 5min, inactivating reverse transcriptase, and terminating reaction.

4.2.2 PCR

According to the sequence presumed by the TeLTF4 gene obtained in example 1, the Primer sequences designed by the Primer Premier 5.0 software are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the specific sequences are as follows:

TeLTF4F1:5’cattatttctactcttcatagc 3’

TeLTF4R1:5’gcgtcgacgtaatttattagtctagtaagaaga 3’

TeLTF4F2:5’cggggtaccatggatgacttttttatcc 3’

the cDNA of leaf tissue of marigold obtained in step 4.2.1 of this example was cloned from the gene of TelTF 4.

Place 200 μ L EP tube on ice, add reagents:

amplification was performed according to the following procedure: 2min at 98 ℃ (pre-denaturation); 10s at 98 ℃ (denaturation), 20s at 55 ℃ (renaturation), 60s at 72 ℃ (extension), and the denaturation-renaturation-extension is carried out for 30 cycles; 5min at 72 ℃ (total extension).

A second round of PCR was performed using the above PCR product as a template and primers TeLTF4F2 and TeLTF4R1, and the other conditions were the same as above.

By the above operation, a PCR amplification product of the TeLTF4 gene was obtained.

4.3 ligation of PCR amplification products to pEASY-Blunt vector

The PCR amplification product of the TeLTF4 gene obtained in step 4.2 of this example was ligated to the Cloning Vector pEASY-Blunt Simple Cloning Vector in a molar ratio of 1:4 (25 ℃, 15min) in the following manner:

pEASY-Blunt Simple Cloning Vector(50μg/μL) 4μL

PCR product (. about.150. mu.g/. mu.L) 1. mu.L

4.4 transformation of E.coli

1) Taking out the frozen Escherichia coli (Escherichia coli) strain DH5 alpha competent cells for ice bath thawing;

2) gently mixing the ligation product of 4.3 with escherichia coli competent cells uniformly, and carrying out ice bath for 30 min;

3) thermally shocking for 90s at 42 ℃, and immediately carrying out ice bath for 1-2 min;

4) adding 0.8mL of SOC, mixing uniformly, and carrying out mild shaking culture at 37 ℃ for 1 h;

5) after centrifugation at 13000rpm for 1min at room temperature, a part of the supernatant was discarded to leave about 200. mu.L of the supernatant, which was then mixed with the cells by a pipette tip, spread on LB plates containing ampicillin (100. mu.g/mL), and cultured at 37 ℃ for 12 hours.

4.5 colony PCR identification

The E.coli strain described in step 4.4 of this example was further subjected to colony PCR assay to confirm that the insert was the target fragment, and the reaction system was as follows:

reaction conditions are as follows: 3min at 94 ℃ (pre-denaturation); 30s at 94 ℃ (denaturation), 20s at 55 ℃ (renaturation), 60s at 72 ℃ (extension), and 26 cycles of denaturation-renaturation-extension; 5min at 72 ℃ (total extension).

The recombinant vector identified by colony PCR, designated pEASY-TeLTF4, was sequenced. As a result of sequencing, the full-length sequence of the TelTF4 gene ligated to the pEASY-Blunt cloning vector was obtained. Wherein, the nucleotide sequence of the TelTF4 gene is shown as SEQ ID NO.1, and specifically comprises the following steps:

the amino acid sequence of the TelTF4 gene is shown as SEQ ID NO.2, and specifically comprises the following steps:

example 3: analysis of the expression of the TelTF4 Gene

The full-length sequence of the TelTF4 gene was obtained by cloning according to example 2, and quantitative PCR primers were designed using Primer design software Primer Premier 5.0, total RNAs were extracted from marigold roots, leaves, and flowers, respectively, and reverse transcription was performed to obtain cDNAs, and quantitative analysis of the expression level of the TelTF4 gene was performed.

1. Reagent

RNA extraction, reverse transcription reagents as described in example 2; a real-time quantitative PCR reagent TransStart Green qPCR SuperMix is purchased from Beijing all-purpose gold biotechnology, Inc.; the primers are synthesized by the company of Biotechnology engineering (Shanghai) and other reagents are imported split charging or domestic analytical pure products.

2. Bacterial strains

Pseudomonas syringae pathogenic strain Pst DC3000(Pseudomonas syringae pv. tomato DC3000) was from the University of Idaho Fangming doctor laboratory, United states of America.

3. Method of producing a composite material

Taking marigold plant root, leaf and flower tissue samples, grinding by liquid nitrogen, extracting RNA, and carrying out reverse transcription, wherein the operation steps are as described in 3.1 in example 1 and 4.2 in example 2.

Based on the high throughput sequencing results, quantitative PCR analysis of the expression level of TeLTF4 was performed using transformation Initiation Factor 6(TIF6), which is stably expressed in different tissues of marigold, as an internal control gene. The primers for TeLTF4 were TeLTF4RTF and TeLTF4RTR, and the primers for TIF6 were TIF6F and TIF 6R. The primer sequences are respectively shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, and the specific sequences are as follows:

TeLTF4RTF:5’gatgtcgtttgctgggttgcc 3’

TeLTF4RTR:5’tggcagtcggaatctgaatgt 3’

TIF6F:5’taagacctggtggtggaaataga 3’

TIF6R:5’cagcaccatgaggacgaaga 3’

the quantitative PCR reaction system is as follows:

reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃, 15s at 60 ℃, 10s at 72 ℃ and 40 cycles. The cDNA was diluted 30 times to the cDNA template obtained by the method of 4.2.1 in example 2 and used for quantitative PCR. After amplification, the lysis curve was analyzed at 65 ℃ for 5s, with 0.5 ℃ increase per cycle, and 60 cycles. Each sample was replicated three times. The PCR reaction was run on Bio-Rad CFX 96.

4. Results

The real-time quantitative PCR analysis result shows that the expression level of the TelTF4 gene in marigold leaves is higher, the expression level of the TelTF4 in the leaves is about 4.5 times of that in roots, and the expression level of the leaves is about 12.8 times higher than that of the TelTF4 in mature flower tissues; the expression level was significantly increased in leaves of marigold plants inoculated with Pseudomonas syringae pathogenic strain PstDC3000 (fig. 1). The protein coded by the TelTF4 gene is shown to play a role in regulation and control of the disease-resistant defense response of marigold.

Example 4: construction of plant expression vector of TeLTF4 gene

The pEASY-TelTF4 plasmid and the plant expression vector pBTEX-HA vector are subjected to enzyme digestion purification and connection, transformed into escherichia coli, and transferred into agrobacterium after being sequenced and identified correctly.

1. Reagent

The plasmid extraction kit and the gel recovery kit are purchased from Tiangen Biotechnology GmbH, the high-fidelity DNA polymerase, the dNTP and the T4 DNA ligase are purchased from Beijing Quanyujin Biotechnology GmbH, the restriction enzymes Kpn I and Stu I are purchased from Fermentas, and other reagents are imported subpackaged or domestic analysis pure products.

2. Vectors and strains

Plant expression vector pBTEX-HA, Agrobacterium (Agrobacterium tumefaciens) strain GV2260 was obtained from the University of Idaho Fangming doctor Xiao laboratory, University of United states. Escherichia coli strain DH5 α was purchased from Beijing Quanjin Biotechnology Ltd.

3. Culture media and solutions

LB medium, SOC medium formulation was as described in example 3.

1000 kanamycin (Kan): 100mg/mL, dissolved in sterile deionized water and stored at-20 ℃.

500 × rifampicin (Rif): dissolving in 50mg/mL of methanol, sterilizing, diluting with deionized water to constant volume, and storing at-20 deg.C.

4. Method of producing a composite material

4.1 plasmid extraction

Plasmid extraction was performed on pEASY-TelTF4 plasmid obtained in example 2 and the plant expression vector pBTEX-HA vector, and the experimental procedures were performed as described in the kit instructions.

1) Column balancing: adding 500 μ L of balance liquid BL into adsorption column, centrifuging at 12000rpm for 1min, and discarding waste liquid;

2) centrifuging at 12000rpm for 1min, collecting bacterial precipitate, and discarding supernatant; adding 250 mu L P1 (RNase A is added), blowing, sucking and mixing evenly until the bacterial sediment is suspended completely;

3) adding 250 mu L P2, and gently turning the centrifugal tube up and down for 8 times to fully crack the thalli;

4) adding 350 mu L P3, gently turning the centrifuge tube up and down for 8 times at 12000rpm, and centrifuging for 10 min;

5) sucking out the supernatant to a new centrifuge tube, centrifuging at 12000rpm for 5 min;

6) carefully transferring the supernatant to an adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

7) adding 500 μ L PD at 12000rpm, centrifuging for 1min, and discarding the waste liquid;

8) adding 600 μ L PW (added with anhydrous ethanol), 12000rpm, centrifuging for 1min, discarding waste liquid, and repeating the operation once;

9) centrifuging at 12000rpm for 2min to remove residual PW;

10) transferring the adsorption column into a new centrifuge tube, adding 50 μ L of sterile deionized water into the center of the column, standing at room temperature for 2min, 12000rpm, centrifuging for 2min, and eluting DNA;

11) the plasmid concentration was determined. Storing at-20 deg.C for use.

4.2 plasmid cleavage

pEASY-TeLTF4, pBTEX-HA were digested with Kpn I and Stu I.

The reaction system is as follows, 37 ℃,1 h:

4.3 glue purification

The sequence fragment of the coding sequence of the TELTF4 gene and the pBTEX-HA plasmid after the enzyme digestion in the step 4.2 of the embodiment were subjected to gel recovery, and the experimental steps were performed as described in the kit instructions.

1) Adding 500 μ L of balance liquid BL into gel recovery adsorption column CA2, centrifuging at 12000rpm for 1min, and discarding column bottom waste liquid;

2) recovering the DNA fragments separated by electrophoresis on an ultraviolet gel cutting instrument, and putting the cut gel block into an EP tube;

3) adding sol solution PN according to the volume of 1:1 according to the mass of the sol. Putting the EP tube on a heater at 50 ℃, and dissolving for 10-15 min until the glue is completely dissolved;

4) after the sol is completely dissolved, cooling to room temperature, transferring the dissolved liquid into a gel recovery adsorption column CA2, and standing for 3min to make the dissolved liquid and the adsorption film fully contact;

5) centrifuging at 12000rpm for 1min, and discarding the waste liquid at the bottom of the column. Adding 600 μ L of PW into the adsorption tube, and standing for 3 min;

6) centrifuging at 12000rpm for 1min, and discarding the waste liquid at the bottom of the column. Repeating the previous step after finishing;

7) placing the empty adsorption column into a centrifuge for 3min at 12000rpm, standing for 15min until all alcohol is completely volatilized;

8) adding 30 μ L eluent EB onto the central adsorption film of adsorption column CA2, standing for 3min, and centrifuging at 12000rpm for 3min to obtain recovered gel product.

4.3 connection

Connecting the sequence fragment of the coding sequence of the digested and recovered TelTF4 gene with pBTEX-HA plasmid by T4 ligase, wherein the reaction system is as follows, and the reaction system is at 25 ℃ for 3 h:

4.4 transformation of E.coli

The ligation products were transformed into E.coli, and the experimental procedure was as described in 4.4 of example 2.

4.5 colony PCR identification

Coli colonies obtained in step 4.4 of this example were subjected to monoclonal picking, culture and colony identification, and the experimental procedure was as described in 4.5 of example 2.

The recombinant vector identified by colony PCR, named pBTEX-TELTF4, was sequenced. Sequencing results showed that a plant expression vector of the TELTF4 gene linked to pBTEX and carrying an HA tag was obtained.

4.6 Agrobacterium transformation

1) The pBTEX-TELTF4 plasmid was extracted according to the procedure described in 4.1 of this example;

2) adding the extracted plasmid pBTEX-TELTF4 into 50 μ L competent cells of Agrobacterium strain GV2260, mixing gently, and ice-cooling for 30 min;

3) placing in liquid nitrogen for cold shock for 1 min;

4) moving the EP pipe to a constant temperature heater at 37 ℃ and heating for 5 min;

5) adding 800 mu L of SOC culture solution, placing in a shaking table at 28 ℃, and culturing for 4-5 h at 200 rpm;

6) centrifuging the bacterial liquid at 4000rpm for 5 min;

7) sucking the supernatant in a super clean bench, slightly blowing the thalli to suspend and mix evenly, wherein the residual volume is about 100 mu L;

8) uniformly coating the bacterial liquid on an LB + Rif + Kan solid culture medium by using a sterilized glass ball, and culturing for 48h in a constant-temperature incubator at 28 ℃;

9) colony PCR was performed according to the same procedure as described in 4.5 of this example, and the positive Agrobacterium transformed with the recombinant plasmid was stored at-80 ℃.

Example 5: expression of the TelTF4 gene in tobacco leaves

The TeLTF4 gene is transiently expressed in tobacco leaves by an agrobacterium-mediated method, the expression condition of the protein coded by the TeLTF4 is detected, and the occurrence condition of leaf hypersensitivity is observed.

1. Reagent

Acetosyringone, mouse HA-tag monoclonal antibody anti-HA, anti-mouse antibody anti-mouse were purchased from Sigma; PVDF membranes are available from Merck Millipore; ECL western blot substrates were purchased from GE corporation; RNA extraction, reverse transcription reagents as described in example 2; real-time quantitative PCR reagents were as described in example 3; the primers are synthesized by the company of Biotechnology engineering (Shanghai) and other reagents are imported split charging or domestic analytical pure products.

2. Plant material

Nicotiana benthamiana (Nicotiana benthamiana) was obtained from the Boeisu Thompson institute, Connell university, USA, and was planted in a phytotron.

3. Culture medium and reagent

IM solution: 4.88g of 2-morpholine ethanesulfonic acid (MES); 2.5g of glucose; NaH₂PO₄0.126 g. MES is first added into deionized water to regulate pH value to 5.6, and glucose and NaH are then added₂PO₄Stirring uniformly, fixing the volume to 475mL, and sterilizing at high temperature.

20 × AB salt solution: NH (NH)₄Cl 20g；MgSO₄6g；KCl 3g；FeSO₄0.05g，CaCl₂2g of the total weight. Sequentially adding the components, completely and uniformly dissolving, diluting to a constant volume of 1L, and sterilizing at high temperature.

200mM acetosyringone (1000 ×): 39mg of the powdered acetosyringone was dissolved in 1mL of dimethyl sulfoxide and stored at-20 ℃ in the dark.

Induction medium: 19mL of IM solution; 1mL of 20 × AB salt solution; 20 mu L of 200mM acetosyringone; kanamycin (20 mu L) with the concentration of 25mg/mL is added with deionized water to be 20 mL.

Protein extraction buffer: 50mM Tris-HCl (Tris-HCl, pH 7.5); 150mM NaCl; 5mM ethylene diamine tetraacetic acid; 10% glycerol; 1% polyvinylpyrrolidone; 20 μ M dithiothreitol; 1mM phenylmethylsulfonyl fluoride; plant protease inhibitors (100. mu.L/10 mL extraction buffer).

5 XSDS-PAGE Loading buffer: 0.6mL of 1M Tris-HCl (pH 6.8); 5mL of 50% glycerol; 2mL of 10% SDS; 0.5mL of beta-mercaptoethanol; 1mL of 1% bromophenol blue, and deionized water is added to the solution to a constant volume of 10 mL.

10 XSDS-PAGE electrophoresis buffer: 120g of Tris; 576g of glycine; 40g of sodium dodecyl sulfate is dissolved in deionized water, and the volume is fixed to 4L. Adding deionized water to dilute to 1X before use.

10 × western blot membrane transfer buffer: 144g of glycine and 30.2g of Tris are dissolved in 0.9L of deionized water, stirred and mixed uniformly, and the volume is adjusted to 1L. When 1L of 1 Xmembrane buffer solution is prepared, 100mL of 10 Xmembrane buffer solution and 100mL of methanol are added, and deionized water is added to the mixture to make a constant volume of 1L.

10 × TBS (Tris-buffered saline) buffer: 80g of NaCl, 2g of KCl and 30g of Tris are dissolved in 0.8L of deionized water, the pH value is adjusted to 7.4, and the deionized water is added to the volume of 1L. When 1 XTSST (Tris-buffered saline with Tween) buffer was prepared, 100mL of 10 XTSST and 202.5mL of 20% Tween were added, and deionized water was added to the volume of 1L.

4. Method of producing a composite material

4.1 tobacco transient expression

1) Marking out agrobacterium transferred to pBTEX-TELTF4 and pBTEX-HA vectors on LB culture plates with Rif and Kan respectively, and culturing for 48h in a thermostat at 28 ℃;

2) selecting a monoclonal, culturing for 12h at 28 ℃, taking 300 mu L of bacteria, transferring into 2.7mL of LB + Rif + Kan culture solution, and culturing for 6-8 h at 28 ℃;

3) centrifuge at 3000rpm for 6min at room temperature, and discard the supernatant. Add 3mL of IM solution to resuspend the cells, and repeat centrifugation to collect the cells once. Resuspending the thallus with 3mL of IM solution, and culturing at 28 ℃ and 250rpm for 5-14 h;

4) centrifuging at 3000rpm for 6min, discarding supernatant, adding 10mM MES 2mL (pH 5.7) and 200mM acetosyringone 200 μ L, resuspending thallus, and vortexing;

5) repeating the step 4) once;

6) the suspension solution was used as a blank control to determine the concentration (OD) of the bacterial liquid₆₀₀) Preparing a staining solution;

7) injecting the infection liquid into the tobacco leaves from the lower epidermis of the tobacco leaves by using a disposable injector, and marking the infection range;

8) and (3) placing the injected plant in the shade for 0.5h, then placing the plant in the light for growing for 36-48 h, collecting a leaf sample, and quickly cooling the leaf sample in liquid nitrogen and storing the leaf sample at-80 ℃.

4.2Western hybridization

1) Taking out tobacco leaves respectively transferred to pBTEX-TELTF4 and pBTEX-HA carriers from a refrigerator at-80 ℃, grinding the tobacco leaves into powder in a mortar by using liquid nitrogen, and transferring the powder into a precooled 1.5mL EP tube;

2) adding 300 μ L protein extract, shaking on vortex instrument to mix the extract and sample, and standing for 10 min;

3) centrifuging at 12000rpm at 4 deg.C for 10min in a low temperature centrifuge;

4) pipette 200. mu.L of supernatant into a new EP tube, add 40. mu.L of 5 XProte loading buffer, and heat at 95 ℃ for 5 min.

5) Subjecting the sample to 10% polyacrylamide gel electrophoresis;

6) performing PVDF film transfer printing on the gel for 100V and 1 h;

7) sealing the PVDF membrane by 5% skimmed milk for 1 h;

8) adding anti-HA antibody to react for 1h at room temperature;

9) washing the membrane with 1 × TBST buffer solution for 3 times, adding horseradish peroxidase-linked anti-mouse antibody, and reacting at room temperature for 1 h;

10) the membrane was washed 3 times with 1 × TBST buffer, and a reaction substrate (western blotting ECL substrate) was added to detect the protein expression in a chemiluminescence apparatus.

4.3 quantitative determination of tobacco disease resistance related gene PR1

The tobacco leaves transferred into pBTEX-TELTF4 and pBTEX-HA vectors are taken, ground by liquid nitrogen, extracted with RNA and subjected to reverse transcription, and the operation steps are as 3.1 in example 1 and 4.2 in example 2. The cDNA obtained by reverse transcription is taken, and the expression level of the disease-resistant marker gene PR1 is detected by taking NtUBI3 as an internal reference gene. The primer sequences are respectively shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, and the specific sequences are as follows:

NtPRF:5’aacctttgacctgggacgac 3’

NtPRR:5’gcacatccaacacgaaccga 3’

NtUBI3F:5’gccgactacaacatccagaagg 3’

NtUBI3R:5’tgcaacacagcgagcttaacc 3’

the quantitative PCR reaction system and reaction conditions were as described in method 3 of example 3.

5. Results

The experimental results showed (FIG. 2) that, in tobacco leaves transiently transfected with the TeLTF4 gene, a protein band encoding TeLTF4 of about 60kD expected size was obtained by Agrobacterium-mediated transformation, whereas no expression product of the tag protein was detected in the control sample of pBTEX empty vector not transfected with the foreign gene. Meanwhile, cell necrosis appears at the leaf part of the TeLTF4 expression, and the control sample without the exogenous gene has no abnormality, which indicates that the TeLTF4 can activate an immune pathway to enable the leaf cells to generate programmed death hypersensitivity when the plant leaf is expressed. In addition, the expression level of the disease resistance marker gene PR1 in the tobacco leaves transferred with the TelTF4 gene is increased by about 4.5 times. Experimental results show that when the cloned TelTF4 gene in marigold leaf is expressed in tobacco, the immune hypersensitivity reaction of the leaf can be activated, and the transcription level of downstream disease-resistant genes is increased. Therefore, the TeLTF4 gene shown in SEQ ID NO.1 plays a role in regulating and controlling the plant immune response and can be used for enhancing the disease resistance of plants.

The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Sequence listing

<110> university of fertilizer industry

<120> a plant gene and application

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1484

<212> DNA

<213> marigold (Tagetes erecta)

<400> 1

cattatttct actcttcata gcttcacccc aaacccttct ccataaacca tggatgactt 60

ttttatccct ctttcacctt caacttcttc ttccatcatc tccgatcacc ttcaaaccac 120

tccaccgcca ccgtcttcgt gccacaataa actccaacac ctcctccaat cccaacccca 180

ccacccttgg gcctatgcca ttttctggaa aaacagcaac aacaacagca acagcaacaa 240

caacaactac tgttttcaag ctttaacatg gtttgatggt tacttcttac aaaaccctaa 300

caaaccacta atcaacaaaa cttacaaaac tcaattaccc gatgactcgg attggtttta 360

cttcatttca ttgaccaaat cattcacttt aggcgatggc tcggctccaa caaaggctta 420

tgagtccaac tccgtggttt ggttcaccgg ggctcataat ctcttatctt tcgattgtga 480

tcgagctaaa gaagctcata ttcatgggtt agaaacactt gtttatgttc caacggctaa 540

tggtgttgta gaaatcggtt cgtttcattt gattgatcaa acggagtcgg atttagctca 600

taaagctcag tcacttttcg gtgccggaac ttcttcttca tcatcttctt ctccacctcc 660

tccggaggct ataatattga tgcaagagaa ggaaacatta agcggtttga tgtcgtttgc 720

tgggttgccg gaagaggtga cgttggacgt tatagatctc ggagaaccca cgccggaaca 780

accaccggag aaatttggaa agattgaccg gatcaccaaa gcaagtatac ccgtatccga 840

tacagcatca gaacattcag attccgactg ccaactgatt ttcacaacca agaaaaaatt 900

agggttaaaa aagaaatcaa gagatccacc gttaaaccac gttgaggcgg agcgacaacg 960

tcgcgagaag ctcaaccaac gattctacac tctacgatca gtcgtcccaa atgtgtccaa 1020

aatggacaag gcctcgttgt tagccgacgc cgtaagttac ataaatgaac tgaaacaaaa 1080

agtggaagaa ttggaatcga aagttaatga tgatcagagg atagtaaaga taccgaaggc 1140

ggaaccgacg gaaatacggg tcgggaaata taaatcgaca agggtttgta aaaaagggag 1200

tgtagataaa ttattaacag cggatgtgaa aatggtggga gaagatgtga tgataagagt 1260

acaatccgaa aacgaaaatt ggcctgtggc gagattaatg ggggcattgc gagaaatgaa 1320

agcaaaggtt catcatgcaa gtatgatgtg tgttaatgac atgatgcttc aagatgttgt 1380

tgctaaaatt aatggtgtca cggaagatga agttaaatct tatcttctta ctagactaat 1440

aaattactaa ctaattaaat attaattaat atttagcttt tcat 1484

<210> 2

<211> 466

<212> PRT

<213> marigold (Tagetes erecta)

<400> 2

Met Asp Asp Phe Phe Ile Pro Leu Ser Pro Ser Thr Ser Ser Ser Ile

1 5 10 15

Ile Ser Asp His Leu Gln Thr Thr Pro Pro Pro Pro Ser Ser Cys His

20 25 30

Asn Lys Leu Gln His Leu Leu Gln Ser Gln Pro His His Pro Trp Ala

35 40 45

Tyr Ala Ile Phe Trp Lys Asn Ser Asn Asn Asn Ser Asn Ser Asn Asn

50 55 60

Asn Asn Tyr Cys Phe Gln Ala Leu Thr Trp Phe Asp Gly Tyr Phe Leu

65 70 75 80

Gln Asn Pro Asn Lys Pro Leu Ile Asn Lys Thr Tyr Lys Thr Gln Leu

85 90 95

Pro Asp Asp Ser Asp Trp Phe Tyr Phe Ile Ser Leu Thr Lys Ser Phe

100 105 110

Thr Leu Gly Asp Gly Ser Ala Pro Thr Lys Ala Tyr Glu Ser Asn Ser

115 120 125

Val Val Trp Phe Thr Gly Ala His Asn Leu Leu Ser Phe Asp Cys Asp

130 135 140

Arg Ala Lys Glu Ala His Ile His Gly Leu Glu Thr Leu Val Tyr Val

145 150 155 160

Pro Thr Ala Asn Gly Val Val Glu Ile Gly Ser Phe His Leu Ile Asp

165 170 175

Gln Thr Glu Ser Asp Leu Ala His Lys Ala Gln Ser Leu Phe Gly Ala

180 185 190

Gly Thr Ser Ser Ser Ser Ser Ser Ser Pro Pro Pro Pro Glu Ala Ile

195 200 205

Ile Leu Met Gln Glu Lys Glu Thr Leu Ser Gly Leu Met Ser Phe Ala

210 215 220

Gly Leu Pro Glu Glu Val Thr Leu Asp Val Ile Asp Leu Gly Glu Pro

225 230 235 240

Thr Pro Glu Gln Pro Pro Glu Lys Phe Gly Lys Ile Asp Arg Ile Thr

245 250 255

Lys Ala Ser Ile Pro Val Ser Asp Thr Ala Ser Glu His Ser Asp Ser

260 265 270

Asp Cys Gln Leu Ile Phe Thr Thr Lys Lys Lys Leu Gly Leu Lys Lys

275 280 285

Lys Ser Arg Asp Pro Pro Leu Asn His Val Glu Ala Glu Arg Gln Arg

290 295 300

Arg Glu Lys Leu Asn Gln Arg Phe Tyr Thr Leu Arg Ser Val Val Pro

305 310 315 320

Asn Val Ser Lys Met Asp Lys Ala Ser Leu Leu Ala Asp Ala Val Ser

325 330 335

Tyr Ile Asn Glu Leu Lys Gln Lys Val Glu Glu Leu Glu Ser Lys Val

340 345 350

Asn Asp Asp Gln Arg Ile Val Lys Ile Pro Lys Ala Glu Pro Thr Glu

355 360 365

Ile Arg Val Gly Lys Tyr Lys Ser Thr Arg Val Cys Lys Lys Gly Ser

370 375 380

Val Asp Lys Leu Leu Thr Ala Asp Val Lys Met Val Gly Glu Asp Val

385 390 395 400

Met Ile Arg Val Gln Ser Glu Asn Glu Asn Trp Pro Val Ala Arg Leu

405 410 415

Met Gly Ala Leu Arg Glu Met Lys Ala Lys Val His His Ala Ser Met

420 425 430

Met Cys Val Asn Asp Met Met Leu Gln Asp Val Val Ala Lys Ile Asn

435 440 445

Gly Val Thr Glu Asp Glu Val Lys Ser Tyr Leu Leu Thr Arg Leu Ile

450 455 460

Asn Tyr

465

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

cattatttct actcttcata gc 22

<210> 4

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gcgtcgacgt aatttattag tctagtaaga aga 33

<210> 5

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cggggtacca tggatgactt ttttatcc 28

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

gatgtcgttt gctgggttgc c 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

tggcagtcgg aatctgaatg t 21

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

taagacctgg tggtggaaat aga 23

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cagcaccatg aggacgaaga 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

aacctttgac ctgggacgac 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gcacatccaa cacgaaccga 20

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gccgactaca acatccagaa gg 22

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

tgcaacacag cgagcttaac c 21

Claims (3)

1. A plant gene has a nucleotide sequence shown as SEQ ID NO. 1.

2. The gene of claim 1, wherein the amino acid sequence of the gene is shown as SEQ ID NO. 2.

3. Use of the gene according to claim 1 for modulating immune hypersensitivity in plants.

Images