CN118813700B

CN118813700B - Application of HCR1 gene in improving phytophthora resistance

Info

Publication number: CN118813700B
Application number: CN202411230989.5A
Authority: CN
Inventors: 李国田; 肖永欣; 孙鹏; 杨磊; 李翔
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2024-09-03
Filing date: 2024-09-03
Publication date: 2025-07-18
Anticipated expiration: 2044-09-03
Also published as: CN118813700A

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to the application of HCR1 gene in improving plant resistance to Phytophthora. The present invention provides the application of HCR1 gene in improving plant resistance to Phytophthora or cultivating transgenic plants resistant to Phytophthora, and the amino acid sequence encoded by the HCR1 gene is shown in SEQ ID NO.1. The present invention introduces the mechanism of "accumulation on membrane - formation of death - membrane lysis - bacterial cell necrosis" of bacteriophage HCR1 gene into plants, and finds that HCR1 gene can improve plant resistance to Phytophthora, and achieves the purpose of plant disease resistance by domesticating exogenous genes, which can not only expand the source of disease resistance gene mining, but also the disease resistance trait can be stably inherited.

Description

Application of HCR1 gene in improving phytophthora resistance

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of an HCR1 gene in improving phytophthora resistance.

Background

Disease-resistant corpuscles in plant cells can be divided into two types, one of which is combined with a specific effector to form a polymer, pore is formed in a cell membrane, and calcium ion flow is initiated, so that plant immune response occurs. The other is combined with effector and needs help of helper gene to polymerize and pore on cell membrane to cause disease-resistant reaction of plant. However, the current plant disease-resistant corpuscles still have great inconvenience in transformation and design, and are simultaneously infected by various pathogenic matters such as fungi, bacteria, viruses and the like to face the situation of losing the resistance of the corpuscles. Meanwhile, the current disease-resistant genes are limited to excavate, clone and discover more disease-resistant corpuscles from plants, a large amount of manpower, material resources and financial resources are required to be consumed, and the traditional plant disease-resistant varieties are easy to lose resistance of the seeds after being planted for successive years, so that some plant endogenous disease-resistant genes cannot exert lasting disease resistance.

Disclosure of Invention

In order to solve the problems, the invention provides application of HCR1 gene in improving phytophthora root rot resistance. The invention discovers that the HCR1 gene can improve the phytophthora root rot resistance and the disease resistance property can be inherited stably.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides application of an HCR1 gene in improving phytophthora resistance of plants or cultivating transgenic plants resistant to phytophthora, wherein an amino acid sequence coded by the HCR1 gene is shown as SEQ ID NO. 1.

Preferably, the plant comprises tobacco.

Preferably, the pathogenic bacteria of phytophthora capsici comprises phytophthora capsici (Phytophthora capsici).

Preferably, the nucleotide sequence of the HCR1 gene is shown as SEQ ID NO. 2.

The invention provides a recombinant vector for improving phytophthora resistance, which comprises an original vector, a RBOH promoter and an HCR1 gene positioned at the downstream of the RBOH promoter, wherein the nucleotide sequence of the RBOH promoter is shown as SEQ ID NO.22, and the amino acid sequence encoded by the HCR1 gene is shown as SEQ ID NO. 1.

Preferably, the original vector comprises a pCAMBIA1381 vector.

The invention provides a construction method of the recombinant vector, which comprises the following steps:

Carrying out PCR amplification by using Arabidopsis genome DNA as a template and using primers NbRBOHpro-F and NbRBOHpro-R to obtain a RBOH promoter containing enzyme cutting sites, wherein the nucleotide sequences of the primers NbRBOHpro-F and NbRBOHpro-R are shown as SEQ ID NO.18 and SEQ ID NO. 19;

The HCR1 gene is used as a template, and primers p1381-HCR1-F and p1381-HCR1-R are used for carrying out PCR amplification to obtain an HCR1 gene sequence containing an enzyme cutting site, wherein the nucleotide sequences of the primers p1381-HCR1-F and p1381-HCR1-R are shown as SEQ ID NO.20 and SEQ ID NO. 21;

connecting the RBOH promoter containing the cleavage site with the linear pCAMBIA1381 vector subjected to EcoR I cleavage to obtain a recombinant vector pCAMBIA1381-RBOH;

And (3) carrying out enzyme digestion on the recombinant vector pCAMBIA1381-RBOH by using restriction enzyme Hind III, and connecting with the HCR1 gene sequence containing the enzyme digestion site to obtain the recombinant vector.

The invention provides an engineering bacterium for improving phytophthora resistance, which is characterized by comprising the recombinant vector disclosed by the technical scheme or a recombinant vector constructed by utilizing the construction method disclosed by the technical scheme.

Preferably, the initial strain for constructing the engineering bacteria comprises agrobacterium GV3101.

The invention provides a method for improving phytophthora resistance, which comprises the steps of introducing a recombinant vector or engineering bacteria into plants, wherein the recombinant vector is the recombinant vector according to the technical scheme or is constructed by using the construction method according to the technical scheme, and the engineering bacteria are engineering bacteria according to the technical scheme.

The beneficial effects are that:

The invention provides application of an HCR1 gene in improving phytophthora resistance of plants or cultivating transgenic plants resistant to phytophthora, wherein an amino acid sequence coded by the HCR1 gene is shown as SEQ ID NO. 1. The invention introduces the mechanism of accumulation-formation of death on the membrane-membrane cleavage-bacterial cell necrosis of phage HCR1 gene into plants, discovers that HCR1 gene can improve phytophthora resistance of plants, achieves the purpose of plant disease resistance by domesticating exogenous genes, not only can expand the excavation source of disease resistance genes, but also can inherit disease resistance characters stably.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 shows the results of transient expression of exogenous genes in Nicotiana benthamiana to induce allergic necrosis reaction in plants, wherein Bright light and UV light represent white light and ultraviolet light, respectively, and the scale is 1cm;

FIG. 2 shows the result of phytophthora capsici infection after 18h of transient expression of HCR1 gene by Nicotiana benthamiana, and the statistical result of lesion area and relative pathogenic bacteria biomass;

FIG. 3 shows infection results and disease area statistics of T0 generation phytophthora capsici of different transgenic lines;

FIG. 4 shows statistical results of infection effects, disease areas and relative pathogenic biomass of T1-generation phytophthora capsici of different transgenic lines.

Detailed Description

The invention provides application of an HCR1 gene in improving phytophthora resistance of plants or cultivating transgenic plants resistant to phytophthora. In the invention, the plant preferably comprises tobacco, the pathogenic bacteria of phytophthora capsici preferably comprises phytophthora capsici (Phytophthora capsici), the amino acid sequence coded by the HCR1 gene is shown as SEQ ID NO.1, the nucleotide sequence of the HCR1 gene is preferably shown as SEQ ID NO.2, and specific sequence information is as follows:

SEQ ID NO.1:

MKMPEKHDLLAAILAAKEQGIGAILAFAMAYLRGRYNGGAFTKTVIDAT MCAIIAWFIRDLLDFAGLSSNLAYITSVFIGYIGTDSIGSLIKRFAAKKAGVEDG RNQ*;

SEQ ID NO.2:

5'-ATGAAGATGCCGGAGAAGCACGACCTCCTCGCCGCTATTCTCGCCG CTAAGGAGCAAGGCATCGGCGCTATCCTCGCCTTCGCTATGGCCTACCTCCGCGGCAGATACAACGGCGGCGCTTTCACAAAGACAGTGATCGACGCCACCATGTGCGCCATCATCGCCTGGTTCATCAGAGACCTCCTCGACTTCGCGGGCCTCTCTTCTAACCTCGCCTACATCACCTCCGTGTTCATCGGCTACATCGGCACCGACTCCATCGGCTCTCTCATCAAGCGCTTCGCCGCCAAGAAGGCCGGCGTTGAGGATGGCAGAAACCAGTGA-3'.

The invention introduces a mechanism of 'film accumulation-formation death valve-film lysis-bacterial cell necrosis' of phage HCR1 gene (namely, after phage injects DNA into bacterial cytoplasm, HCR1 gene can be continuously expressed on cell membrane, and a large amount of HCR1 gene is accumulated to form 'death valve' within 2min, then cracking factors are rapidly released to cause bacterial cell cracking death), and the invention proves the effect of HCR1 gene in plant disease resistance by introducing HCR1 gene into plant disease resistance, and the HCR1 gene can rapidly cause immune reaction of plant after being introduced into plant, timely inhibit pathogen infection, and has the advantages of remarkable disease resistance effect and stable inheritance of disease resistance property. The invention enhances the disease resistance of plants by domesticating exogenous genes of the plants, and becomes broad-spectrum resistance genes which can aim at various pathogenic bacteria.

Based on the advantages, the invention provides a recombinant vector for improving phytophthora root rot resistance, which comprises an original vector, a RBOH promoter and an HCR1 gene positioned at the downstream of the RBOH promoter, wherein the nucleotide sequence of the RBOH promoter is shown as SEQ ID NO.24, and the amino acid sequence coded by the HCR1 gene is shown as SEQ ID NO. 1. In the present invention, the original vector preferably includes a pCAMBIA1381 vector.

Based on the advantages, the invention provides an engineering bacterium for improving phytophthora resistance, which comprises the recombinant vector disclosed by the technical scheme or a recombinant vector constructed by using the construction method disclosed by the technical scheme. In the present invention, the initial strain for constructing the engineering bacterium preferably includes agrobacterium GV3101.

Based on the advantages, the invention provides a method for improving phytophthora root rot resistance of plants, which is to introduce a recombinant vector or engineering bacteria into the plants, wherein the recombinant vector is the recombinant vector according to the technical scheme or is constructed by using the construction method according to the technical scheme, and the engineering bacteria are engineering bacteria according to the technical scheme.

For further explanation of the present invention, the use of the HCR1 gene provided by the present invention for improving phytophthora resistance will be described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

S1, vector construction

1. Total RNA of rice was extracted by Trizol method (R401-01-AA, noruzan, nanjing, china) with reference to the instructions, and cDNA was obtained by reverse transcriptase (R312-01-AB, noruzan, nanjing, china) with reference to the instructions.

2. The synthetic HCR1 gene (shown as SEQ ID NO. 2) is used as a template to amplify 3 HCR1 gene full-length fragments respectively, a HindIII enzyme cutting site and a carrier homologous 14bp sequence are added at the 5' end of an upstream primer and a downstream primer, the downstream primer comprises a stop codon TAA sequence for independent expression of HCR1, a HindIII enzyme cutting site and a carrier homologous 14bp sequence are added at the 5' end of the upstream primer and the downstream primer, the downstream primer does not comprise a stop codon TAA sequence for fusion expression of HCR1-eGFP, a Sma I enzyme cutting site and a carrier homologous 14bp sequence are added at the 5' end of the upstream primer and the downstream primer comprises a stop codon TAA sequence for fusion expression of eGFP-HCR1, and the Xa23 gene fragment is obtained by PCR amplification of the whole Xa23 gene fragment by using the extracted rice cDNA as a template. The reaction conditions were 95℃pre-denaturation for 3min, 95℃denaturation for 15sec,60℃annealing for 15sec,72℃extension for 15sec,35 cycles, 72℃final extension for 5min. The PCR product was recovered using an agarose gel recovery kit (DC 301-01, northenan, nanjing, china). The primer sequences were as follows:

HCR1-F:5'-AAATCGACTCTAGAAAGCTTATGAAGATGCCGGAGAAGCA-3',SEQ ID NO.3;

HCR1-R:5'-CTCACCATGGTCTCAAGCTTTCACTGGTTTCTGCCATCCTC-3',SEQ ID NO.4;

HCR1-eGFP-F is shown in SEQ ID NO. 3;

HCR1-eGFP-R:5'-CTCACCATGGTCTCAAGCTTCTGGTTTCTGCCATCCT CAAC-3',SEQ ID NO.5;

eGFP-HCR1-F:5'-ACGAGCTGTACAAGCCCGGGATGAAGATGCCGGAG AAGCA-3',SEQ ID NO.6;

eGFP-HCR1-R:5'-TAGTCCATGTCGACCCCGGGTCACTGGTTTCTGCCAT CCTC-3',SEQ ID NO.7;

Xa23-F:5'-AAATCGACTCTAGAAAGCTTATGTTGCATCATCTCAAGGAG CT-3',SEQ ID NO.8;

Xa23-R:5'-CTCACCATGGTCTCAAGCTTTTAAACAGGGAGAATAACCAT CTTGTCG-3',SEQ ID NO.9;

3. The pCAMBIA1300 vector is subjected to enzyme digestion by using restriction enzymes Hind III and Sma I respectively, and the enzyme digestion products are recovered and purified by using an agarose gel recovery kit to obtain a Hind III linearization pCAMBIA1300 vector and a Sma I linearization pCAMBIA1300 vector;

The HindIII linearization pCAMBIA1300 vector is respectively connected with the full-length fragment of the HCR1 gene and the Xa23 gene fragment with HindIII restriction enzyme sites under the action of homologous recombinase (#RDA01, primary severe selection and Chinese Wuhan), and then the connection products are converted into escherichia coli DH5 alpha to obtain recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP and pCAMBIA1300-Xa23;

and (3) connecting the SmaI linearization pCAMBIA1300 vector with the HCR1 gene fragment with the SmaI restriction enzyme cleavage site under the action of homologous recombinase (#RDA01, boyuanshan, chinese Wuhan), and then converting the connection product into escherichia coli DH5 alpha to obtain the recombinant vector pCAMBIA1300-eGFP-HCR1.

4. The combined length of the full-length sequence of the HCR1 gene or Xa23 gene and a partial empty vector sequence is taken as a positive cloning target band, and the positive cloning of the recombinant vector is detected in three PCR amplification systems respectively.

The detection primers for detecting the recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP and pCAMBIA1300-Xa23 are as follows:

p1300-Hind III-F:5'-CGCCATTTCGCCTTTTCA-3',SEQ ID NO.10;

p1300-Hind III-R:5'-TGCAGATGAACTTCAGGGTCAG-3',SEQ ID NO.11;

the detection primers for detecting the recombinant vector pCAMBIA1300-eGFP-HCR1 are as follows:

p1300-Sma I-F:5'-GCAAAGACCCCAACGAGAAG-3',SEQ ID NO.12;

p1300-Sma I-R:5'-CCGGCAACAGGATTCAATCT-3',SEQ ID NO.13;

detection of cell lethality of S2 and HCR1 genes

The transient expression of exogenous genes in Nicotiana benthamiana induces allergic necrosis reaction (HR) of plants, and recombinant vectors containing exogenous genes are CAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, pCAMBIA1300-eGFP-HCR1, pCAMBIA1300-Xa23 and empty vector pCAMBIA1300 constructed by S1, wherein the pCAMBIA1300 empty vector is used for negative control containing eGFP genes and pCAMBIA1300-Xa23 containing Xa23 genes can induce HR reaction as positive control. The method comprises the following specific steps:

4 recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, pCAMBIA1300-eGFP-HCR1 and pCAMBIA1300-Xa23 and empty vector pCAMBIA1300-eGFP are respectively transferred into agrobacterium GV3101 by adopting a freeze thawing method. After 18h of shaking culture at 28℃and 200rpm, the cells were resuspended using a centrifuge (1500 g) for 5min, then the concentration value OD _600nm was measured using a visible light spectrophotometer and the OD _600nm value of each component was adjusted to 1.0 again using the infecting solution (10 mM MgCl ₂, 10mM MES and 100. Mu.M acetosyringone). Agrobacteria heavy suspension containing HCR1 gene, eGFP-HCR1 gene or HCR1-eGFP gene, empty vector negative control eGFP gene and positive control Xa23 gene are injected into 4 different parts of the same leaf of 6-leaf stage Nicotiana benthamiana. 48h after injection, the tobacco leaves are observed and photographed under white light and ultraviolet light. The results are shown in FIG. 1.

The results in FIG. 1 show that the HCR1 gene, eGFP-HCR1 gene and positive control Xa23 gene are capable of eliciting an HR response in tobacco cells, whereas the HCR1-eGFP gene and empty vector negative control eGFP gene are not capable of eliciting an HR response. As can be seen from FIG. 1, the HCR1 gene has the essential characteristics of plant disease-resistant proteins.

Example 2HCR1 Gene inhibits Phytophthora capsici infection of tobacco leaves

The test is divided into 2 groups, agrobacterium GV3101 containing vectors pCAMBIA1300-HCR1, pCAMBIA1300-eGFP and pCAMBIA1300-Xa23 are subjected to shaking culture at 28 ℃ and 200rpm for 18 hours, and then centrifuged for 5min by using a centrifuge 1500g, and the bacterial cells are resuspended by using an infecting solution (10 mM MgCl ₂, 10mM MES and 100 mu M acetosyringone), and the OD _600nm value is measured by a visible light spectrophotometer to adjust the OD _600nm value of each component to 1.0. The Xa23 gene was used as a positive control, and the agrobacterial suspensions containing the HCR1 gene and the empty vector negative control eGFP gene were injected into 2 symmetrical parts of the same leaf of Nicotiana benthamiana in the 6-leaf stage. After culturing for 18h at 24 ℃, inoculating phytophthora capsici (Phytophthora capsici) LT263 bacterial blocks at an agrobacterium injection site, observing and photographing tobacco leaves under ultraviolet light after culturing for 18h in a dark and moisture-preserving manner at 24 ℃, counting the infection area of the phytophthora capsici, and detecting the biomass of pathogenic bacteria by adopting PCR. The results are shown in FIG. 2. The phytophthora capsici LT263 is preserved in the rice disease-resistant laboratory of the university of agricultural science and technology college of China. Are disclosed in the literature 【Sha G,SunP,KongXJ,HanXY,Sun QP,Fouillen L,et al.Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.Nature,2023,618(7967):1017-1023.https://doi.org/10.1038/s41586-023-06205-2】.

The PCR detection primers for the biomass of pathogenic bacteria are as follows:

Internal reference primer:

NbEF1a-F:5'-AGAGGCCCTCAGACAAAC-3',SEQ ID NO.14;

NbEF1a-R:5'-TAGGTCCAAAGGTCACAA-3',SEQ ID NO.15;

Phytophthora capsici detection primer:

Pc-tubulin-F:5'-CAGAGGGTGCTGAGCTTATTGACT-3',SEQ ID NO.16;

Pc-tubulin-R:5'-GAGAGTGGGTGATCTGGAAACCC-3',SEQ ID NO.17;

The results of fig. 2 show that the phytophthora capsici infection can be obviously inhibited after the transient expression of the HCR1 gene of the cigarette, the area of the disease spots is obviously smaller than that of an empty vector negative control eGFP gene (p < 0.01), and the biomass content of the infected part of the phytophthora capsici after the expression of the HCR1 gene is obviously smaller than that of the negative control eGFP gene (p < 0.01).

As can be seen from FIG. 2, the HCR1 gene has plant disease resistance.

EXAMPLE 3 acquisition of HCR1 transgenic tobacco

S1, vector construction

The full-length fragments of RBOH promoter and HCR1 gene were amplified by PCR and constructed into the EcoR I and Hind III cleavage sites of the plant stable expression vector pCAMBIA1381, respectively, by homologous recombination.

1. The primer NbRBOHpro-F and NbRBOHpro-R are used for amplifying RBOH promoter by taking Arabidopsis genome DNA as a template, and the synthesized HCR1 gene (shown as SEQ ID NO. 2) is used for amplifying the full length of the HCR1 gene sequence by taking the primers p1381-HCR1-F and p1381-HCR1-R as the template. The reaction conditions were 95℃for 3min, 95℃for 15sec,60℃for 15sec, 72℃extension at an amplification efficiency of 30sec/1kb, 35 amplification cycles, and 72℃for 5min. The primer sequences were as follows:

NbRBOH pro-F:5'-ATCCAGATCCAGTGGGATCCATAATAATGTAATAGC GAGAATACCTCTATTG-3',SEQ ID NO.18;

NbRBOH pro-R:5'-CCGAGCTCACCCGGGGATCCCGGATGATGATTTTC AGAATTTTGCA-3',SEQ ID NO.19;

p1381-HCR1-F:5'-GTCGACCTGCAGCCAAGCTTATGAAGATGCCGGAG AAGCA-3',SEQ ID NO.20;

p1381-HCR1-R:5'-GTGGACTCCTCTTAAAGCTTTCACTGGTTTCTGCCA TCCTC-3',SEQ ID NO.21;

The nucleotide sequence of RBOH promoter is shown as SEQ ID NO.22, and specific sequence information is as follows:

SEQ ID NO.22:

5'-ATAATAATGTAATAGCGAGAATACCTCTATTGAGATATTGAAGAGTCA TTCCACGACCTTATATGAAGATGTTATATATCTATCAAGCATGATAATTTCAAGGTGCAGAATATTCATTCAAGTCAATATGGTTGATTTATTTATGAAATCTCTAACAACGATCTCTTGAAGATGGTGCACAAGATTGGAATGCAAATGCTTAAAGATGTGAATTGAGACTCTCATCAGGGGGAGTTAATGCGTGTTGCACTCTTTTTCCCTTACAAGGTTTTGTCCCACTGGATTTTTCTTGCAAGGTTTTCAATGAGGCAACCAAAAGGCATATTATTAGATATTGTACTCTTTTTTCTTTTCTAGATTTTTTCCCATTGGATTTTATTTTAGTTAGGTTTTAATGAGGCACATTATCTGTTGAATAGACATTCAAGGGGGAGTGTCATAAATAGAATTTTATTTATGCTGAATGTCTATATTTAAAGAATTCTAGGGTTTATTACTTGGTGGCTAAGTCACATTTTCCCTATAAATAAAGGGTTCCGTTCTATTGGAAATCATCTCATATCAATAAGAATTCTCTTTCCCTACTTTTCTCTGCAATATTCGTCCTCTTCTTTTCTTGTTTCATAACATAATTTAGGTTCTTATTTTTTTCCTTTTTAATTAACTAGACTAGATAGATTAATTCGCATTATCCGGGCTTCTATTTTTTTTTCCCCTTCCTTTTTTTGACGGAGAATAGTCTCTGATCTAGTGGGCTAGTGGCTTTGCCACCTTATTATACAAGGTGATGCGTATGGGTTGTGTGTGAATATTTACCAATACAAAACGGACCAATTCAATAATACCTGCTAAAAATAAAGATTTCTTGAAATTTCAATTTCGACCATATGCACTAAATTTAAAGGATGCCTAGGTAACTACTCATGAAATATGGTATTTCTATAAAAATAAATTGTTTCCCAGGACAACTGGACTAGTCTTAAATTGAATCATATATCAAATAAATAAGGTGTAAAGTTAGGTCTTTTTTTAAATTTTAATAAGGTAAATACAATCATATCATACCCATATTTTCTCTCCTATTCATTCAACAAAATAAAAAATAAATAAATGAATATTTGTTCACTTTTATCTCATCATAAATATTTGGTCGGATGCAAATTATAAATTTCGATCCCCTGATTGCGACCACTTTATCTTCAGAAAAATCTTCGATGAGTAGTTTTCTACGTCGTTGCGGCGACTGTGGAGTATAAATTATTGATAACCTTAGTCTTAAAATAAATTTATTTGAAGAAAAATAAGAAAAATGGTAGTATATATTATATAATAACGCCATGCATGCGGTAGTGGATCTATATATATAATTTGGGTGCTCGAGCGCCCATTAACCTTGATGCATGTATAGTTATATAGAAAATTTGAGAAAACATATAAAAATTTACAAAAATATCCAATAATTTTATGTACAAAAATTTTTAAGCACCCACAAACTTAAATTCCTAAATCCGACACTTCATAAATTATGCATAATGATATAAACAAAATATTCTTTGGTCAAACAAAGACCTAACCCCTTCATTTCCTAGCTCTCGCCTTTTTTCTTCCTTCTTTTTTTTCAATAGAATGCGGCTTCTTCTCACCATTATATAACTAAAGTTGAAGGTCTTGTTCTTGATAGAGAAAGAACGACCACTTCTGCATTCTGCTTGGAACAAGAAAAATCTCCAATCTTTTATTTTTTTATTAAAATTAAGCACACAAAAAAAGAAGTAAGACTCGATCTTCTTTCTTCTCTTGGTCTGAATCTCCAAATTAGAATACCAATTAATCTTTTGTCATTCTCGCGTTCATATATACTGGAATATACATCTTTTTTTCAACCTATCTTCTTTCATTTTCAAGAATTCGAGTTACATAAATAGTAGGTTCACTACGTTTACTTCAACCTCCTTAAAGTTTATTCATTCATATTATTTCTCAAAGAAAAAACTATAGAA-3'.

2. and (2) carrying out enzyme digestion on the pCAMBIA1381 vector by using restriction enzyme EcoRI, recovering and purifying the enzyme digestion product by using an agarose gel recovery kit, connecting the enzyme digestion product with the RBOH promoter fragment obtained by amplification in the step (1) under the action of homologous recombinase, and then converting the connection product into escherichia coli DH5 alpha to obtain the recombinant vector pCAMBIA1381-RBOH containing the RBOH promoter.

3. And (2) carrying out enzyme digestion on the recombinant vector pCAMBIA1381-RBOH by using restriction enzyme Hind III, recovering and purifying the enzyme digestion product by using an agarose gel recovery kit, connecting the enzyme digestion product with the HCR1 gene fragment amplified in the step (1) under the action of homologous recombinase, and then converting the connection product into escherichia coli DH5 alpha to obtain the recombinant vector pCAMBIA1381-RBOH-HCR1 containing RBOH promoter and HCR1 gene.

4. The combined length of the HCR1 gene sequence and the empty vector sequence is taken as a positive cloning target band, and the PCR detection primers of the recombinant vector are as follows:

p1381-F:5'-GCCCTTTGGTCTTCTGAGACTG-3',SEQ ID NO.23;

p1381-R:5'-CTGCATCGGCGAACTGATC-3',SEQ ID NO.24;

Preparation of S2 and HCR1 gene transgenic tobacco material

Transgenic Bentonite was prepared using recombinant vector pCAMBIA1381-RBOH-HCR 1. The transgenic method is as follows:

(1) The method comprises the steps of infecting tobacco with agrobacterium, cutting leaf of the benthamiana into leaves with the square of 2cm ², and then placing the explant into agrobacterium GV3101 bacterial liquid (OD _600nm value is 0.4-0.6) containing recombinant vector pCAMBIA1381-RBOH-HCR1 for soaking for 10min. The explants were then transferred to sterile filter paper to blotter dry and placed in a differentiation medium, the basal medium of which was MS medium, also containing only 0.5mg/LNAA and 2 mg/L6-BA, for 3d in the dark.

(2) Differentiation and screening of transgenic tobacco explants were transferred to differentiation medium containing 300mg/L cephalosporin and 50mg/L and cultured at 25℃under light. And cutting off buds from a basal part when the length of the selected resistant buds is 1-1.5cm after the cluster buds grow out after the cluster buds are cultured every 3-4 Zhou Jidai times, and transferring the buds to a strong seedling culture medium for culturing, wherein the basic culture medium of the strong seedling culture medium is an MS culture medium and also only contains 0.1mg/L6-BA, 0.01mg/LNAA, 30mg/L hygromycin and 100mg/L cephalosporin.

(3) Rooting of transgenic tobacco, namely, picking out tobacco seedlings with grown roots, transferring the tobacco seedlings to a rooting culture medium for culture and detection, wherein the basic culture medium of the rooting culture medium is an MS culture medium and also only contains 30mg/L hygromycin and 100mg/L cephalosporin.

(4) The CTAB method is used for extracting genome DNA small samples.

(5) PCR identification of positive transgenic plants, namely, PCR detection is carried out by using specific primers, the size of the obtained target fragment is 350bp, and the HCR1 transgenic tobacco is obtained and named NbRBOH-HCR1.

The PCR detection primers are shown as SEQ ID NO.20 and SEQ ID NO. 21.

After the NbRBOH-HCR1 transgenic tobacco obtained above is cultured for one month, disease resistance detection is carried out.

Example 4 analysis of resistance of HCR1 transgenic tobacco to Phytophthora capsici

Resistance detection of S1, to generation NbRBOH-HCR1 transgenic tobacco

After NbRBOH-HCR1 transgenic tobacco is cultured for one month, wild tobacco (marked as WT) is used as a control, leaves at the same position are taken, then cross wounded spots are gently scratched at the inoculated positions by a needle, phytophthora capsici LT263 bacterial pieces are placed at the centers of the wounded spots, after 24 ℃ dark and moisture-retaining culture for 28 hours, phytophthora infestations are observed under an ultraviolet lamp and the disease areas are counted, 5 tobacco strains with obvious resistance (p < 0.05) are obtained through screening, and the tobacco strains are marked as RBOH:HCR1-3, RBOH:HCR1-6, RBOH:HCR1-9, RBOH:HCR1-14 and RBOH:HCR1-15 (see figure 3).

Resistance detection of S2 and T1 generation NbRBOH-HCR1 transgenic tobacco

The T0 generation NbRBOH-HCR1 transgenic tobacco seeds with obvious resistance to phytophthora capsici are collected, the resistance verification is carried out according to the method described in S1, the infection condition of the phytophthora capsici is observed, the disease incidence area and the biomass of relative pathogens are counted, and the result is shown in figure 4, wherein the leaves with the same number are different leaves of the same plant.

The results in fig. 3 and 4 show that NbRBOH-HCR1 transgenic tobacco lines 3, 6, 9, 14 and 15 show a reduced incidence of phytophthora capsici after inoculation, and that the phytophthora capsici disease area and relative pathogen biomass are significantly smaller than the control, indicating that HCR1 transgenic tobacco is significantly resistant to phytophthora capsici infection.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

The application of the HCR1 gene in improving phytophthora capsici resistance or cultivating transgenic plants resistant to phytophthora capsici is disclosed, wherein the amino acid sequence coded by the HCR1 gene is shown as SEQ ID NO.1, the plants are tobacco, and pathogenic bacteria of the phytophthora capsici comprise phytophthora capsici (Phytophthora capsici).
2. The use according to claim 1, wherein the nucleotide sequence of the HCR1 gene is shown in SEQ ID No. 2.