CN112501332B - Primer for detecting specific molecules of wheat take-all germs as well as detection method and application thereof - Google Patents
Primer for detecting specific molecules of wheat take-all germs as well as detection method and application thereof Download PDFInfo
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Abstract
The invention aims to search a new molecular detection target gene in wheat take-all pathogen, and designs a specific detection primer of the take-all pathogen by using the gene to develop a real-time quantitative PCR detection method of the take-all pathogen. The detection technology of real-time quantitative PCR of the take-all pathogen is developed, the minimum infection threshold of the take-all pathogen under the proper condition is researched and developed, the qualitative and quantitative detection of the residual pathogen in the soil is developed, the basis is provided for the early warning of diseases, the technical support is provided for the early prevention and control of the diseases, and the guarantee is provided for the high yield of wheat.
Description
Technical Field
The invention belongs to the field of biotechnology. In particular to a primer for detecting wheat take-all germ specific molecules and a detection method and application thereof.
Background
Wheat take-all is a devastating fungal disease caused by the infestation of wheat by the Gaeumannomyces graminis var.tritici, which was first discovered in italy in 1852 and is now common in major wheat production areas throughout the world and is one of the serious threats to wheat production. The disease is reported for the first time in 1931 in China and occurs in each main wheat production area. In recent years, the disease incidence is remarkably increased, the damage degree is also increased, the yield is reduced by 10% -20% in general diseased fields, and large-area withering of wheat can be caused in parts of regions with serious damage, so that the wheat yield and the grain safety are seriously affected.
The wheat take-all pathogen can be infected in seedling stage and adult stage, in winter wheat planting area of Huang-Huai-Hai area, the pathogenic bacteria can be infected before winter when the temperature is higher and the humidity is higher, after the wheat enters into the winter period, the pathogenic bacteria are hidden in the plant body, and the topping heading stage is quickly expanded in the plant body. Also, in spring with rising air temperature, wheat turning green and jointing stage can cause infection, which is a main time point for disease occurrence. The wheat take-all disease firstly invades from the wheat fibrous root, then forms black disease spots on the root, blocks the water and nutrition transportation of the root, causes the root to rot, causes the disease part to become black, is commonly called as 'black foot disease', the plant shows dwarf symptom, further influences the plant growth, and the whole plant shows withered white ear symptom.
The wheat take-all pathogen is mainly reserved in soil for a long time with disease residues as mycelia, and in recent years, the accumulated amount of pathogenic bacteria residues in fields is increased along with the change of cultivation modes such as straw returning and the like, so that the incidence and the harm degree of the wheat take-all pathogen are correspondingly increased. Disease infection is not obvious in the early stage, once germs invade plants, the germs can be rapidly expanded and cannot be saved, so that the wheat take-all disease is not treated by a method, and only early prevention is taken as the main point, and therefore, early detection, prediction and prediction of the disease are particularly critical and urgent. The early disease diagnosis is generally judged by disease symptoms, such as the typical symptoms of 'black feet' and 'dry ears' caused by take-all disease. However, the damage and the loss can not be saved only when the wheat is observed in the middle and later period of the onset of the disease. Therefore, establishing qualitative and quantitative detection technology of wheat take-all pathogen, quantitative detection of residual bacteria sources in soil and rapid detection of asymptomatic plants after early infection provides technical support for early prevention of diseases, and is a scientific problem to be solved urgently.
The invention aims to search a new molecular detection target gene in wheat take-all pathogen, and designs a specific detection primer of the take-all pathogen by using the gene to develop a real-time quantitative PCR detection method of the take-all pathogen. Ykt6p is an R-SNARE protein essential for cell survival, Ykt6 is very conserved in the evolution of eukaryotes, Ykt6 amino acid sequences in different species are analyzed by alignment, and the sequences have conserved exon coding sequences and intron base sequences with larger variation. Therefore, Ykt6 is used as a new molecular detection target, a specific molecular detection primer of wheat take-all pathogen is designed through sequence comparison analysis, and experiments prove that the primer can only specifically amplify a 90bp strip under a DNA template containing the wheat take-all pathogen and cannot amplify strips of healthy wheat plants, other microorganisms in soil and other diseases of wheat, so that the primer has good specificity and avoids the generation of false positive. On the basis, a detection technology of real-time quantitative PCR of the take-all pathogen is developed, a minimum infection threshold of the take-all pathogen under a proper condition is researched and developed, qualitative and quantitative detection of residual pathogenic bacteria in soil is carried out, a basis is provided for early warning of diseases, technical support is provided for early prevention and control of the diseases, and guarantee is provided for high yield of wheat.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems that the wheat take-all pathogen is relatively common in disease, serious in harm, difficult to prevent and control and the like, the invention provides a new molecular detection target gene of the wheat take-all pathogen, designs a specific detection primer of the take-all pathogen by utilizing the gene, develops a real-time quantitative PCR detection method of the take-all pathogen, researches and develops a minimum infection threshold value of the take-all pathogen under a proper condition, develops qualitative and quantitative detection of residual pathogenic bacteria in soil, and provides a basis for early warning of diseases.
2. Technical scheme
In order to establish a prediction mechanism of wheat take-all disease, the invention adopts the following technical scheme.
The primer for detecting the specific molecules of the wheat take-all pathogen takes an R-SNARE protein coding gene GgYkt6 as a target, designs the primer through gene sequence comparison, verifies the amplification efficiency and specificity by using an experiment, and is characterized in that: the sequences of the primer pairs are as follows:
upstream (19bp) GgF1: 5'-CGATTCCAGGGGACTCTGA-3'
Downstream (19bp) GgR1: 5'-TCGGGACGAGCGACAGATA-3'
A method for detecting the specificity molecular detection primer of wheat take-all pathogen in wheat take-all pathogen includes such steps as extracting the DNA of the bacterial strain to be detected, the plant with disease and the microbe in soil, PCR amplification by molecular detection primer, dyeing the amplified product, agarose gel electrophoresis, and detecting under ultraviolet light if a specific band of 90bp is present.
The specific application of the wheat take-all pathogen detection primer is verified by common PCR amplification, the primer can specifically amplify a 90bp strip only under a DNA template containing the wheat take-all pathogen, and cannot amplify a strip under a template only containing healthy wheat plants, other microorganisms in soil, other diseases of wheat and other pathogen DNAs, so that the primer can be used for qualitatively detecting whether separated pathogens, diseased wheat plants and soil contain the wheat take-all pathogen.
The application of the molecular detection primer specific to wheat take-all pathogen in real-time quantitative fluorescent PCR molecular detection is based on a standard curve equation made by a quantitative standard DNA sample, the detection efficiency is 95.5 percent, and the correlation coefficient R between the logarithm value of the DNA concentration of the standard sample and the Ct value2> 0.99, and the quantitative linear regression equation is y-3.553 x +30.93, where y is Ct value and x is lg [ DNA concentration ═ lg []Provides a theoretical basis for the quantification of the DNA of unknown samples.
The prediction and prediction can be made on the incidence condition of the wheat take-all disease, and the method is characterized in that: detecting the genome content of wheat take-all pathogen with the minimum quantity of about 100fg in the soil by real-time fluorescent quantitative PCR; the minimum inoculum size, as measured by indoor virulence, that causes take-all disease in wheat is approximately 10pg genome per mg of soil.
3. Advantageous effects
According to the invention, an indispensable gene encoding the membrane vesicle transport regulatory protein Ykt6 in an organism is screened out through bioinformatics analysis and comparison, the encoding gene has a base sequence consisting of a conserved exon and a differentiated intron, and a molecular detection primer with strong specificity and high amplification efficiency on wheat take-all pathogen is further developed through experimental screening. Through reference of documents and experimental verification, the primers used in the method and the corresponding two-step quantitative amplification technical mode have better specificity and higher sensitivity, and the standard curve and the regression equation have high reliability. And the content of residual pathogenic bacteria in the soil can be more conveniently calculated by matching with a soil and plant genome extraction kit. The invention provides a molecular detection target and a primer of pathogenic bacteria, establishes a qualitative and quantitative detection technology and a method, and provides a technical platform for disease identification, early diagnosis of pathogenic plants in a latent period and detection of the residual quantity of soil pathogenic bacteria, aiming at the wheat production problems that the residual quantity of field wheat take-all pathogens in soil is increased, the diseases are aggravated, the early latent period of the diseases is long, the diseases are not obvious and the disease becomes 'dead' after the diseases occur due to straw returning to fields in recent years. Further, by using the primer and the technology, a minimum infection threshold value of wheat take-all germs under a proper condition is researched and developed, qualitative and quantitative detection of residual pathogenic bacteria in soil is carried out, a basis is provided for early warning of diseases, technical support is provided for early prevention and control of the diseases, and guarantee is provided for high yield of wheat.
Drawings
FIG. 1 is an alignment chart of YKT6 genome sequences of different species, with the primer sequence in the lower part of the line;
FIG. 2 is the specificity verification of the primers, 1-10 are wheat take-all pathogens collected in different areas, 11-17 are other common pathogens on crops, and M is 2000 mark;
FIG. 3 shows specificity detection of wheat take-all genome, other pathogenic genome and mixed wheat genome.
FIG. 4 shows the sensitivity verification of primers, and the concentrations of PCR templates corresponding to 1-8 are 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, and 10 fg/. mu.L, respectively;
FIG. 5 is an amplification curve and corresponding regression equation for quantitative PCR diluted according to gradient concentration;
FIG. 6 shows the disease condition of wheat take-all pathogen indoor inoculation;
FIG. 7 is a graph showing the curves and lysis curves of soil genome extraction for quantitative amplification after indoor inoculation of wheat take-all pathogen.
Detailed description of the invention
The present invention will be further described with reference to the following examples, but the spirit of the present invention is not limited to the following examples.
EXAMPLE 1
Genome sequence alignment of molecular targets and primer design.
Genome sequences of the gene Ykt6 encoding a vesicle transport regulatory protein of pathogenic bacteria such as wheat take-all (Gaeumannomyces graminis), helminthosporium tritici (Bipolaris sokiniana), Fusarium graminearum (Fusarium graminearum), Fusarium pseudograminearum (Fusarium pseudograminearum), rice blast (Magnaporthe grisea), Sclerotinia sclerotiorum (sclerotiorum), Phytophthora sojae (Phytophthora sojae), and Phytophthora capsici (Phytophthora capsici) were obtained from genomic databases such as Genbank and ensembl fungi by bioinformatics. And then genome sequence comparison is carried out by using Bioedit sequence comparison software. Through sequence alignment, we screened a group of intron region sequences with large sequence differences, the corresponding sequences are shown in FIG. 1, and the bases under the red line are the primer sequences.
Upstream (19bp) GgF1: 5'-CGATTCCAGGGGACTCTGA-3'
Downstream (19bp) GgR1: 5'-TCGGGACGAGCGACAGATA-3'
EXAMPLE 2
And (3) verifying the common PCR detection condition and specificity.
Respectively selecting 10 wheat Fusarium graminearum strains separated from different areas in fields, wherein common pathogenic bacteria on wheat are common crop pathogenic bacteria such as wheat helminthosporium umbiliciformis (Bipolaris sorokiniana), wheat Fusarium graminearum (Fusarium graminearum), wheat pseudo Fusarium graminearum (Fusarium pseudo graminearum), sclerotinia sclerotiorum (rhizoctonia cerealis), Fusarium solani (Fusarium solani), rice blast (Magnaporthe grisea), Phytophthora sojae (Phytophthora sojae) and the like. The above pathogenic bacteria are subjected to liquid culture on PDA or V8 medium, and mycelium is collected for extracting genome. The genome was extracted using a kit (cat # DP171206) from TIANGEN, and the detailed procedures were as described in the kit.
The primers are used for PCR amplification, and the used reagents and concentrations are as follows: 25mmol Tris.Cl (pH 8.3), 125mmol KCl, 3.75mmol MgCl20.25mmol dNTPs, 1 muL of specific primer GgF1/GgR1(10 mumol/L), 0.1mg BSA and 50U of Taq DNA polymerase, taking 1 muL of DNA solution of a substance to be detected as a reaction template, adding deionized water into the reaction template until the volume is increased to 25 muL, uniformly mixing, and performing PCR amplification, wherein the procedures are (1) denaturation at 95 ℃ for 30 seconds and (2) denaturation at 95 ℃ for 5 seconds; (3) annealing at 61 ℃ for 10 seconds; (5) the procedures 2-3 were cycled 35 times. And (3) carrying out electrophoretic detection on the amplification product, namely taking 10 mu L of PCR amplification product, carrying out electrophoretic detection on the PCR amplification product by using 1% (mass-volume ratio) agarose gel at the voltage of 120V, and detecting the result under ultraviolet light after 20 minutes. If the band with the molecular weight of about 90bp exists, the detected pathogen is proved to be wheat take-all pathogen. The results of the experiments showed that the target bands were detected in all the amplification products containing the genome of Ustilaginoidea tritici (FIGS. 2, 1-10), while the target bands were not detected in the amplification products containing the genomes of the other pathogenic bacteria (FIGS. 2, 11-17). The result shows that the primers and the method used in the experiment can specifically detect the wheat take-all pathogen.
Mixing different pathogenic bacteria genomes and wheat genomes, carrying out PCR amplification, and detecting whether the amplification is interfered. The combinations of mixed genomes were: 1. gaeumannomyces graminis + Fusarium graminearum; 2. gaeumannomyces graminis + Bipolaris sorokiniana; 3. gaeumannomyces graminis + Wheat; 4. gaeumannomyces graminis + Fusarium graminearum + Bipolar Sorokiniana + Fusarium pseudograminearum + Rhizotonia cerealis + Wheat; 5. fusarium graminearum + Bipolar sookiniana + Fusarium pseudograminearum + Rhizotonia cerealis + Wheat; 6. and (5) negative control. The experimental results show that in the combination of 1-4, the mixture containing the wheat take-all pathogen genome can specifically amplify a target strip, while in the combination of 5, the mixture containing no wheat take-all pathogen genome and only containing other wheat main pathogen genomes cannot amplify a detection strip, and the negative control 6 cannot amplify a strip. Experiments show that the primers and the amplification method can specifically detect the object to be detected without interference (FIG. 3).
EXAMPLE 3
Primer sensitivity verification
The DNA of the wheat take-all bacterium strain is diluted by 10 times of gradient, and the concentrations of the obtained template DNA are respectively 10 ng/mu L, 1 ng/mu L, 100 pg/mu L, 10 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L and 1 fg/mu L. mu.L of the DNA was used as a template for quantitative PCR amplification in the same manner as above, and 10. mu.L of the PCR product was amplified and subjected to agarose gel electrophoresis. The amplification result shows that the primer and the method used in the ordinary PCR amplification can detect the genome quantity of about 10fg at the lowest (figure 4, the number of the corresponding concentration is 1-8), and the experimental result shows that the primer and the method have the lowest detection concentration of about 1 pg/muL under the ordinary PCR amplification mode.
EXAMPLE 4
And (3) establishing real-time quantitative PCR and a regression equation.
And calculating the copy number of the target gene of the sample to be detected by adopting a SYBR Green I method real-time fluorescence quantitative PCR (absolute quantification). The quantitative PCR kit comprises: takara' TB
Premix Ex TaqTMII (Tli RNaseH plus) "(No.: RR 820A); a detection platform: roche LightCycler 96 fluorescent quantitative PCR instrument. Taking a quantitative genome of the wheat take-all pathogen as a standard substance, then diluting according to a 10-fold gradient, and in a quantitative PCR reaction system, adding 1uL of diluted genome into each hole as a template, namely, in each reaction system, the corresponding original genome concentration is respectively 1.8 multiplied by 103pg，1.8×102pg，1.8×10pg，1.8×100pg，1.8×10-1And pg. The reaction system is as follows: 10 μ L of 2 XPromix Ex Taq Mix buffer, primers GgF1/GgR1 (1)0. mu. mol/L) of each 0.5. mu.L and 1. mu.L of the genome template, and the system was supplemented with sterilized ultrapure water to 20. mu.L. The reaction conditions were as follows: (1) denaturation at 95 ℃ for 30 seconds, (2) denaturation at 95 ℃ for 5 seconds; (3)60 ℃ annealing for 45 seconds, program 2-3 cycle 40 times, (4)97 ℃ for 10 seconds; (5) the dissolution curves were 65 ℃ for 1 minute and 97 ℃ for 1 second.
The experimental result shows that the primer for wheat take-all and the amplification method thereof have excellent amplification efficiency and specificity, the detection efficiency is 95.5%, R2 is 1.0, and the obtained standard curve equation is as follows: y-3.553 lgX +30.93, Y being the CT value of the quantitative PCR amplification and X being the original genomic concentration content per well. The establishment of the standard equation can provide a platform for the quantitative detection of wheat take-all pathogen (figure 5).
EXAMPLE 5
Artificially inoculating wheat take-all pathogen and detecting infection threshold.
After the sorghum rice is soaked in purified water for 12 hours, 150g of the sorghum rice is respectively weighed and placed in a triangular flask to be sterilized for 3 times under high pressure (121 ℃, 20 min). Activating and culturing wheat take-all pathogen in PDA culture medium for 5 days, taking 6 pieces of hypha with the size of about 2 × 2cm, placing in a sterilized sorghum rice culture beaker, performing contact culture at 25 ℃ for 7 days, and shaking uniformly once every day so that the hypha can grow uniformly. After 7 days, drying the sorghum rice which is full of hypha in an oven at 28 ℃, crushing the wheat grains into dry powder by using a crusher, uniformly mixing the dry powder with sterilized soil, uniformly mixing 2.5g, 5g, 10g and 20g of bacterial powder with 500g of sterilized soil respectively, and putting the mixture into a culture pot (the name of a coded sample is 1-4). The inoculated wheat variety is 'Shannong 17' sown wheat, and the condition of the disease is observed after 2 weeks. The experimental result shows that the disease symptoms are not seen and the root is not blackened when the soil is inoculated with 2.5g of bacterial powder; the plant inoculated with 5g of bacterial powder has no obvious disease symptoms, but part of fibrous roots are infected, and blackening symptoms appear; the plant inoculated with 10g of the bacterial powder obviously shows dwarfing symptom, and the main roots, fibrous roots and stem bases show blackening; the emergence rate of the plants inoculated with 20g of fungal powder was significantly reduced, plant death occurred very early, and the roots and stem bases of the plants were significantly blackened (fig. 6).
Uniformly mixing the inoculated and diseased soil, and then taking 250mg of soil for DNA extraction, wherein the use kit comprises: the "magnetic bead method soil genome DNA extraction kit" (No. B618763) of the biological organism, the extraction method is described in the specification, and finally 50. mu.L of TE buffer is used for dissolving. The genome in soil microorganisms is extracted by using the genome kit, 1 mu L of the extracted genome is used for quantitative PCR detection, the detection result and the corresponding genome quantity of the wheat take-all pathogen contained in each gram of soil are shown in Table 1, and the amplification curve and the dissolution temperature are shown in figure 7. Experimental results show that the wheat plant disease can be caused by the soil with the content of wheat take-all germs being inoculated indoors being larger than 10pg/g under the condition of proper temperature and humidity.
EXAMPLE 6
And (4) performing field sampling detection on wheat take-all germs.
Collecting soil at fixed points in wheat planting areas along rivers, between rivers and the Chinese yams and in the north of the Chinese yams in Anhui, wherein the collecting time is that after the land is ploughed and leveled and before sowing, each soil is in the range of about 1 mu of land, about 100g of soil is respectively collected according to a 5-point sampling method, after 5 points are uniformly mixed, 250mg of soil is used for DNA extraction, and the method is the same as the method. Then, the quantitative PCR method is used for quantitative detection, and the regression equation is used for calculating the content of wheat take-all germs in the soil. Meanwhile, 200g of soil is used for planting wheat, and whether the wheat is attacked is observed after 2-3 weeks, and specific data are shown in table 2. The experimental result shows that the incidence of the disease is obviously improved in the field with the wheat take-all disease genome content of more than 10pg/g soil in the soil detected by the field.
Table 1, the amount of the genome of pathogenic bacteria contained in each gram of soil is calculated by using a regression equation after the genome of soil is extracted for quantitative PCR amplification after wheat take-all pathogen is inoculated indoors
TABLE 2 quantitative determination of take-all germs in soils collected from different regions and disease incidence of indoor wheat cultivation using the same
Claims (5)
1. A specific molecular detection primer for wheat take-all is characterized in that R-SNARE protein coding gene is usedGgYkt6As a target, designing a primer through gene sequence comparison, and verifying the amplification efficiency and specificity through experiments, wherein the sequence of the primer is as follows:
GgF1: 5’-CGATTCCAGGGGACTCTGA-3’
GgR1: 5’-TCGGGACGAGCGACAGATA-3’ 。
2. the method for detecting the primer for detecting the specificity molecules of the wheat take-all pathogen in the wheat take-all pathogen as claimed in claim 1, which is characterized in that the DNA of the microorganism in the strain to be detected, the diseased plant or the soil is extracted and is used as a template to carry out PCR amplification, the amplified product is dyed and is subjected to agarose gel electrophoresis, and the detection result is carried out under ultraviolet light, if a specificity strip of 90bp exists, the detection result proves that the detected object contains the wheat take-all pathogen.
3. The application of the primer for detecting the specific molecules of the wheat take-all germs as claimed in claim 1 in PCR detection, wherein PCR amplification is carried out, the primer can specifically amplify a 90bp strip only under a DNA template containing the wheat take-all germs, and can not amplify strips for healthy wheat plants, other microorganisms in soil and other diseases of wheat, and can be used for qualitatively detecting whether separated pathogens, diseased wheat plants or soil contain the wheat take-all germs.
4. The application of the primer for detecting the specific molecules of the wheat take-all pathogen in the real-time quantitative fluorescent PCR molecular detection according to claim 1, wherein the detection efficiency is 95.5% based on a standard curve equation made by a quantitative standard DNA sample, and the correlation coefficient R between the logarithm value of the DNA concentration of the standard substance and the Ct value is the same as the correlation coefficient R2> 0.99, quantitative linear regression equation y = -3.553x +30.93, where y is Ct value, x = lg [ DNA concentration = -3.553x +30.93Degree of rotation]。
5. The use of claim 4, wherein the prediction of the onset of wheat take-all is made, the minimum concentration of real-time quantitative PCR detection is 10fg/μ L, the sensitivity is 1000 times higher than that of ordinary PCR, and the minimum amount of wheat take-all is 10pg genome per gram of soil as determined by indoor pathogenicity.
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