CN114703197B

CN114703197B - MeHsf23 gene for improving disease resistance of cassava and application thereof

Info

Publication number: CN114703197B
Application number: CN202210297846.0A
Authority: CN
Inventors: 李琳琳; 王超群; 耿梦婷; 陈银华; 张子奇; 郑婉茹
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2024-02-06
Anticipated expiration: 2042-03-25
Also published as: CN114703197A

Abstract

The invention provides a MeHsf23 gene for improving disease resistance of cassava and application thereof, wherein the nucleotide sequence of the MeHsf23 gene is shown as SEQ ID NO. 1, and the amino acid sequence of protein encoded by the gene is shown as SEQ ID NO. 2; the MeHsf23 gene and the protein thereof regulate and control the resistance of cassava to bacterial wilt bacteria, and the resistance of the cassava to the bacterial wilt bacteria can be changed through over-expression or silencing of the expression of the MeHsf23 gene, so that the method has important significance in researching the mechanism of resisting the bacterial wilt bacteria of the cassava and cultivating disease-resistant varieties.

Description

MeHsf23 gene for improving disease resistance of cassava and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a MeHsf23 gene for improving disease resistance of cassava and application thereof.

Background

The cassava (Manihot esculenta Crantz) tubers are rich in starch, the fresh tubers contain 30-40% dry matter, and the starch accounts for 85% of these dry matter. Cassava is a major food crop (Rossin et al, 2011) of 5 hundred million population in tropical regions and is mainly used for food, industry, fuel ethanol production and the like in China. It is an important source of heat for about billions of people in the hot zone and is an emerging biomass energy plant. In production, diseases are always one of important biological stress factors for limiting the yield of cassava, wherein bacterial wilt (Cassava Bacterial Blight, CBB) of cassava brings destructive attack to cassava production in a plurality of countries, and is one of internationally important quarantine diseases. At present, the main cultivated varieties of cassava in China are not resistant to bacterial wilt, and if the disease is developed in a large area, the cassava production in China is greatly influenced. Therefore, research on the mechanism of bacterial wilt resistance of cassava and cultivation of disease-resistant varieties become a real problem to be solved urgently in the continuous healthy development of the cassava industry, and an effective method for preventing and treating the bacterial wilt of cassava is not available at present.

In the process of resisting pathogen infection of plants, the expression of disease resistance function genes is regulated by transcription factors, which is a key link of plant immune response. Heat shock protein transcription factor Hsf (Heat shock transcription factors) is a very important class of stress-related transcription factors, encoded by a multiple gene family. Hsf not only can regulate and control the expression of Hsp genes through specifically combining HSE (Heat shock element) cis-acting elements in heat shock protein (Heat shock protein, hsp) gene Hsp promoters, so as to enhance the heat resistance of plants, but also can regulate and control the expression of a plurality of stress-resistant genes, so that the capability of the plants for resisting biological and abiotic stress is improved.

Plant Hsf proteins are encoded by a gene family of about 16-56 members, which has been found in many plant species. Hsf is an evolutionarily conserved transcription factor, which can be classified into class A, class B and class C, and plant Hsf is used as a terminal component of signal transduction to mediate activation of various genes on various stresses. There have been many studies on the function of the HsfA subfamily in thermal, drought, salt and oxidative damage reactions. However, the HsfB and HsfC subfamilies are much less known.

Hsf generally comprises several conserved domains: an N-terminal DNA Binding Domain (DBD) responsible for the recognition of the thermal stress-inducing gene promoter of several (HSES, 50-GAAnnTTC-30); (two) two-part oligomerization domain (HR-A/B); (III) other domains, including Nuclear Export Signal (NES), nuclear Localization Signal (NLS) and C-terminal activator AHA. In plant stress responses, transcription factors play an important role by linking upstream protein kinases to downstream gene expression. Currently, there is a great deal of research showing that HSF can significantly improve the ability of plants to cope with abiotic stresses, such as: the rice over-expression TaHSFA4a gene can improve the tolerance of plants in a high-concentration heavy metal environment, the tomato over-expression SlHsfA1 gene can improve the heat resistance of plants, the corn over-expression ZmHSF04 gene can improve the tolerance of plants to high-salt stress, and the Arabidopsis over-expression of different HSF genes can improve the tolerance of plants to different adversity. In arabidopsis, the overexpression line of the AtHSFA1b shows larger seed yield and water productivity, and tolerance to drought and biological stress, the AtHsfA6a of the arabidopsis has remarkable induction on exogenous abscisic acid (ABA) and drought, the ABA-dependent signal path has positive regulation effect on salt and drought stress, and the overexpression of the HSFA2 gene of the arabidopsis can improve the tolerance of plants to high-salt, osmotic stress and high-oxidation environments. Overexpression of the lily HsfA3s gene in Arabidopsis can change the metabolism level of proline in Arabidopsis plants to improve the tolerance of the plants to high salt stress. It can be seen that HSF has different stress resistance in different plants, and that different HSF proteins also have different stress resistance in the same plant.

Disclosure of Invention

Therefore, the invention provides a MeHsf23 gene for improving disease resistance of cassava and application thereof.

The technical scheme of the invention comprises the following contents:

in a first aspect of the invention, the invention provides a MeHsf23 gene for improving disease resistance of cassava, the nucleotide sequence of the MeHsf23 gene is shown as SEQ ID NO. 1, and the amino acid sequence of protein coded by the MeHsf23 gene is shown as SEQ ID NO. 2.

In a second aspect, the invention also provides an application of the MeHsf23 gene and the protein encoded by the same in regulating and controlling the resistance of cassava to cassava bacterial wilt bacteria (Xanthomonas axonopodis pv. Manihot HN11, xamHN 11)

Preferably, the regulation of the resistance of the cassava to the bacterial wilt bacteria is to increase or decrease the resistance of the cassava to the bacterial wilt bacteria.

Preferably, the method for improving the resistance of cassava to cassava bacterial wilt bacteria is to construct a MeHsf23 expression vector to transform cassava plant materials.

Preferably, the method for reducing the resistance of cassava to bacterial wilt bacteria is to construct a silencing vector of MeHsf23 to transform cassava plant material.

Further, the plant may also be an arabidopsis plant material.

Preferably, the silencing vector is a VIGS vector.

Preferably, the vector is a recombinant plasmid, recombinant bacterium or transgenic cell.

Preferably, the transformation method is a method of transforming plant cells, which is conventional in the art, without limitation, and the transformation method used in one embodiment of the present invention is an agrobacterium-mediated method.

Preferably, the plant material is protoplasts, suspension cells, leaf discs, leaves and/or stem segments of a plant.

In a third aspect of the invention, the invention also provides the application of the MeHsf23 gene in biological breeding.

Preferably, the biological breeding is cultivation of transgenic plants.

Preferably, when the transgenic plant is a disease resistant transgenic plant, the breeding method comprises the steps of: enhancing the expression of the MeHsf23 gene in the target plant to obtain the transgenic plant with disease resistance higher than that of the target plant.

Preferably, when the transgenic plant is a disease-susceptible transgenic plant, the breeding method comprises the steps of: inhibiting the expression of the MeHsf23 gene in the target plant results in a transgenic plant having a lower disease resistance than the target plant.

Experiments prove that after the MeHsf23 gene is silenced, the area of the disease spots is obviously increased under the infection of bacterial wilt bacteria, which indicates that silencing the MeHsf23 gene can cause the reduction of the resistance of cassava to bacterial wilt, and the expression of the MeHsf23 gene is regulated to change the resistance of cassava to bacterial wilt.

Compared with the prior art, the invention has the beneficial effects that:

the MeHsf23 gene can directly change the resistance of cassava to bacterial wilt germs by regulating and controlling the expression of the MeHsf23 gene, and an over-expression or silencing vector can be constructed based on the MeHsf23 gene and is directly used for biological breeding in the aspect of cassava bacterial wilt resistance, so that the MeHsf23 gene has important significance in researching the mechanism of cassava bacterial wilt resistance and cultivating disease-resistant varieties.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only preferred embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a cloning and amplifying band of a cassava MeHsf23 gene;

FIG. 2 shows a hydrophilic analysis of cassava MeHsf23 protein; and (3) injection: negative values indicate hydrophilicity, positive values indicate hydrophobicity;

FIG. 3 is a schematic diagram of the structure of introns and exons of the cassava MeHsf23 gene sequence;

FIG. 4 shows the conserved structure and analysis result of the cassava MeHsf23 protein;

FIG. 5 is a model for predicting the advanced structure of the cassava MeHsf23 protein;

FIG. 6 shows subcellular localization of cassava MeHsf23 in cassava in accordance with the present invention; and (3) injection: confocal observation of luminescence: green fluorescence represents fluorescence emitted by GFP-MeHsf23 fusion proteins;

FIG. 7 shows the results of detection of the expression level of the target gene at various times after infection with XamHN11 of the present invention;

FIG. 8 is a graph showing the change in cassava leaf phenotype following VIGS silencing according to the present invention, wherein part A is a positive control, part B is a negative control, and part C is a pCsCMV-MeHsf23 panel;

FIG. 9 shows the results of detecting the expression level of a target gene after the silencing of VIGS according to the present invention, as follows: * Indicating significant differences (P < 0.05);

FIG. 10 shows cassava leaf phenotypes at various time points after infection with XamHN11 according to the present invention;

fig. 11 is a bar graph of the plaque area of cassava leaf at various time points after infection with xahn 11 according to the present invention, notes: * Indicating significant differences (P < 0.05).

Detailed Description

In order to better understand the technical content of the present invention, a specific embodiment is provided below to further explain the present invention.

The plant materials used in the following examples of the present invention are the cassava variety SC8, the tobacco variety burley.

The strain used in the following examples of the present invention was E.coli strain Trans5α; agrobacterium strain GV3101; cassava bacterial wilt bacteria (Xanthomonas axonopodis pv. Manihot HN11, xamh 11).

The plasmid vectors used in the following examples of the present invention are subcellular localization vectors pCAMBIA1300-GFP-MeHsf23, pCAMBIA1300-GFP; the VIGS silencing vector pCsCMV-B, pCsCMV-A, pCsCMV-MeHsf23.

EXAMPLE 1 cloning of the MeHsf23 Gene and bioinformatics analysis

1.1 Cloning of MeHsf23 Gene

Extracting total RNA of cassava SC8 leaves by adopting a TRIzol method, detecting the concentration and purity of the extracted total RNA by using a micro-spectrophotometer, detecting the quality of the extracted total RNA by 1% agarose gel electrophoresis, reversely transcribing the total RNA into cDNA by adopting a RevertAidTM First Strand cDNA Synthesis Kit kit, designing an amplification primer MeHsf23-F by adopting Premier 5.0 software according to the sequence information of the cassava MeHsf23 gene: GTCGAATTCCCCTCCCCCTTAATTTAATT, meHsf23-R: CATGGATCCGAGAAAAGTGTCGAACCATT. And (3) performing PCR amplification by taking cDNA obtained by reverse transcription of the total RNA as a template, cloning the coding region sequence of the cassava MeHsf23 gene, and obtaining a target fragment with the size of about 750bp after the amplification is finished (figure 1). The reaction system is as follows: taKaRa LA Taq 0.2. Mu.L, 10xLA PCR Buffer 2. Mu.L, dNTP mix 4. Mu.L, upstream and downstream primers each 0.5. Mu.L, template 2. Mu.L, and ddH added ₂ O was added to 20. Mu.L. The reaction procedure is: pre-denaturation at 94℃for 3min; cycling for 35 times at 94 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 1 min; and then extending at 72 ℃ for 10min. And (3) recovering a target fragment by using a PCR product purification recovery kit, connecting the target fragment to a pEASY-Blunt vector, transforming into escherichia coli DH5 alpha competent, selecting positive monoclonal to perform colony PCR detection, and extracting the pEASY-Blunt-MeHsf23 plasmid by using a plasmid small-scale rapid extraction kit after sequencing by using a biological biotechnology company. According to the sequencing result, NCBI online websites are utilized for sequence alignment, and the sequence alignment is dividedThe open reading frame was analysed and showed that the full length 729bp of the MeHsf23 gene was encoded by 2 exons.

1.2MeHsf23 Gene protein bioinformatics analysis

1.2.1 Physiological and biochemical properties of MeHsf23 Gene proteins

Physicochemical properties of the MeHsf23 protein such as amino acid number, molecular formula, molecular weight, isoelectric point, instability coefficient, fat index and average hydrophilicity coefficient were predicted from on-line website ExPASy (https:// web. ExPASy org/protparam /), and the results showed that the MeHsf23 protein theory formula: c (C) ₁₂₂₄ H ₁₉₃₄ N ₃₅₀ O ₃₈₃ S ₉ Theoretical relative molecular mass: 27.9kDa, theoretical isoelectric point pI:7.59. the protein is rich in: glutamic acid Glu (10.0%), leucine Leu (9.1%), lysine Lys (8.7%), serine Ser (8.3%), arginine (7.5%), threonine (6.6%), asparagine Asn (5.8%). The hydrophilicity index ranged from-2.744 to 2.222 (negative values for hydrophilicity and positive values for hydrophobicity), and the average hydrophilicity index of-0.880, indicating that the protein was better in water solubility (FIG. 2). Theoretical instability coefficient: 56.86, which is an unstable protein. The fat index was 65.98.

The structural features of MeHsf23 gene were analyzed using GSDS online website and the results showed that the gene contained 2 exons and 1 intron (fig. 3).

Domain analysis of MeHsf23 (FIG. 4) was performed using the NCBI database on-line website CDD (https:// www.ncbi.nlm.nih.gov/Structure/bwrpsb/bgi), and the protein tertiary Structure was predicted by the SWISS-MODEL (https:// swissmodel. Expasy/interactive) website (FIG. 5) and found that MeHsf23 contains the conserved domain of the HSF gene family.

Subcellular localization prediction was performed on MeHsf23 protein using on-line website Plant-mPloc SEfvEf (http:// www.csbio.sjtu.edu.cn/bioif/Plant-multi /), predicting that MeHsf23 protein might be localized to the nucleus.

1.2.2 subcellular localization

The website prediction result shows that the cassava MeHsf23 protein is positioned in the cell nucleus. To further verify the accuracy of the predicted results, recombinant vectors pCAMBIA1300-GFP-MeHsf23, pCAMBIA1300 were usedGFP empty transformed Agrobacterium competent cell GV3101. Single colonies were picked and cultured in 5mL LB liquid medium containing kana and rifampicin at 28℃for 18h at 200 rpm. mu.L of overnight culture broth was taken and cultured to OD at 28℃in 50mL of LB liquid medium containing kana and rifampicin antibiotics at 200rpm ₆₀₀ The cells were collected by centrifugation at 4000rpm for 5min at a value of about 0.8, and the supernatant was discarded. 20mL of buffer (10 mmol/L MgCl) was added ₂ 10mmol/L MES) was resuspended, centrifuged at 4000rpm for 5min and the supernatant was discarded. 20mL of buffer (10 mmol/L MgCl) was added again ₂ 10 mmol/LMES) was resuspended, centrifuged at 4000rpm for 5min, and the supernatant was discarded. Finally adding plant injection (10 mmol/L MgCl) ₂ 10mmol/L MES, 150. Mu. Mol/L acetosyringone) to re-suspend the bacterial cells, and adjusting the OD ₆₀₀ The value is about 0.8, and the mixture is kept stand for 2 to 3 hours at room temperature to obtain bacterial liquid containing GFP-MeHsf23 fusion protein. The empty plasmid pCAMBIA1300-GFP was used as a control.

Selecting good-growth tobacco herb smoke growing for 4-5 weeks, taking a proper amount of bacterial liquid by using a 1mL sterile injector, injecting the bacterial liquid into the back of tobacco leaves, and marking an injection area. Culturing the injected tobacco at 24 ℃ for 24 hours, taking tobacco leaves in an injection area, placing the leaves on a glass slide, dripping less than clean water for tabletting, observing the presence or absence of fluorescence under a laser confocal microscope, and photographing and recording.

Subcellular localization results As shown in FIG. 6, the localization of the MeHsf23 protein in cassava was determined by observing the distribution of GFP-MeHsf23 fusion protein in cells under LUYOR-3415RG and laser confocal microscope. Under a laser confocal microscope, the green fluorescence emitted by GFP in no-load is observed in cytoplasm and cell nucleus of tobacco, and the green fluorescence emitted by GFP in pCAMBIA1300-35S-MeHsf23-GFP is only distributed in cell nucleus of tobacco leaf, so that the localization of MeHsf23 protein in cell nucleus is indicated.

EXAMPLE 2 analysis of expression Pattern of MeHsf23 Gene after XamHN11 treatment

And (3) taking cassava inoculated with the cassava bacterial wilt bacteria XamHN11, extracting total RNA of the cassava 3h, 6h, 1d, 3d and 6d after inoculation, and carrying out reverse transcription to obtain cDNA. Fluorescent quantitative PCR primers qRT-MeHsf23-F were designed using Premier 5.0 software: GACATGTTTCGAAAGGGTGAAA; qRT-MeHsf23-R: TAGTGAGCTCCGAACTCAATAC, the fluorescent quantitative PCR reaction system is as follows: TB Green Premix Ex Taq II (2X) 10. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 2. Mu.L of cDNA, and ddH were added ₂ O was added to 20. Mu.L. The reaction procedure is: pre-denaturation at 95 ℃ for 30s;40 cycles included denaturation at 95℃for 5s, annealing at 58℃for 30s, extension at 72℃for 30s, and extension at 72℃for 5min. Three replicates were set for each sample, with EF1 alpha as the reference gene. The gene expression value was calculated using the 2- ΔΔCt method.

As shown in the figure 7, the results of the cassava SC8 leaves inoculated with XamHN11 bacteria show that the expression level of the 6d MeHsf23 gene is obviously improved after inoculation, and the MeHsf23 gene is involved in regulating and controlling the bacterial wilt of cassava.

EXAMPLE 3 pCsCMV mediated silencing of MeHsf23 gene

3.1 VIGS vector pCsCMV-NC infects cassava

The competent cells of Agrobacterium GV3101 (pSoup-p 19) were transformed with the recombinant vector pCsCMV-MeHsf23, the positive control pCsCMV-B, which was Sup>A recombinant plasmid containing the viral gene and the indicator gene, and the negative control pCsCMV-A, which was Sup>A plasmid containing only the viral gene, and then positive clones identified by colony PCR were picked up into 5mL LB (50. Mu.g/mL kanamycin+25. Mu.g/mL rifampin) liquid medium, cultured at 28℃overnight at 220 rpm. mu.L of overnight culture broth was taken in 50mL of LB (50. Mu.g/mL kanamycin+25. Mu.g/mL rifampicin) liquid medium, and incubated at 28℃and 220rpm to OD ₆₀₀ The value was 0.75. The bacterial solution was transferred to a 50mL centrifuge tube, centrifuged at 4000rpm for 5min, and the supernatant was discarded. Adding proper amount of buffer solution (10 mmol/L MgCl) ₂ 10mmol/L MES) was resuspended, centrifuged at 4000rpm for 5min, the supernatant discarded and repeated twice. 25mL of plant injection (10mM MES+10mM MgCl) was added ₂ +200. Mu.M acetosyringone) was resuspended and allowed to stand for 3h at room temperature under dark conditions. And selecting cassava SC8 with good growth vigor and consistent state, pressing and infiltrating the bacterial liquid to the back of cassava leaves by using a 1mL syringe, and starting to observe the phenotype change after 30d, and photographing and recording.

As shown in FIG. 8, the positive control containing the indicator gene resulted in a plant with impaired chlorophyll synthesis, a significantly blushing phenotype of the new leaf, and a pCsCMV-B-dependent plant ⁺ The new cassava leaf appeared to have a green-turning phenotype after about 30d infection, and the negative control and experimental group without the indicator gene did not change, which indicates that the pCsCMV-NC system can effectively produce silencing effect on cassava.

3.2 Silencing effect detection of MeHsf23 gene

The 3.1 experimental group and negative control cassava leaves after 30d infection are taken, RNA is respectively extracted, cDNA is obtained through reverse transcription, the template is used for detecting the expression quantity of the MeHsf23 gene by using a qRT-PCR technology, and the primer is qRT-MeHsf23-F, qRT-MeHsf23-R, and the reaction system and the reaction program of the qRT-PCR technology are the same as those of example 2. Three replicates were set for each sample, with EF1 alpha as the reference gene. The gene expression value was calculated using the 2- ΔΔCt method.

The results are shown in fig. 9, and the expression level of MeHsf23 in the experimental group is significantly reduced, which indicates that the MeHsf23 gene is effectively silenced.

3.3 Bacterial wilt and disease statistics inoculated on VIGS silencing plants

The bacterial wilt pathogen XamHN11 of cassava stored at-80℃was streaked on LPGA solid medium and cultured upside down at 28℃for 2d. With 10mM MgCl ₂ Washing off the cells, centrifuging at 4000rpm for 5min to collect the cells, and using 10mM MgCl ₂ The cells were washed two to three times and the bacterial concentration was adjusted to 1108CFU/mL (OD ₆₀₀ 0.1). The negative control was 10mM MgCl ₂ Sucking bacterial liquid by a 1mL syringe with a needle removed, propping the front surface of the blade with a finger, pressing and infiltrating the bacterial liquid to the back surface of the blade by the syringe, observing the spreading condition of the water stain-like disease spots of the cassava blade at different time points after inoculation is completed, and photographing and recording.

Removing 0d, 3d and 6d cassava leaves inoculated with XamHN11 with a 1cm diameter puncher, placing in a sterilized mortar, adding 1mL MgCl ₂ Grinding thoroughly, and diluting different concentrations. 100 mu L of the supernatant of 3 serially diluted concentration gradients is coated on the LPGA solid culture medium, three plates are coated on each gradient, the colony number is counted after inversion culture is carried out for 24 hours at 28 ℃, and the viable bacteria number in the leaf is calculated according to the colony number on the plates.

As shown in fig. 10 and 11, the cassava leaf has water-stain-like disease spots at the time of inoculation for 3d, the disease spot spreading area of the experimental group is larger than that of the control group, and the disease spot spreading area of the experimental group is further enlarged at the time of 6d and is obviously larger than that of the control group. The change of leaf phenotype after inoculation shows that silencing of the MeHsf23 gene can result in reduced resistance of cassava to bacterial wilt, which indicates that expressing the MeHsf23 gene can improve resistance of cassava to bacterial wilt.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

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Claims

The application of the MeHsf23 gene or the protein coded by the MeHsf23 gene in reducing the resistance of cassava to bacterial wilt bacteria is characterized in that the nucleotide sequence of the MeHsf23 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein coded by the MeHsf23 gene is shown as SEQ ID NO. 2;

the method for reducing the resistance of cassava to cassava bacterial wilt bacteria is to construct a silencing vector of MeHsf23 to transform cassava plant materials.
2. The use according to claim 1, wherein the vector is a recombinant plasmid, a recombinant bacterium or a transgenic cell.
3. The use according to claim 1, wherein the plant material is protoplasts, suspension cells, leaf discs, leaves and/or stem segments of a plant.