Primer and method for detecting expression characteristics of corn hydrolase EH II gene

Abstract

The invention provides primers and methods for detecting the expression characteristics of corn hydrolase EH II gene. The primers include two pairs, and the sequences are shown in SEQ ID NO.1-4. The invention establishes a fluorescence quantitative PCR method for detecting the expression amount of corn hydrolase EH II gene at different time points after corn resistant and susceptible materials are inoculated with corn leaf blight pathogen, and performs transcriptome sequencing, and compares the expression amount trend of EH II gene fluorescence quantitative expression with the expression amount trend of transcriptome, so as to lay a foundation for studying the resistance defense mechanism generated when corn is infected by pathogens and the expression characteristics of EH II gene.

Description

Technical Field

[0001] The invention relates to a primer and a method for detecting the expression characteristics of corn hydrolase EH II gene, belonging to the field of biotechnology. Background Art

[0002] Corn is an important food crop and cash crop in my country. Cochliobolus heterostrophus ) is an important foliar fungal disease of corn in the world's corn producing areas. Different corn varieties have obvious differences in resistance to corn leaf blight. Research and production practice have shown that using variety resistance to control corn leaf blight is an economical and effective measure. However, there is currently little research on the resistance defense mechanism of corn and the expression of genes related to corn leaf blight resistance.

[0003] Hydrolases are a general term for a class of enzymes that catalyze hydrolysis reactions in animals, plants, and microorganisms, and are usually named in the format of substrate plus hydrolase. There is an exhydrolase II (EH II) hydrolase in corn, which contains 634 amino acids. The exhydrolase II (EH II) gene is involved in corn starch and sucrose metabolism, phenylpropanoid biosynthesis, and amino acid metabolism. Among them, the phenylpropanoid biosynthesis pathway is usually closely related to plant disease resistance. Previous studies have found that the expression level of this gene varies significantly in different resistant corn materials, with the resistant materials being significantly higher than the susceptible materials. The expression level changes with the infection time of corn leaf blight. 24 hours after the pathogen inoculation, the expression level of the EH II gene in the corn disease-resistant material is significantly induced and increased, indicating that this gene is involved in the resistance defense mechanism of corn against leaf blight infection, and early infection of the pathogen can affect the expression regulation of the EH II gene in different materials or varieties.

[0004] There are few reports on maize EH II gene at home and abroad, and no reports on its expression. Therefore, real-time fluorescence quantitative PCR was used to study the expression characteristics of maize EH II gene at different time points after inoculation of corn leaf blight. The results showed that the expression levels of EH II gene in resistant and susceptible materials were different with different inoculation times, and the change trend was consistent with the change trend of transcriptome expression. Therefore, if the hydrolase gene is used as a target for variety resistance screening, rapid screening of maize resistant materials at the gene expression level will have important scientific significance for opening up new areas of variety resistance detection, guiding the rational layout of varieties and disease prevention and control. Summary of the invention

[0005] The purpose of the present invention is to provide a primer and method for detecting the expression characteristics of corn hydrolase EH II gene, and different treatments designed for fully understanding the expression characteristics of corn hydrolase EH II gene.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The conidia suspension of Microblight spores of corn was inoculated (concentration of about 5×10 6 The two resistant and susceptible maize materials (100 μg / mL) were sampled at 0 h, 12 h and 24 h after inoculation, divided into three biological replicates, with a total of 18 groups of samples.

[0008] Design a pair of primers for detecting the expression characteristics of corn hydrolase EH II gene, including upstream and downstream primers that meet the characteristics of fluorescent quantitative PCR reaction:

[0009] EH II-F: 5'-CGGCATCAACGAGAACAACAC-3',

[0010] EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3';

[0011] And the upstream and downstream primers as internal reference genes:

[0012] GAPDH-F: 5'-GAAGCACATCCAGGCGG-3',

[0013] GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3'

[0014] The fluorescent quantitative PCR method for detecting the expression characteristics of the corn hydrolase EH II gene of the present invention is carried out according to the following steps:

[0015] (1) First-strand cDNA synthesis: Extract total RNA from the sample and purify it, and use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA;

[0016] (2) Conventional PCR detection using the following primers

[0017] Fluorescence quantitative upstream and downstream primers of corn hydrolase EH II gene:

[0018] EH II-F: 5'-CGGCATCAACGAGAACAACAC-3',

[0019] EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3';

[0020] And the upstream and downstream primers as internal reference genes:

[0021] GAPDH-F: 5'-GAAGCACATCCAGGCGG-3',

[0022] GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3';

[0023] The conventional PCR amplification system was 25 µL: 10× buffer 2.5 µL, dNTP 1.0 µL, upstream primer 0.5 µL, downstream primer 0.5 µL, 100 ng / µL cDNA template 1.0 µL, Ex-TaqE 0.15 µL, ddH2O 19.35 µL;

[0024] The conventional PCR reaction program for the EH II gene was as follows: pre-denaturation at 94 °C for 5 min, followed by denaturation at 94 °C for 30 s, annealing at 59.4 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for another 10 min;

[0025] The conventional PCR reaction program for GAPDH gene was as follows: pre-denaturation at 94 °C for 5 min, followed by denaturation at 94 °C for 30 s, annealing at 57.2 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for another 10 min;

[0026] (3) Real-time fluorescence quantitative PCR reaction:

[0027] Using the cDNA obtained in step (1) as a template, adding the primers described in step (2), performing a fluorescent quantitative PCR reaction, setting 3 replicates for each sample, and taking the average value after amplification;

[0028] The real-time fluorescence quantitative PCR amplification system was 20 μL: masterMix 10 μL, upstream primer (10 μM) 0.6 μL, downstream primer (10 μM) 0.6 μL, 100 ng / μL cDNA template 4.0 μL, ddH2O 4.8 μL;

[0029] The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95 °C for 90 s, followed by denaturation at 95 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s, for 40 cycles;

[0030] The more detailed test method of the present invention is as follows:

[0031] The real-time fluorescence quantitative PCR detection method of corn hydrolase EH II gene can be achieved by the following steps:

[0032] (1) Primer design: Based on the sequence of the corn hydrolase EH II gene obtained from the transcriptome sequencing results, DNAMAN software was used to design specific primers suitable for fluorescent quantitative PCR detection. The primer sequences are as follows:

[0033] EH II-F: 5'-CGGCATCAACGAGAACAACAC-3',

[0034] EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3';

[0035] At the same time, according to the sequence of the maize energy metabolism GAPDH gene obtained from the transcriptome sequencing results, primers for the internal control of fluorescent quantitative PCR were designed. The primer sequences are as follows:

[0036] GAPDH-F: 5'-GAAGCACATCCAGGCGG-3',

[0037] GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3';

[0038] (2) Inoculation test of corn materials with different resistance: When corn leaves were 5-6 leaves, conidia suspension of Microsporum spp. (concentration of about 5×10 6 The corn plants were treated with uniform spray of 1% ethanol (0.1% ethanol / mL) and samples were taken at 0 h, 12 h and 24 h after inoculation, divided into 3 biological replicates, with a total of 18 groups of samples. The samples were quick-frozen in liquid nitrogen for 30 min and then stored in a -80 ℃ refrigerator for later use. The samples taken at 0 h after inoculation were used as control samples.

[0039] (3) Synthesis of the first strand of cDNA: Total RNA was extracted from corn leaves and reverse transcribed to synthesize the first strand of cDNA according to the instructions of the Fast Quantity RTKit (KR106) kit of TIANGEN.

[0040] (4) Real-time fluorescence quantitative PCR reaction: The real-time fluorescence quantitative PCR reagent used was a product of TRAN. The first strand of the cDNA synthesized above was used as a template, and the above EH II-F, EH II-R and GAPDH-F, GAPDH-R were used as specific primers to perform fluorescence quantitative PCR. Three parallel replicates were set for each sample, and the average of the parallel Ct values ​​obtained after amplification was taken.

[0041] Real-time fluorescence quantitative PCR amplification system 20 μL: masterMix 10 μL, upstream primer (10 μM) 0.6 μL, downstream primer (10 μM) 0.6 μL, 100 ng / μL cDNA template 4.0 μL, ddH2O 4.8 μL;

[0042] The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95 °C for 90 s, followed by denaturation at 95 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s, for 40 cycles;

[0043] (5) The expression level of corn hydrolase EH II gene relative to the internal reference GAPDH gene is calculated as: relative mRNA expression = 2 -ΔΔCt ×100%, where Ct value = average Ct value of target gene - Ct value of internal reference GAPDH gene, △△Ct = (△Ct of target gene in test sample - △Ct of target gene in control sample).

[0044] (6) The results showed that the expression levels of the corn hydrolase EH II gene in the disease-resistant material at the three time points were significantly higher than those in the susceptible material. As the inoculation time increased, the expression level of the corn hydrolase EH II gene in the disease-resistant material after 24 h of inoculation was significantly increased compared with the control after 0 h of inoculation, while the gene expression level in the susceptible material was significantly decreased. The gene expression levels of the two corn materials at different time points were consistent with the expression trend of the transcriptome, which provided important basic data for our in-depth research on the corn hydrolase EH II gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :Electrophoresis of conventional PCR products of maize EH II gene and GAPDH gene with fluorescent quantitative primers. Lane M is the DNA relative molecular weight standard; Lane 1 is the maize hydrolase EH II gene, with a product length of 156 bp; Lane 2 is the GAPDH gene, with a product length of 221 bp. Figure 2 : Specific primers for fluorescence quantitative PCR detection of corn hydrolase EH II gene (left) and melting curve of GAPDH gene (right).

[0046] Figure 3 :The relative expression levels of maize hydrolase EH II gene at different time points after Luo31-S and MO17-R were inoculated with Microsporum zeae based on fluorescence quantitative PCR. The columns and the left axis are the expression levels of fluorescence quantitative PCR. Among them, Luo31-S0, Luo31-S12, and Luo31-S24 represent the samples of maize susceptible material Luo31-S inoculated with Microsporum zeae at 0 h, 12 h, and 24 h, respectively; MO17-R0, MO17-R12, and MO17-R24 represent the samples of maize resistant material MO17-R inoculated with Microsporum zeae at 0 h, 12 h, and 24 h, respectively.

[0047] Figure 4:The relative expression of maize hydrolase EH II gene at different time points after Luo31-S and MO17-R were inoculated with Microsporum fusca based on transcriptome level. The column and the left axis are the expression of transcriptome. Among them, Luo31-S0, Luo31-S12, and Luo31-S24 represent the samples of maize susceptible material Luo31-S inoculated with Microsporum fusca at 0 h, 12 h, and 24 h, respectively; MO17-R0, MO17-R12, and MO17-R24 represent the samples of maize resistant material MO17-R inoculated with Microsporum fusca at 0 h, 12 h, and 24 h, respectively. DETAILED DESCRIPTION

[0048] The present invention is explained below in conjunction with specific embodiments and drawings. The schemes of the embodiments described here do not limit the invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0049] Example 1

[0050] Handling of test materials

[0051] In the greenhouse, when the susceptible corn material Luo 31-S and the resistant corn material MO17-R were at the 5-6 leaf stage (about 30 days old), they were treated with conidia suspension of Microscopium spp. (concentration of about 5×10 6 30 corn plants were treated with a uniform spray of 1% ethanol / mL. After the inoculated corn plants were moisturized with plastic film for 24 h, they were continued to be cultivated in the greenhouse, and the disease environment temperature in the greenhouse was maintained at 22-32 ℃ and the relative humidity was above 80%. Samples were taken at 0 h, 12 h and 24 h after inoculation. Each sample consisted of the fourth leaf of 10 corn plants, divided into 3 biological replicates, for a total of 18 groups of samples. The samples were quick-frozen in liquid nitrogen for 30 min and then stored in a -80 ℃ refrigerator for later use.

[0052] Example 2

[0053] Primer design

[0054] (1) Based on the sequence of the corn hydrolase EH II gene obtained by transcriptome sequencing, DNAMAN software was used to design specific primers suitable for fluorescence quantitative PCR detection. The primer sequences are as follows:

[0055] EH II-F: 5'-CGGCATCAACGAGAACAACAC-3',

[0056] EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3';

[0057] The length of the conventional PCR product of corn hydrolase EH II gene fluorescence quantitative was 156 bp. The results of agarose gel electrophoresis showed that the amplified product had the same length and was a single band ( Figure 1 ), indicating that the designed primers have strong specificity and are suitable for real-time fluorescence quantitative PCR detection.

[0058] (2) Based on the sequence of the maize energy metabolism GAPDH gene obtained by transcriptome sequencing, primers for internal control of fluorescent quantitative PCR were designed. The primer sequences are as follows:

[0059] GAPDH-F: 5'-GAAGCACATCCAGGCGG-3',

[0060] GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3';

[0061] The length of the conventional PCR product of corn GAPDH gene fluorescence quantitative analysis was 221 bp. The agarose gel electrophoresis results showed that the amplified product had the same length and was a single band ( Figure 1 ), indicating that the designed primers have strong specificity and are suitable for real-time fluorescence quantitative PCR detection.

[0062] Example 3

[0063] Extraction of total RNA

[0064] Refer to TransZol TM Extract total RNA according to the Up Plus RNA Kit instructions. Grind corn leaf tissue samples in liquid nitrogen until powder, immediately add 1 mL Trizol, mix thoroughly and lyse. Add 200 uL chloroform, shake vigorously and mix thoroughly, centrifuge at 4 ℃ 12000 rpm for 10 min. Take the supernatant, add an equal volume of phenolform, shake thoroughly and mix thoroughly, centrifuge at 4 ℃ 12000 rpm for 10 min. Take the supernatant, add an equal volume of chloroform, shake thoroughly and mix thoroughly, centrifuge at 4 ℃ 12000 rpm for 10 min. Take the supernatant, add an equal volume of isopropanol, mix thoroughly, and precipitate at -20 ℃ for 1 h. Centrifuge at 4 ℃ 12000 rpm for 15 min. Discard the supernatant, add 0.5 mL 75% ethanol, and centrifuge at 4 ℃ 8000-10000 rpm for 5 min. Repeat the previous step. After discarding the supernatant, centrifuge briefly, drain the ethanol with a pipette, and vacuum dry for 2 min. Add 20 uL RNA-Freewater, dissolve at room temperature for 5 min, mix well, and centrifuge briefly to obtain RNA solution. The obtained RNA is stored at -80 ℃ for later use.

[0065] Example 4

[0066] Synthesis of the first strand of cDNA: According to the instructions of the Fast Quantity RT Kit (KR106) of TIANGEN, the RNA in Example 3 was used as a template to synthesize the first strand of cDNA by reverse transcription. The specific steps are as follows:

[0067] According to the instructions of Fast Quant RT Kit (with gDNase), 50 ng-2 μg of total RNA was used to establish a 20 μL reaction system.

[0068] (1) Thaw the template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×Fast RT Buffer, and RNase-Free ddH2O at room temperature (15-25°C) and quickly place on ice after thawing. Vortex each solution to mix well before use and briefly centrifuge to collect the liquid remaining on the tube wall.

[0069] (2) Prepare a mixture according to the genomic DNA removal system in Table 1 and mix thoroughly. Centrifuge briefly and incubate at 42°C for 3 min. Then place on ice.

[0070] Table 1 gDNA removal reaction system

[0071]

[0072] (3) Prepare the mixed solution according to the reverse transcription reaction system in Table 2.

[0073] Table 2 Reverse transcription reaction system

[0074]

[0075] (4) Add the Mix from the reverse transcription reaction to the reaction solution from the gDNA removal step and mix thoroughly.

[0076] (5) Incubate at 42°C for 15 min.

[0077] (6) Incubate at 95°C for 3 min. The resulting cDNA product can be used for subsequent experiments. The product can be stored at -20°C or -80°C.

[0078] Example 5

[0079] Real-time fluorescence quantitative PCR reaction was performed. The real-time fluorescence quantitative PCR reagent was a product of TRAN. The cDNA in Example 4 was used as a template and the primers in Example 2 were used to perform real-time fluorescence quantitative PCR amplification reaction. Three parallel replicates were set for each sample, and the average of the parallel Ct values ​​obtained after amplification was taken.

[0080] (1) The fluorescence quantitative PCR amplification system is as follows:

[0081] Real-time fluorescence quantitative PCR amplification system 20 μL: masterMix 10 μL, upstream primer (10 μM) 0.6 μL, downstream primer (10 μM) 0.6 μL, 100 ng / μL cDNA template 4.0 μL, ddH2O 4.8 μL;

[0082] (2) The fluorescence quantitative PCR reaction procedure was as follows: 95°C pre-denaturation for 90 s, 95°C denaturation for 5 s, 60°C annealing for 15 s, 72°C extension for 20 s, and 40 cycles;

[0083] (3) After the fluorescence quantitative PCR is completed, the ratio of the relative expression levels of the corn material at different time points of inoculation is calculated based on the Ct value. -ΔΔCt The expression level of corn hydrolase EH II gene relative to GAPDH gene was calculated as: relative mRNA expression = 2 -ΔΔCt ×100%, where △Ct value = average Ct value of target gene - average Ct value of internal reference GAPDH gene, △△Ct = (△Ct of target gene in test sample - △Ct of target gene in control sample), △Ct mean = AVERAGE (Ct value of all sample replicates), Ct SD = STDEV (Ct value of all sample replicates).

[0084] Table 3 C(T) values, mean values, standard deviations, fluorescence quantitative expression levels and transcriptome expression levels of EH II gene at different time points after inoculation of susceptible material Luo 31-S with X.

[0085]

[0086] Table 4 C(T) values, mean values, standard deviations, fluorescence quantitative expression levels and transcriptome expression levels of EH II gene at different time points after inoculation of disease-resistant material MO17-R with X.

[0087]

[0088] (4) Figure 2 Shown are the specific primers for fluorescent quantitative PCR detection of corn hydrolase EH II gene and the melting curve of GAPDH gene. Figure 3 The relative expression of EH II gene at different time points (0 h, 12 h, 24 h) after inoculation of Y. maydis in susceptible material (Luo 31-S) and resistant material (MO17-R) based on fluorescence quantitative PCR. Figure 4This study is based on the transcriptome level to compare the differential expression of maize EH II genes at different time points (0 h, 12 h, and 24 h) after Luo 31-S and MO17-R were inoculated with corn leaf blight. The results showed that the expression levels of maize hydrolase EH II genes at three time points in the disease-resistant materials were significantly higher than those in the susceptible materials. With the extension of the inoculation time, compared with the control inoculated at 0 h, the expression level of maize hydrolase EH II genes in the disease-resistant materials inoculated at 24 h was significantly increased, while the gene expression level in the susceptible materials was significantly decreased. The gene expression levels at different time points in the two maize materials were consistent with the expression level changes in the transcriptome, which provides important basic data for our in-depth study of the maize hydrolase EH II gene.

[0089] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention. SEQUENCE LISTING <110> Institute of Plant Protection, Fujian Academy of Agricultural Sciences <120> Primer and method for detecting expression characteristics of corn hydrolase EH II gene <130> <160> 4 <170> PatentIn version 3.3 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <400> 1 cggcatcaac gagaacaaca c 21 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 atgaggtcgt ggttggcg 18 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <400> 3 gaagcacatc caggcgg 17 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 tgtaggagtg ggtggtggtc 20

Claims

1. A fluorescent quantitative PCR primer for detecting the expression characteristics of corn hydrolase EH II gene, characterized in that: Including upstream and downstream primers that meet the characteristics of fluorescent quantitative PCR reaction: EH II-F: 5'-CGGCATCAACGAGAACAACAC-3', EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3'; And the upstream and downstream primers as internal reference genes: GAPDH-F: 5'-GAAGCACATCCAGGCGG-3', GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3'.

2. A fluorescent quantitative PCR method for detecting the expression characteristics of corn hydrolase EH II gene using the primers described in claim 1, characterized in that: Follow these steps: (1) First-strand cDNA synthesis: Extract total RNA from the sample and purify it, and use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; (2) Perform conventional PCR detection using the following primers: Fluorescence quantitative upstream and downstream primers of corn hydrolase EH II gene: EH II-F: 5'-CGGCATCAACGAGAACAACAC-3', EH II-R: 5'-ATGAGGTCGTGGTTGGCG-3'; And the upstream and downstream primers as internal reference genes: GAPDH-F: 5'-GAAGCACATCCAGGCGG-3', GAPDH-R: 5'-TGTAGGAGTGGGTGGTGGTC-3'; The conventional PCR amplification system was 25 µL: 10× buffer 2.5 µL, dNTP 1.0 µL, upstream primer 0.5 µL, downstream primer 0.5 µL, 100 ng / µL cDNA template 1.0 µL, Ex-TaqE 0.15 µL, ddH2O 19.35 µL; The conventional PCR reaction program for the EH II gene was as follows: pre-denaturation at 94 °C for 5 min, followed by denaturation at 94 °C for 30 s, annealing at 61.2 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for another 10 min; The conventional PCR reaction program for GAPDH gene was as follows: pre-denaturation at 94 °C for 5 min, followed by denaturation at 94 °C for 30 s, annealing at 57.2 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for another 10 min; (3) Real-time fluorescence quantitative PCR reaction: Using the cDNA obtained in step (1) as a template, adding the primers described in step (2), performing a fluorescent quantitative PCR reaction, setting 3 replicates for each sample, and taking the average value after amplification; The real-time fluorescence quantitative PCR amplification system was 20 µL: master mix 10 µL, 10 µM upstream primer 0.6 µL, 10 µM downstream primer 0.6 µL, 100 ng / µL cDNA template 4.0 µL, ddH2O 4.8 µL; The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95 °C for 90 s, followed by denaturation at 95 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s, for 40 cycles.

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