CN116479166A - Internal reference gene for stable expression of verticillium dahliae, and primer and application thereof - Google Patents
Internal reference gene for stable expression of verticillium dahliae, and primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene for stable expression of verticillium dahliae, and a primer and application thereof. According to the invention, the Verticillium dahliae H3 gene, the Tubulin gene, the EF1a gene and the GAPDH gene are taken as candidate genes, real-time fluorescence quantitative PCR is adopted for amplification, the stability analysis is carried out by using the programs of the gemum and NormFinder algorithms, the comprehensive analysis is carried out by using a refFinder online analysis tool, and finally the most stable expression of the Tubulin gene in the Verticillium dahliae is found. The Tubulin gene is used as an internal reference gene in the fluorescent quantitative PCR detection of the verticillium dahliae, so that the aim of calibration can be fulfilled, and the related gene expression of the verticillium dahliae can be accurately and quantitatively detected.
Description
The present application claims priority from the chinese patent office filed at month 17 of 2023, application number CN202310264690.0, chinese patent application entitled "internal reference gene stably expressed by verticillium dahliae and primers and applications thereof", the entire contents of which are incorporated herein by reference.
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an internal reference gene for stable expression of verticillium dahliae, and a primer and application thereof.
Background
The verticillium wilt of cotton caused by verticillium dahliae is a soil-borne disease which seriously affects the yield and quality of cotton, and can cause 15-20 hundred million yuan of economic loss in cotton areas every year. The verticillium dahliae has strong variability and often shows pathogenic differentiation in the process of co-evolution with hosts and environments. According to different symptoms of cotton in the disease period, verticillium wilt can be divided into fallen leaf type and non-fallen leaf type, the fallen leaf type refers to that most of cotton wilts after the cotton is ill, even leaves fall off, the whole plant is withered, the non-fallen leaf type refers to that pathogenic bacteria infects veins and leaf edges of cotton plants to form yellow disease spots, the edges of the disease spots are gradually burnt up, but leaves do not fall off, the fallen leaf type strain proportion is increased year by year, and pathogenicity is enhanced year by year. The molecular mechanism of verticillium dahliae in various biological or physiological processes is understood by researching the expression condition of related genes in verticillium dahliae, and the method has important significance in preventing and treating cotton verticillium wilt.
Real-time fluorescent quantitative PCR (RT-qPCR) is a common method in gene expression and transcription analysis, and has high sensitivity, good repeatability and strong specificity. However, some experimental factors, such as batch-to-batch differences in RNA extraction, efficiency of cDNA synthesis and PCR reactions, etc., all of these abiotic variations can affect the accuracy of real-time fluorescent quantitative PCR. In order to reduce the impact of these factors, the data needs to be subjected to a normalization process to eliminate non-biological variations, with the most common strategy being to use a stably expressed reference gene as an internal reference for data normalization, commonly referred to as an internal reference gene.
The reference gene is also called housekeeping gene, and is a gene that can be stably expressed in a cell. The expression of the reference gene is affected to different degrees under different test materials and test conditions. Under certain experimental conditions, if the most stable reference genes and the least stable reference genes are respectively selected for quantitative experiments, the expression of the target genes can be dynamically changed. Thus, selection of reference genes was determined to have a crucial impact on the results of related experimental studies. There is no report on reference genes that can be stably expressed in verticillium dahliae.
Disclosure of Invention
The invention aims to solve the defects of the prior art, obtain the internal reference gene with stable expression in the verticillium dahliae, and accurately and quantitatively detect the related gene expression of the verticillium dahliae.
The invention provides an application of a Tubulin gene as an internal reference gene in fluorescent quantitative PCR detection of Verticillium dahliae, wherein the Verticillium dahliae comprises Verticillium dahliae and Verticillium dahliae with non-fallen leaves.
Preferably, the verticillium dahliae comprises a strain XJ592; the non-verticillium dahliae includes a strain XJ511.
The invention also provides a primer for amplifying the Tubulin gene in the application of the technical scheme, which comprises an upstream primer Tub-F1 and a downstream primer Tub-R1;
the upstream primer Tub-F1 comprises a nucleotide sequence shown as SEQ ID NO. 1;
the downstream primer Tub-R1 comprises a nucleotide sequence shown as SEQ ID NO. 2.
The invention also provides application of the primer in the technical scheme in the fluorescent quantitative PCR detection of the verticillium dahliae.
Preferably, the reaction system for fluorescent quantitative PCR detection comprises the following components in 25 mu L: 12.5. Mu.L of 2X UltraSYBR Mixture, 1. Mu.L of 2.5. Mu.M to 25. Mu.M upstream primer, 1. Mu.L of 2.5. Mu.M to 25. Mu.M downstream primer, 1 to 10ng of template cDNA and the balance of sterile water.
Preferably, the reaction conditions for the fluorescent quantitative PCR detection are: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing at 58℃for 1min, extension at 72℃for 10s,40 cycles
The invention also provides application of the primer in the technical scheme in preparation of a verticillium dahliae fluorescent quantitative PCR detection kit
The invention takes Verticillium dahliae H3 gene (histone gene), tubulin gene (Tubulin gene), EF1a gene (translation elongation factor) and GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase gene) as candidate genes, designs primers, adopts real-time fluorescence quantitative PCR for amplification, uses the programs of the gemm and NormFinder algorithm for stability analysis, and uses a refFinder online analysis tool for comprehensive analysis, and finally discovers that the Tubulin gene expression in the Verticillium dahliae is the most stable. The Tubulin gene is used as an internal reference gene in the fluorescent quantitative PCR detection of the verticillium dahliae, so that the aim of calibration can be fulfilled, and the related gene expression of the verticillium dahliae can be accurately and quantitatively detected.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the result of agarose gel electrophoresis after PCR amplification using the cDNA mixture of sample No. 1-4 of example 1 as a template;
FIG. 2 shows a dissolution curve obtained by RT-qPCR using cDNA of sample No. 1-4 of example 1 as a template and H3-F/H3-R as amplification primers;
FIG. 3 shows a dissolution curve obtained by RT-qPCR using cDNA of sample 1-4 of example 1 as a template and Tub-F1/Tub-R1 as amplification primers;
FIG. 4 shows a dissolution curve obtained by RT-qPCR using cDNA of samples 1 to 4 as a template and Tub-F2/Tub-R2 as amplification primers in example 1, respectively;
FIG. 5 shows a dissolution curve obtained by RT-qPCR using cDNA of samples 1 to 4 as a template and EF1a-F/EF1a-R as amplification primers in example 1, respectively;
FIG. 6 shows a dissolution curve obtained by RT-qPCR using cDNA of samples 1 to 4 as a template and GADPH-F/GADPH-R as amplification primers in example 1, respectively;
FIG. 7 is a distribution box diagram of Ct values obtained by amplification using cDNA of samples 1 to 4 as a template in example 1.
Detailed Description
The invention provides an application of a Tubulin gene as an internal reference gene in fluorescent quantitative PCR detection of Verticillium dahliae, wherein the Verticillium dahliae comprises Verticillium dahliae and Verticillium dahliae with non-fallen leaves.
In the present invention, the verticillium dahliae preferably includes a strain XJ592; the non-verticillium dahliae preferably includes strain XJ511.
In the present invention, the Tubulin gene has accession number 20711537 at NCBI.
According to the invention, a verticillium dahliae H3 gene (histone gene), a Tubulin gene (Tubulin gene), an EF1a gene (translation elongation factor) and a GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase gene) are taken as candidate genes, stability analysis is performed by using a gemm algorithm program and a NormFinder algorithm program, comprehensive analysis is performed by using a refFinder online analysis tool, and finally, the fact that the expression of the Tubulin gene in the candidate genes is the most stable under two conditions of culture medium culture and cotton induction is found, and the EF1a gene is the next time. In the invention, the Tubulin gene is taken as an internal reference gene, and the expression of VDAG_07740, VDAG_09898 and VDAG_10381 genes of the XJ592 and XJ511 strains under shaking culture conditions and cotton root induction conditions is detected. The fluorescent quantitative PCR result is consistent with the transcriptome sequencing result, which shows that the fluorescent quantitative PCR detection method can achieve the aim of calibrating by taking the Tubulin gene as an internal reference gene in the fluorescent quantitative PCR detection of the verticillium dahliae, and accurately and quantitatively detect the related gene expression of the verticillium dahliae.
The invention also provides a primer for detecting the Tubulin gene by fluorescent quantitative PCR, which comprises an upstream primer Tub-F1 and a downstream primer Tub-R1;
the upstream primer Tub-F1 comprises a nucleotide sequence shown as SEQ ID NO. 1;
the downstream primer Tub-R1 comprises a nucleotide sequence shown as SEQ ID NO. 2.
The nucleotide sequences of the upstream primer Tub-F1 and the downstream primer Tub-R1 are specifically as follows:
upstream primer Tub-F1 (SEQ ID NO. 1): 5'-GCTCACCCAGCAGATGTTC-3';
downstream primer Tub-R1 (SEQ ID NO. 2): 5'-CGACCTCCTTCATGGCAAC-3'.
The invention also provides application of the primer in the technical scheme in the fluorescent quantitative PCR detection of the verticillium dahliae. In the present invention, the application preferably comprises preparing a verticillium dahliae fluorescent quantitative PCR detection kit.
According to the invention, the Tubulin gene is taken as an internal reference gene, and amplification primers are designed, so that the upstream primer Tub-F1 and the downstream primer Tub-R1 have strong specificity and stable expression quantity, and can be used as amplification primers of the Tubulin gene for fluorescent quantitative PCR detection of Verticillium dahliae and accurate quantitative detection of the expression of the related gene of Verticillium dahliae.
For further explanation of the present invention, reference genes and primers and applications thereof stably expressed by Verticillium dahliae provided by the present invention will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
In the implementation process of the invention, the culture medium is a conventional culture medium unless specified otherwise.
Example 1
1. Test materials and methods
1.1 experimental materials: the cotton variety to be tested is Ji cotton 11, and the verticillium dahliae is deciduous strain XJ592 and non-deciduous strain XJ511 (both XJ592 and XJ511 are collected, identified and stored in a disease-planting textroom of Xinjiang university).
1.2 sample preparation
1.2.1 sample preparation under Medium culture conditions
The two strains XJ592 and XJ511 are respectively activated and cultured on a PDA plate for one week, and respectively the bacterial cakes are picked and inoculated into a PDB culture medium for shaking culture, wherein the rotation speed of the shaking culture is 175rpm, and the temperature is 25 ℃. Mycelium was collected after 48h shaking, and collected XJ592 mycelium was used as sample No.1 and collected XJ511 mycelium was used as sample No.2 for further use.
1.2.2 sample preparation under Induction conditions of Cotton root extract
The two strains XJ592 and XJ511 are respectively activated and cultured on a PDA plate for one week, and respectively picking fungus cakes and inoculating the fungus cakes into a cotton induction culture medium for shake culture, wherein the rotation speed of shake culture is 175rpm, and the temperature is 25 ℃. Mycelium was collected after 48h shaking, collected XJ59 mycelium was used as sample No. 3, and collected XJ511 mycelium was used as sample No. 4 for later use.
Wherein, the cotton induction culture medium consists of 5mL cotton root extract and 50mL LPDB culture medium; the preparation method of the cotton root extract comprises the following steps: 2.5g of cotton roots are weighed, ground to powder in liquid nitrogen, extracted with 50ml of pure water for 2h, centrifuged at 5000rpm for 10min, and the supernatant is filtered and sterilized with a 0.22 μm bacterial filter.
1.3 extraction of Total RNA and Synthesis of cDNA
1.3.1 respectively quick-freezing samples 1 to 4 in liquid nitrogen, grinding, extracting total RNA of the samples by using a Trizol total RNA extraction kit (Thermo Fisher Scientific of Seeimer Feishier technology Co.), and putting the samples in a refrigerator at-80 ℃ for specific steps, wherein the specific steps refer to the instruction book;
1.3.2 detection of the resulting RNA concentration using a NanoDrop 2000 spectrophotometer, all samples were adjusted to 1. Mu.g/. Mu.L RNA concentration according to PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara) reverse transcription kit instructions, RNA was reverse transcribed to obtain cDNA, and the specific reaction system and reaction conditions are shown in Table 1 below.
TABLE 1 reaction System and reaction conditions
1.4 selection of candidate reference genes and primer design
The H3 gene (accession number 20711498), the Tubulin gene (accession number 20711537), the EF 1. Alpha. Gene (accession number 20704022) and the GAPDH gene (accession number 20710379) were selected as candidate reference genes, and primers were designed, and specific information is shown in Table 2.
TABLE 2 candidate reference genes and RT-PCR primers therefor
1.5 analysis of specificity of candidate reference genes
1.5.1PCR and agarose gel electrophoresis detection
The cDNA of samples 1 to 4 obtained in the step 1.3 is prepared according to the following steps: 1:1:1, and using the primers in step 1.4 as amplification templates, respectively, PCR amplification was performed using 2 XTaq Master mix (Dye).
The PCR amplification reaction system is as follows: 25. Mu.L of 2 XTaqMastermix, 2. Mu.L of upstream primer (10. Mu.M), 2. Mu.L of downstream primer (10. Mu.M), 1. Mu.L of amplified template cDNA (500 ng/. Mu.L) and 20. Mu.L of sterile water;
the PCR amplification reaction procedure was: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 10s, and performing 35 cycles; final extension at 72℃for 2min. After the amplification, agarose gel electrophoresis was performed, and the results are shown in FIG. 1. Wherein, lanes from left to right in FIG. 1 are DL2000, the primer pair H3-F/H3-R amplification products in sequence; primer pair Tub-F1/Tub-R1 amplification products; primer pair Tub-F2/Tub-R2 amplification products; primer pair EF1a-F/EF1a-R amplification products; the primer pair GAPDH-F/GAPDH-R amplification product.
As can be seen from FIG. 1, under both conditions of culture in medium and cotton induction, the candidate internal reference gene primers in both deciduous strain XJ592 and non-deciduous strain XJ511 amplified a single band, with no other non-specific bands and primer dimers.
1.5.2 real-time fluorescent quantitative PCR analysis
(1) The cDNAs of samples 1 to 4 obtained in step 1.3 were subjected to gradient dilution to give final concentrations of 15 ng/. Mu.L, 1.5 ng/. Mu.L, 0.15 ng/. Mu.L, 0.015 ng/. Mu.L, and 0.0015 ng/. Mu.L, respectively, as amplification templates, and real-time fluorescent quantitative PCR was performed for each sample, and three repeated experiments were performed. Preparing a real-time fluorescent quantitative PCR reaction system on ice: 12.5. Mu.L of 2 XUltraSYBRMixture, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 1. Mu.L of amplification template, 9.5. Mu.L of sterile water, and 25. Mu.L in total.
The real-time fluorescence quantitative PCR reaction procedure is: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing at 58℃for 1min and extension at 72℃for 10s were performed for 40 cycles. By using480 system carries out amplification reaction to obtain a dissolution curve, wherein the dissolution curve is shown in figures 2-6, and figure 2 shows the analysis result of the dissolution curve of all templates amplified by using H3-F/H3-R as primers; FIG. 3 shows the result of analysis of the dissolution profile of the primer Tub-F1/Tub-R1 amplified for all templates; FIG. 4 shows the result of analysis of the dissolution profile of amplified total template using Tub-F2/Tub-R2 as primer; FIG. 5 shows the result of analysis of the dissolution profile of the amplification of all templates using EF1a-F/EF1a-R as primers; FIG. 6 shows the results of a dissolution profile analysis of amplification of all templates using GAPDH-F/GAPDH-R as primers.
As can be seen from FIGS. 2 to 6, the dissolution curves of the five pairs of primers are single peaks, indicating that the primers have stronger specificity.
(2) The graph pad software was used to construct a distribution box graph of Ct values of the dissolution curve obtained in step (1), and the result is shown in FIG. 7.
As can be seen from FIG. 7, there are different differences in expression levels of the 4 candidate reference genes in different samples, the candidate reference genes have Ct values between 18.85 and 37.66, the range of variation of H3 is the largest, the Ct value of Tubulin amplified by Tub-F1/Tub-R1 is between 23.35 and 32.66, the Ct value of Tubulin amplified by Tub-F2/Tub-R2 is between 20.83 and 30.83, EF1a is between 23.55 and 33.83, GAPDH is between 27.92 and 37.66, the range of variation is the smallest, the average Ct value of H3 is the highest, the average Ct value of GAPDH is the largest, the abundance of expression is the lowest, and the 4 candidate reference genes have different differences in expression levels in different samples.
(3) Exporting the Ct value obtained in the step (1) to sort data in Excel and making a standard curve to obtain the standard curveIs of the correlation coefficient (R) 2 ) And amplification efficiency (E), wherein the primer amplification efficiency is calculated as: e=10 -1/K -1, K is the slope and the results are shown in Table 3.
TABLE 3 primer set amplification results of RT-qPCR on candidate genes in Mixed cDNAs of samples No.1 to No. 4
Note that: in the table, tub1 represents the detection result of Tubulin gene amplification by Tub-F1/Tub-R1; in the following table, tub2 represents the results of detection of Tub-F2/Tub-R2 for Tubulin gene amplification.
As can be seen from Table 3, the standard curve R 2 The amplification efficiency is calculated according to a formula, and the amplification efficiency of five pairs of primers is between 0.9 and 1.1 (namely, between 90 and 110 percent), which indicates that the primers designed by the invention have strong specificity and high amplification efficiency, and the primer pair Tub-F1/Tub-R1 is better than Tub-F2/Tub-R2.
1.6 stability analysis of candidate reference genes
1.6.1 get Norm analysis and NormFinder analysis
The Ct values were analyzed using gemum software and NormFinder, respectively, to obtain stable values M for each reference gene, and the results are shown in Table 4.
TABLE 4 stability analysis results of candidate genes by gemm and NormFinder
The gemum software is a program written by Vandesompele et al and used for screening internal reference genes in real-time fluorescence quantitative PCR, and the analysis of the gemum program obtains a stable value M of the internal reference genes, and the smaller the value M is, the better the stability of the internal reference genes is, and the worse the stability is. As can be seen from Table 4, the M values of the candidate reference genes are less than 1.5, indicating higher stability, wherein Tubulin and EF1a are the two most stable genes.
normFinder is a procedure for screening stable reference genes, the smaller the expression stability value M, the more stable the gene expression. As can be seen from Table 4, the stability of each gene was ranked as Tubulin > EF1a > GAPDH > H3, with Tubulin being the most stable gene and the primer pair Tub-F2/Tub-R2 being superior to Tub-F1/Tub-R1.
1.6.2RefFinder analysis
Reffilter is a network tool for screening reference genes, has geNorm, normFinder, bestKeeper software functions, analyzes the Ct value amplified by all samples by using reffilter software, and compares the stability of each candidate gene, and the results are shown in table 5.
TABLE 5 stability analysis ranking results of RefFinder on candidate genes
Note that: the numbers 1-5 in the table represent the ranking.
As can be seen from Table 5, the stability rank of the candidate reference gene is named Tubulin (Tub-F1/Tub-R1) > Tubulin (Tub-F2/Tub-R2) > EF1a > GAPDH > H3.
As can be seen from the above examples, the two strains XJ592 and XJ511 have the best stability of the Tubulin gene under both conditions of culture in a medium and induction in cotton, respectively, and the primer pair Tub-F1/Tubu-R1 has better detection stability as an amplification primer than Tub-F2/Tub-R2. The invention uses the fluorescent quantitative PCR detection of the internal reference gene of the Tubulin gene verticillium dahliae, has good stability and can accurately and quantitatively detect the related gene expression of the verticillium dahliae.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
- The application of the Tubulin gene as an internal reference gene in the fluorescent quantitative PCR detection of the Verticillium dahliae is characterized in that the Verticillium dahliae comprises Verticillium lare and Verticillium dahliae.
- 2. The use according to claim 1, wherein the verticillium dahliae comprises strain XJ592; the non-verticillium dahliae includes a strain XJ511.
- 3. Primers for amplifying the Tubulin gene for use according to claim 1 or 2, comprising an upstream primer Tub-F1 and a downstream primer Tub-R1;the upstream primer Tub-F1 comprises a nucleotide sequence shown as SEQ ID NO. 1;the downstream primer Tub-R1 comprises a nucleotide sequence shown as SEQ ID NO. 2.
- 4. Use of the primer of claim 3 in fluorescent quantitative PCR detection of verticillium dahliae.
- 5. The use according to claim 4, wherein the reaction system for fluorescent quantitative PCR detection is 25 μl, comprising: 12.5. Mu.L of 2 XUltraSYBRMixture, 1. Mu.L of 2.5. Mu.M to 25. Mu.M upstream primer, 1. Mu.L of 2.5. Mu.M to 25. Mu.M downstream primer, 1 to 10ng of template cDNA and the balance of sterile water.
- 6. The use according to claim 4 or 5, wherein the reaction conditions for the fluorescent quantitative PCR detection are: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing at 58℃for 1min, extension at 72℃for 10s,40 cycles.
- 7. Use of the primer of claim 3 in the preparation of a verticillium dahliae fluorescent quantitative PCR detection kit.
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