CN117844818A - Corn TIR family gene ZmTIR3 and application thereof - Google Patents
Corn TIR family gene ZmTIR3 and application thereof Download PDFInfo
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Abstract
The invention discloses a corn TIR family gene ZmTIR3, wherein the genome nucleotide sequence of the gene ZmTIR3 is shown as SEQ ID No.1, and the protein coding sequence is shown as SEQ ID No. 2. The invention also discloses application of the corn TIR family gene ZmTIR3 in corn small leaf spot disease resistance breeding and tobacco resistance breeding. The application of the corn TIR family gene ZmTIR3 as an ETI related cell necrosis induction factor in basic research of plant immunity. The activation of similar immune responses of ZmTIR3 in different species indicates that the ZmTIR3 has important application value in crop disease-resistant breeding.
Description
Technical Field
The invention belongs to the field of molecular genetics, and particularly relates to a corn TIR family gene ZmTIR3 and application thereof in improving plant disease resistance.
Background
The people are the national origin and the food is the daily. Grain safety plays an important role in economic development and social stability. With the increase of population, the grain safety problem is particularly remarkable, and how to ensure the stable supply of Chinese grains is a great challenge for researchers. The national statistical office data shows that the corn yield in 2019 reaches 5215 hundred million jin, which is 42.5% of the annual grain yield, and is the first large grain crop in China. Besides being used as grain, corn can also be used as feed and industrial raw materials for processing starch and ethanol, and is an important component of Chinese grain territories. In recent years, the demand of corn is continuously increased, the market supply is not required, the import quantity is rapidly increased, and higher requirements are put on the corn production in China.
Corn production is often severely affected by biotic stress, including bacterial, fungal, and viral diseases, among others. In recent years, corn fungus diseases occur increasingly frequently, resulting in huge yield losses. The prediction data of the national statistical bureau show that the occurrence areas of southern rust and large spot in 2023 can reach 5500 mu and 8000 mu respectively, and the occurrence areas of small spot and brown spot break through 3500 mu. At present, prevention and control of corn fungus diseases are mainly started from two aspects of breeding of disease-resistant varieties and chemical prevention and control, but due to rapid variation of pathogenic bacteria, drug-resistant strains rapidly appear, and meanwhile, excellent disease-resistant genes are overcome. Therefore, the cultivation of novel broad-spectrum disease-resistant varieties is more important. The traditional corn breeding method needs longer time for improving an excellent variety, and consumes huge manpower and material resources. The advent of transgenic and genomic editing techniques has accelerated crop improvement, but the use of both techniques has relied on the discovery of key functional genes and the mechanism resolution. Therefore, the development of new key disease-resistant related genes is important for disease-resistant breeding of corn.
The plant immune system can be divided into two different layers, PTI and ETI. Cell surface Pattern Recognition Receptors (PRRs) in plants can recognize pathogen/injury/microorganism related molecular patterns (PAMPs/DAMPs/MAMPs) and activate plant pattern-triggered immunity (PTI) thereby limiting pathogen infestation. While some pathogens evolve, secreting some effector proteins to evade or inhibit PTI, resulting in the occurrence of effector protein-induced pathogenicity (ETS). Accordingly, plants have evolved intracellular binding and leucine rich repeat receptor (NLR) encoded by the R gene to recognize effector proteins and activate ETI, where it is possible for pathogens to inhibit or evade ETI by producing new effector proteins. These evaded pathogens are in turn likely to be recognized by the NLR encoded by the new R gene, thereby eliciting a new round of ETI immunity.
NLR, which can trigger ETI reactions, is in turn divided into TNL and CNL according to their N-terminal domains, where TNL consists of an N-terminal TIR domain, an intermediate NB-ARC domain and a C-terminal LRR domain. The LRR domain is involved in direct or indirect recognition of effector proteins, and the NB-ARC domain has ATP binding activity and acts as a switch for NLR activation; upon NLR activation, the N-terminal TIR domain acts as the signal domain for downstream response.
TIR domain proteins are integral to the plant immune system, directly or indirectly involved in the plant immune network, and trigger the initiation of downstream EDS 1-dependent immune signals. Classical TNL proteins are present in most dicotyledonous plants, but not in monocotyledonous plants such as maize. There are studies showing that there are 4 classes of conserved TIR proteins in plants, in addition to classical TNL, TNP and sequenced TIR-only, whereas sequenced TIR-only proteins in monocots can trigger EDS 1-dependent cell death in tobacco. The related functions of TIR domain proteins in maize have not been reported so far.
Disclosure of Invention
Aiming at the current research situation, the invention aims to provide a corn TIR family gene ZmTIR3 and application thereof in improving disease resistance.
The invention discloses a corn TIR family gene ZmTIR3, which is characterized in that: the genome nucleotide sequence of the gene ZmTIR3 is shown as SEQ ID No.1, and the protein coding sequence is shown as SEQ ID No. 2.
In view of the fact that the nucleotide sequence of the gene in the patent is easily modified or mutated by a directional optimization method or a point mutation method and the like by a person skilled in the art, the nucleotide sequences which have the homology of more than or equal to 85% with the CDS sequence of the gene provided by the invention and still have the function of the gene after being artificially modified are all sequence derivatives of the gene in the invention, which are equivalent to the sequences in the invention, and belong to the protection category of the patent.
The invention also discloses application of the corn TIR family gene ZmTIR3 in breeding of corn small leaf spot disease resistance.
The invention also discloses application of the maize TIR family gene ZmTIR3 in tobacco disease-resistant breeding.
The invention also discloses application of the corn TIR family gene ZmTIR3 in enhancing the immune response of Nicotiana benthamiana.
The invention also discloses application of the corn TIR family gene ZmTIR3 as an ETI-related cell necrosis induction factor in basic research of plant immunity.
The corn ZmTIR3 gene provided by the invention is a novel gene which has not been reported yet. Based on the fact that ZmTIR3 contains a TIR domain, the gene is presumed to be possibly involved in the immune response of plants, and the applicant discovers through molecular biological means that the heterologous expression of ZmTIR3 in Nicotiana benthamiana can activate disease resistance phenotypes such as cell necrosis, active oxygen burst and the like. The qRT-PCR result shows that the ZmTIR3 gene is obviously induced and expressed by the small spot bacteria, and meanwhile, the small spot bacteria inoculation test is carried out on the EMS mutation mutant of the ZmTIR3, so that the resistance of the mutant to the small spot bacteria is obviously weakened, the positive correlation between the ZmTIR3 and the small spot bacteria resistance of corn is shown, and the application prospect of the mutant in the aspect of corn disease resistance breeding is predicted. Compared with the prior art, the invention can obtain the following technical effects:
1) The function of the corn ZmTIR3 gene provided by the invention is not reported in the related report, the gene belongs to a TIR-domain-containing gene family, the gene contains a TIR domain, the gene is obviously infected and expressed by the small spot bacteria, and mutant analysis shows that mutation of the ZmTIR3 can lead to weakening of corn small spot disease resistance.
2) The heterologous expression of ZmTIR3 in the lamina of the Nicotiana benthamiana activates the immune response of the Nicotiana benthamiana, including ROS burst and the like, and the gene has application value for improving disease-resistant breeding of the Nicotiana benthamiana.
3) The tobacco leaf heterologous expression of ZmTIR3 can induce the cell necrosis of the Nicotiana benthamiana, and the gene can be used as an ETI-related cell necrosis induction factor for basic research.
Drawings
Fig. 1: domain prediction results for ZmTIR3
Fig. 2: zmTIR3 is obviously infected and induced to express by small spot bacteria
Fig. 3: zmTIR3 is positively correlated with the disease resistance of maize plaque
A. Leaf phenotype after wild type and mutant Zmtir3-1 inoculation; B. post-inoculation lesion area statistics
Fig. 4: zmTIR3 elicits a pronounced cell necrosis phenotype on tobacco leaves
GFP served as a negative control. The numbers in brackets below the gene numbers represent the number of necrotic leaves/total injected leaves
Fig. 5: zmTIR3 elicits a distinct active oxygen burst phenotype on tobacco leaves
GFP served as a negative control. Three days after transient expression, the leaves were stained with DAB, bleached and photographed.
Detailed Description
The present invention will be further illustrated by the following specific examples, but the following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
The procedure described in the examples below is a routine experimental procedure unless otherwise specified. The reagents, vectors and strains are all known as sales channels unless otherwise specified.
Example 1, maize TIR3 protein Structure prediction
The present invention predicts the protein structure of maize TIRs using a protein structure prediction website such as Pfam, SMART, CDD, from which ZmTIR3 containing only one TIR domain was selected for subsequent study (fig. 1).
Example 2: extraction and reverse transcription of total RNA of corn
1) Taking 0.1g of corn sample, quickly freezing with liquid nitrogen, vibrating once at 45Hz for 30s, then putting into the liquid nitrogen to quickly freeze again for 1min, repeatedly vibrating once at 45Hz for 30s, and putting back into the liquid nitrogen;
2) Taking out the ground sample from the liquid nitrogen, beating the cover with forceps for several times to make the powder on the cover fall into the bottom of the tube, then rapidly opening the cover, recovering the temperature at room temperature for 1min (the powder cannot be changed into green), adding into 1ml Trizol, vigorously mixing, standing at room temperature for 20min, adding 1/5 volume of chloroform, vigorously mixing to form emulsion, and standing (ice bath) for layering. Centrifuge 12000g for 15min at 4 ℃.
3) Transferring the supernatant to a new centrifuge tube, adding equal volume of (-20 deg. precooled) isopropanol, mixing, standing at-20 deg. C.
4) Taking out, centrifuging for 15min at 12000 g. The isopropyl alcohol was decanted, 80% ethanol was added, the tube was carefully covered with a cap and rolled several times, the ethanol was decanted and the residual liquid was sucked off with a gun head.
5) Blowing on the super clean bench for about 5min (until the sediment becomes colorless and transparent).
6) Adding proper amount of DEPC treated ultrapure water, stirring with a gun head, and mashing to precipitate (or violently flicking the bottom of the tube with fingers).
7) Slightly centrifuging. Taking supernatant into new test tube, packaging, detecting, and storing the rest in-80 deg.C refrigerator.
8) Reverse transcription of the extracted RNA was performed using the reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind) (Northenon) according to the instructions to obtain full-length cDNA for subsequent gene cloning
Example 3: cloning of corn TIR family gene ZmTIR3 and agrobacterium transformation
1) The cDNA sequence of the gene was obtained by searching the maize MaizeGDB database (https:// www.maizegdb.org /) according to the sequence number Zm00001d021820 of the ZmTIR3 gene, and was used for primer design and screening of the gene clone.
2) PCR amplification PRIMERs were designed using PRIMER5.0 software according to the sequences described above.
The upstream primer is 5'AGAGGACACGCTCGAGATGGCGTCGTCCGCGTTGC 3';
the downstream primer was 5'TTAATTAACCCCATAAGCTTCAGCCTCGAAAGGATCATCTGG 3'.
3) The cDNA obtained in example 2 was used as a template for cloning of ZmTIR3 gene.
The reaction system is as follows:
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 57℃for 30s, extension at 72℃for 1min, and cycling for 35 times; extending at 72℃for 5min. The amplified product was subjected to agarose gel electrophoresis.
4) After electrophoresis, the target band was recovered by using a DNA fragment recovery kit (OMEGA), and specific procedures for gel recovery were described in the specification. The recovered fragment of interest was ligated into the plant expression vector pART27-4myc (from literature, available) using the ClonExpress IIOne Step Cloning Kit kit (Norflu). Coli was then transformed, plated on LB plates containing 100ug/mL spectinomycin, and incubated overnight at 37 ℃. After the monoclonal is grown, the monoclonal is selected for colony PCR identification, and all the primers are vector primers at two ends of the insertion site. Clones with the correct size of the PCR detection fragment were selected for sequencing.
5) The vector with correct sequence is transformed into competent cells of agrobacterium GV3101 by a heat shock transformation method, and after resuscitating for 3 hours at 28 ℃ and 200rpm, the vector is coated on an LB plate containing a proper amount of three antibiotics of rifampicin, spectinomycin and gentamicin, and is cultured for about 48 hours at 28 ℃, and after monoclonal growth is carried out, the subsequent experiment is carried out.
Example 4 ZmTIR3 has a positive correlation with resistance to Erysiphe zeylanica
To test whether ZmTIR3 is involved in the disease resistance process of maize, we an EMS mutant ZmTIR3-1 of ZmTIR3 (commercially available, a mutant that was terminated in advance, where the mutant was used only for the disease resistance test of ZmTIR 3) and performed the test for resistance to plaque bacteria.
The specific method comprises the following steps: activating corn small spot bacteria on a solid V8 culture medium, culturing for 5-7 days in an inverted incubator at 28 ℃, preparing spore suspension after hyphae grow on a flat plate basically, uniformly spraying the spore suspension onto corn leaves in a 4-leaf period, culturing for 2-3 days in a moisturizing mode, photographing, and calculating the area of the disease spots by using imageJ. The inoculation test results show that compared with the wild type, the mutant Zmtir3-1 leaf spot is denser, and the resistance to the leaf spot is remarkably reduced (figure 3A). The lesion area statistics showed that the lesion area of the mutant Zmtir3-1 leaf was significantly higher than that of the wild type (fig. 3B). This suggests that ZmTIR3 has a positive correlation with resistance to leptosphaeria zeylanica.
Example 5, expression of ZmTIR3 was significantly induced by plaque infection
The four leaf stage leaves of maize were inoculated with a spore suspension of Spot bacteria according to the inoculation method of example 4, then sampled at time points, followed by extraction of total RNA by the method of example 2, and then reverse transcription of the extracted RNA with a reverse transcription kit. Finally, the expression level of ZmTIR3 at different time points after inoculation is detected by using an ABclonal 2X Universal SYBR Green Fast qPCR Mix kit for RT-qPCR experiment.
qRT-PCR results showed that the expression of ZmTIR3 was significantly induced by the infection with plaque bacteria (FIG. 2).
Example 6 heterologous expression of ZmTIR3 by tobacco lamina can trigger a cell necrosis response
To investigate whether maize ZmTIR3 could trigger a significant cell necrosis response on tobacco leaves, we performed transient expression experiments on tobacco leaves.
The specific operation is as follows: agrobacterium containing the ZmTIR3 gene overexpression vector was grown overnight in 3mL LB (with the addition of the corresponding antibiotic) at 28℃in a shaker at 250 rpm. Then, the mixture was removed, poured into a 2mL centrifuge tube, centrifuged at 4000g for 3min, and the supernatant was discarded, and 1mL MES (containing acetosyringone) solution was added to the 2mL centrifuge tube to resuspend Agrobacterium. 200uL of the resuspension was placed in a new 2mL centrifuge tube, MES solution was added to 2mL, the walls of the flick tube were mixed well, and the OD was measured. According to the measured OD value, calculating the required heavy suspension when the OD value is 0.4, sucking the required heavy suspension into a new 2mL centrifuge tube, adding MES solution to 2mL, uniformly mixing the light bullet tube walls, then injecting the agrobacterium solution with the OD value of 0.4 into Nicotiana benthamiana leaves growing to 4 weeks old by using a syringe, and carrying out normal growth under light after being treated for 12 hours in the dark. Tobacco leaves were observed after 5 days.
The results indicate that one leaf expressing ZmTIR3 exhibited a distinct necrotic phenotype (fig. 4)
EXAMPLE 7 heterologous expression of ZmTIR3 by tobacco lamina can trigger intense bursts of reactive oxygen species
To investigate whether corn ZmTIR3 could trigger a significant reactive oxygen species burst on tobacco leaves, we performed transient expression experiments on tobacco leaves and DAB staining experiments on their injection areas.
The specific operation is as follows: firstly, DAB staining solution is prepared. Adding 50mg DAB (DAB insoluble, grinding DAB into powder after finishing DAB granule, adding into conical flask) and 45mL ddH into 100mL conical flask 2 O, then cover the flask with tinfoil (because DAB is photosensitive), add a small magnetic stirrer to the flask, reduce PH to 3.0 with 0.2M HCl (dissolve DAB). 25ul Tween 20 (0.05% v/v) and 2.5mL 200mM Na were added to the stirred DAB solution 2 HPO 4 10mM Na was obtained 2 HPO 4 DAB staining solution. Secondly, DAB staining is carried out, tobacco leaves after 3 days of agrobacterium injection are taken down, the front side of the tobacco leaves is upwards placed in a culture dish with 90mm, and DAB staining solution is poured into the culture dish until the tobacco leaves are covered. The dishes were then placed on the shaker and left at a shaking speed of 80rpm for 7 hours, and the shaker was completely covered with tinfoil. After incubation, the leaves were transferred to a jar and bleaching solution (ethanol: acetic acid: glycerol = 3:1:1) was poured into the jar until the leaves were completely covered and the jar was placed in a 95 ℃ water bath and boiled for 20 minutes. And then replacing the original bleaching solution with the fresh bleaching solution, standing for 30 minutes at room temperature, directly observing DAB dyeing by the leaves, and taking photos by selecting a pure white background under unified illumination.
Experimental results indicate that one-sided leaf of active oxygen transiently expressing ZmTIR3 accumulates significantly (FIG. 5).
Claims (4)
1. A maize TIR family gene ZmTIR3, characterized in that: the genome nucleotide sequence of the gene ZmTIR3 is shown as SEQ ID No.1, and the protein coding sequence is shown as SEQ ID No. 2.
2. Use of the maize TIR family gene ZmTIR3 of claim 1 in maize leaf spot disease resistance breeding.
3. Use of the maize TIR family gene ZmTIR3 of claim 1 in tobacco disease-resistant breeding.
4. Use of the maize TIR family gene ZmTIR3 of claim 1 as ETI-related cell necrosis-inducing factor in plant immune basic research.
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