CN111705149A

CN111705149A - Burkholderia gladioli fluorescence quantitative PCR reference gene and screening and application of primer thereof

Info

Publication number: CN111705149A
Application number: CN202010595646.4A
Authority: CN
Inventors: 李晓丹; 汪仁; 徐晟�; 王蓉; 江玉梅; 周正雄; 李宜奎; 王松凤
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2020-09-25
Anticipated expiration: 2040-06-28
Also published as: CN111705149B

Abstract

The invention discloses a Burkholderia gladioli real-time fluorescent quantitative PCR (qRT-PCR) reference gene and screening and application of primers thereof. According to the obtained transcriptome analysis result and the reported reference genes, 12 candidate reference genes with relatively stable expression are preliminarily screened, and the gene names are respectively as follows:atpD、clpP、clpX、ftsZ、gyrB、lpxC、pyrG、recA、rpoB、rpoD、thyAand16S(ii) a Aiming at the candidate genes, an amplification primer is designed, the reference genes of Burkholderia gladioli under different culture conditions (temperature, initial pH value of a culture medium, culture time and NaCl treatment with different concentrations) are screened, the qRT-PCR result is analyzed by adopting geNorm, NormFinder and Bestkeeper software, and the reference genes which are most stably expressed under different culture conditions and NaCl treatment conditions are finally screened. The invention is beneficial to the stability and reliability of the Burkholderia gladioli gene expression analysis research under different conditions.

Description

Burkholderia gladioli fluorescence quantitative PCR reference gene and screening and application of primer thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a Burkholderia gladioli real-time fluorescent quantitative PCR reference gene and screening and application of primers thereof.

Background

Burkholderia (Burkholderia) is a gram-negative coryneform bacterium, and is widely present in water, soil, plant bodies, and human bodies. Burkholderia can be used for biodegradation, biocontrol and plant growth promotion in agriculture. Part of Burkholderia can synthesize a small molecular antibacterial substance, toxoflavin, which has good tumor cell inhibitory activity and antifungal activity (especially the inhibitory activity on azole drug-resistant aspergillus fumigatus), is a compound with good application prospect, and can be used as a candidate drug after structural modification. Burkholderia gladioli (Burkholderia gladioli) HDXY-02 has the characteristic of high toxoflavin yield, and has important practical significance as a production strain for biosynthesis of toxoflavin. During the research, the significant difference between the B.gladioli HDXY-02 and the toxoflavin synthesis capacity of another B.gladioli CICC10574 which is purchased from the China Center for Industrial Culture Collection (CICC) is found, and the B.gladioli CICC10574 hardly produces toxoflavin. Transcriptome sequencing shows that the expression levels of the genes related to the synthesis of toxoflavin of the two strains are significantly different, but further verification is required.

The real-time fluorescent quantitative PCR (qRT-PCR) has the advantages of simple operation, low cost and high sensitivity, and is often used for the research of the transcription level of target genes and the verification of transcriptome data. The qRT-PCR experiment requires the expression of a stable housekeeping gene as an internal reference. However, the RNA expression level of the reference gene is not stable under different experimental conditions, and the experimental results may be biased by analyzing the gene expression level only based on the reference gene commonly used in the literature. In order to analyze the expression level of a gene, it is necessary to select an appropriate reference gene according to the need of each study. Therefore, it is necessary to select stably expressed reference genes from various reference genes, namely B.gladioli HDXY-02 and B.gladioli CICC 10574. Determination of internal reference genes is critical to accurately analyze the expression difference research of genes in different strains of B. The invention screens the reference genes of B.gladioli HDXY-02 and B.gladioli CICC10574, aims to find the most stable reference genes in the B.gladioli HDXY-02 and B.gladioli CICC10574, lays a foundation for the subsequent gene expression analysis and the transcriptional regulation and control mechanism research of different B.gladioli strains, and provides a basis for further improving the toxoflavin yield of the B.gladioli HDXY-02.

Disclosure of Invention

The invention aims to provide an internal reference gene of real-time fluorescence quantitative PCR (qRT-PCR) of a B.gladioli strain under different culture conditions and NaCl treatment at different concentrations and primers thereof.

The Burkholderia (Latin classification named Burkholderia sp.) HDXY-02 is preserved in China general microbiological Culture Collection Center (CGMCC), the preservation unit address is No. 3 of Ministry No.1 of West Cheng of the south-oriented region in Beijing, the microbial research institute of China academy of sciences, the preservation date is 4 months and 20 days in 2017, the registration number of the strain is CGMCC No.14054, and the strain is identified as Burkholderia gladioli (Burkholderia gladioli). The other strain is B.gladioli CICC10574 purchased from China Center of Industrial Culture Collection (CICC).

In order to solve the technical problems, the invention adopts the following technical scheme: two strains of B.gladioli bacteria were treated at different culture temperatures, initial pH values of the medium and culture times B.gladioli HDXY-02 and B.gladioli CICC10574, and additionally with different NaCl concentrations. And extracting total RNA after sampling, removing genomic DNA by using a reverse transcription kit, carrying out reverse transcription on the genomic DNA to synthesize cDNA, carrying out qRT-PCR on the candidate internal reference genes, and carrying out data analysis by adopting geNorm, NormFinder and BestKeeper software so as to screen out the internal reference genes which are most stably expressed in B.gladioli under different culture conditions.

The invention relates to a screening method and application of B.gladioli qRT-PCR reference genes and primers, which comprises the following steps:

(1) obtaining a candidate reference gene sequence: selecting 14 stably expressed genes in B.gladioli HDXY-02 and B.gladioli CICC10574 transcriptome sequencing results as candidate genes of the reference genes;

(2) primer synthesis and primary screening:

a. designing a primer (http:// primer3.ut. ee /), controlling the gene amplification length to be between 80 and 250bp, and synthesizing the primer by Shanghai biology;

b. after PCR amplification verification, 12 candidate internal reference genes (atpD, clpP, clpX, ftsZ, gyrB, lpxC, pyrG, recA, rpoB, rpoD, thyA and 16S) which are amplified to a single strip are used for the screening of qRT-PCR internal reference genes in the next step B.gladioli HDXY-02 and B.gladioli CICC 10574;

c. calculating regression coefficients (R) of 12 candidate reference genes²) And amplification efficiency, judging whether the primer meets the requirements of qRT-PCR, wherein the primer sequence and related information are detailed in Table 1;

(3) preparation of qRT-PCR template:

a. sample preparation: inoculating activated B.gladioli strain into LB liquid culture medium for overnight culture, respectively inoculating liquid culture medium according to 1% of inoculation amount, culturing under the conditions of different temperatures (25 ℃, 30 ℃, 37 ℃ and 40 ℃) and different initial pH values (5, 6, 7 and 8), sampling for 24h and 48h, sampling for different growth periods (4h, 8h, 12h, 24h, 36h and 48h), adding NaCl (0%, 1%, 1.5% and 2%) with different concentrations into KMB culture medium for treatment, respectively taking 1mL of bacterial liquid from each sample, centrifugally collecting thalli at 12,000rpm, and storing at-80 ℃ for later use;

b. and (3) extracting total RNA of the thalli and detecting the quality: adding 1mL Trizol to resuspend the lysed thallus, standing the lysed sample at room temperature for 5-10min, adding 0.4mL chloroform, vigorously shaking for 15sec, standing at room temperature for 3min, centrifuging at 12,000rpm for 10min at 4 ℃, sucking the upper aqueous phase, transferring to a clean centrifuge tube, adding isopropanol with the same volume, mixing, standing at room temperature for 20min, centrifuging at 12,000rpm for 10min at 4 ℃, discarding the supernatant, adding 1mL 75% ethanol to wash the precipitate, centrifuging at 12,000rpm for 3min at 4 ℃, discarding the supernatant, drying at room temperature for 5-10min, adding a proper amount of RNase-free ddH₂Dissolving RNA in O, taking 1-2 μ L of RNA sample, adding 10 × Loading Buffer, performing agarose gel electrophoresis, detecting RNA integrity, diluting RNA appropriately, and detecting absorbance (A)₂₃₀、A₂₆₀、A₂₈₀And A₃₂₀) And calculating the concentration of the RNA sample;

c, cDNA synthesis: PrimeScript obtained by Takara^TMAn RT reagent Kit with gDNA Eraser reverse transcription Kit, firstly removing genome DNA, preparing reaction liquid according to the Kit specification, and carrying out 2min at 42 ℃; then RNA is respectively reverse transcribed into cDNA, 2 mug of RNA is added into each reverse transcription system, reverse transcription is carried out for 15min at 42 ℃ and 5s at 85 ℃, and the activity of the reverse transcriptase is removed;

SYBR Green real-time fluorescent quantitative PCR amplification: preparing a PCR reaction system, wherein the reaction system is 20 mu L, qRT-PCR reaction conditions comprise 5min at 95 ℃, 10s at 95 ℃ and 30s at 60 ℃, and the reaction is carried out for 40 cycles.

The invention has the advantages that: in order to detect the relative expression of genes at the transcription level and remove the possible differences in RNA yield, quality and reverse transcription efficiency among different samples to obtain the true difference of the specific expression of the target gene, an appropriate reference gene is usually selected for correction. The primer sequence of the selected B.gladioli internal reference gene has high specificity amplification, near amplification efficiency and good linear correlation, and can be used for detecting the expression quantity change of the gene in different B.gladioli strains.

The invention has the following advantages:

(1) the stability is high: exporting Ct values obtained by experiments, respectively adopting three software geNorm, NormFinder and BestKeeper based on different calculation methods to perform data analysis, finally, comprehensively analyzing results by combining the three software, and comprehensively evaluating 12 candidate internal reference genes to show that the clpX gene is most stable under a common culture condition, taking B.gladioli HDXY-02 and B.gladioli CICC10574 cultured for 24h as samples, taking toxoflavin to synthesize each gene of related tox gene clusters as a target gene, and selecting the clpX gene which can be used as the internal reference gene of B.gladioli qRT-PCR under the common culture condition, and simultaneously verifying the expression rule of each gene of the tox gene cluster in the transcriptional group sequencing results of B.gladioli HDXY-02 and B.gladioli CICC 10574;

(2) the adaptability is wide: determining the most suitable reference genes under different conditions for gene expression difference analysis in the subsequent B.gladioli, selecting different reference genes under different culture conditions to analyze the gene expression level by qRT-PCR, wherein lpxC can be used as the reference gene of B.gladioli qRT-PCR under the NaCl treatment condition, and verifying the expression level difference of each gene of tox gene cluster related to tox synthesis of B.gladioli CICC10574 when NaCl is treated and not treated.

The invention solves the selection problem of the reference gene of the B.gladioli under different culture conditions and during NaCl treatment, and can establish the real-time fluorescent quantitative PCR detection method of the B.gladioli under different culture conditions.

Drawings

FIG. 1 is a graph of growth curves and toxoflavin production for B.gladioli HDXY-02 and B.gladioli CICC10574, in which A is B.gladioli HDXY-02 and B is B.gladioli CICC 10574.

FIG. 2 is an agarose electrophoresis pattern of a PCR amplification product of a part of candidate reference genes.

FIG. 3 is a qRT-PCR verification graph of the gene expression amount analysis of toxoflavin synthesis-related tox gene clusters of B.gladioli HDXY-02 and B.gladioli CICC 10574.

FIG. 4 is a graph showing the analysis of the expression level of each gene in the tox gene cluster related to flavin synthesis in B.gladioli CICC10574 after NaCl treatment by qRT-PCR detection.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1B screening of the gladioli reference Gene

The strains used are B.gladioli HDXY-02 separated and preserved in the laboratory and B.gladioli CICC10574 purchased from the China center for culture Collection of Industrial microorganisms (CICC). Gladioli HDXY-02 was found to produce a clear yellow pigment when LB or KMB solid and liquid media were used. After separation, extraction and identification, the pigment is determined to be toxoflavin. Further studies found that no production of yellow pigment was observed in the standard strain b.gladioli cic 10574 purchased from cic, compared to b.gladioli HDXY-02, whether in solid or liquid culture conditions. The growth curve and the analysis result of the toxoflavin synthesis amount (shown in figure 1) show that the two strains have no obvious difference in growth, but have obvious difference in toxoflavin synthesis capacity (histogram is the yield of toxoflavin), and B.gladioli HDXY-02 starts to synthesize a large amount of toxoflavin at about 20h of culture (figure 1A), but B.gladioli CICC10574 hardly produces toxoflavin (figure 1B).

Transcriptome sequencing finds that the expression quantity of the toxoflavin synthesis related genes of the two strains has a significant difference, but further qRT-PCR verification is required. Since the reference gene has a great influence on the results of gene expression analysis, it is necessary to screen reference primers for B.gladioli HDXY-02 and B.gladioli CICC10574 qRT-PCR.

1. Primer design

The primers for the 14 candidate reference genes required for the experiment were designed on-line (http:// primer3.ut. ee /), and the amplification length was controlled to be between 80 and 250bp, and synthesized by Shanghai Biotechnology Ltd.

2. Extraction and quality detection of total RNA of thallus

(1) Selecting a B.gladioli strain single colony, and extracting RNA;

(2) taking 1mL of bacterial liquid, centrifuging at 12,000rpm for 1min, and collecting cells;

(3) adding 1mL of Trizol into a centrifuge tube;

(4) placing the lysed sample or homogenate at room temperature for 5-10min to completely separate the nucleoprotein and the nucleic acid;

(5) 0.4mL of chloroform was added thereto, followed by vigorous shaking for 15sec and standing at room temperature for 3 min. Centrifuging at 12,000rpm at 4 deg.C for 10 min;

(6) absorbing the upper aqueous phase, transferring to a clean centrifuge tube, adding isopropanol with the same volume, mixing uniformly, and standing at room temperature for 20 min;

(7) centrifuging at 12,000rpm at 4 deg.C for 10min, and discarding the supernatant;

(8) the precipitate was washed by adding 1mL of 75% ethanol. Centrifuge at 12,000rpm for 3min at 4 ℃ and discard the supernatant. Drying at room temperature for 5-10 min;

(9) adding 30-50 μ L RNase-free ddH₂O, fully dissolving RNA;

(10) taking a proper amount of RNA sample, adding 10 XLoading Buffer, performing 1% agarose gel electrophoresis under the voltage of 100V, and detecting the integrity of RNA;

(11) RNA was diluted by a reasonable fold and A was measured₂₆₀And A₂₈₀Numerical value, purity of RNA was examined.

After 1% agarose gel electrophoresis, the bands of 23S rRNA and 16S rRNA are very clear, bright and have no tailing phenomenon, and the band of 5S rRNA is provided, which indicates that the integrity of the extracted RNA is better. The OD value (OD) of each RNA sample at different wavelengths was determined by a spectrophotometer₂₃₀、OD₂₆₀、OD₂₈₀And OD₃₂₀) And calculating the concentration of the RNA sample. RNA A of samples₂₆₀/₂₈₀Between 1.8 and 2.0, indicating that there are fewer protein impurities in the RNA, and RThe integrity of the NA is better.

3. cDNA Synthesis

According to the RNA quantitative result, the volume of the added RNA in the reverse transcription system is determined, and the reverse transcription amount of the RNA is ensured to be 2 mu g. Using a reverse transcription kit, firstly removing genome DNA, preparing reaction liquid according to the kit specification, and carrying out reaction for 2min at 42 ℃; the RNAs were then separately reverse transcribed into cDNAs. Reverse transcription was carried out at 42 ℃ for 15min and at 85 ℃ for 5s, and the reverse transcriptase activity was removed.

4. SYBR Green qRT-PCR system and amplification program

The qRT-PCR reaction system used was 20. mu.L, where AceQ qPCR SYBR Green Master Mix 10. mu.L, primers 0.5. mu.L each (10 pmol. mu.L)^-1) Adding template cDNA, and using ddH₂O is added to a constant volume of 20. mu.L. qRT-PCR adopts a two-step method, the reaction is carried out for 40 cycles at 95 ℃ for 5min, 95 ℃ for 10s and 60 ℃ for 30 s.

5. Standard curve and amplification efficiency analysis of reference gene

(1) Taking cDNA of the strain, taking 10 times as a gradient, and carrying out gradient dilution on the cDNA, wherein the cDNA is respectively diluted by 10 times, 100 times, 1,000 times and 10,000 times;

(2) qRT-PCR was performed using these gradient cDNAs as templates, with 3 replicates per group;

(3) after qRT-PCR reaction is finished, the average value of Ct values of each group of three parallel Ct values is calculated, a standard curve of the reference gene under different template concentrations is drawn, and a curve equation, amplification efficiency and R are obtained². After calculating the slope K of the standard curve, the equation E is substituted into (10)^-(1/K)-1) × 100%, the amplification efficiency (E) of the primer was calculated.

Primers were designed based on Burkholderia gladioli genome data. In 12 of the 14 primers (see Table 1 for primer sequences), a single signal peak was observed, indicating no non-specific band and no formation of primer dimer, and the 12 candidate genes were atpD, clpP, clpX, ftsZ, gyrB, lpxC, pyrG, recA, rpoB, rpoD, thyA and 16S.

And (3) taking the cDNA of B.gladioli HDXY-02 and B.gladioli CICC10574 as templates, and detecting the primer amplification efficiency of the qRT-PCR primer of the internal reference gene 12. cDN diluted in a gradientA was used as a template for PCR amplification, and information on the qRT-PCR primers was calculated (Table 1). As can be seen from Table 1, the regression coefficients (R) of the 12 candidate reference gene amplification standard curves²) Between 0.9916 and 0.9999, the amplification efficiency is between 90.5 percent and 109.2 percent, and the method meets the requirements of qRT-PCR primers.

The results of agarose electrophoresis are shown in FIG. 2. The primers amplified fragments of the expected size, the bands were single and no primer dimer appeared.

TABLE 1 reference genes qRT-PCR primer information

Example 2 expression stability analysis of candidate reference genes

1. GeNorm analysis

The geNorm software obtains the relative value of each gene change by pairwise comparison of the genes, and judges the stability value of the reference gene according to the M value, wherein the lower the M value is, the better the stability is, and generally, the genes with the M value less than 1.5 indicate that the expression level is more stable.

And inputting the Ct value into a geonorm program to obtain an expression stability value (M value) of each reference gene, and sequencing the reference genes according to the size of the M value. The results are shown in Table 2, where the pyrG and thyA genes of B.gladioli HDXY-02 were more stable and the 16S and gyrB genes of B.gladioli CICC10574 were more stable at different cultivation stages; the thyA and 16S genes of B.gladioli HDXY-02 are more stable, and the lpxC and pyrG genes of B.gladioli CICC10574 are more stable at different culture temperatures; the thyA and pyrG genes of B.gladioli HDXY-02 are more stable, and the pyrG and clpX genes of B.gladioli CICC10574 are more stable, at different initial pH values.

TABLE 2 GeNorm software analysis results

2. NormFinder analysis

The normafinder software calculates the variability within and between groups and calculates the Stability Value (SV) based on a model based approach. The NormFinder program assesses the stability of reference genes on the basis of stability values (SV values), the smaller the SV value, the higher the stability of the gene, similar to geonorm. The results of the NormFinder on candidate reference gene treatments of B.gladioli HDXY-02 and B.gladioli CICC10574 under different culture conditions are shown in Table 3.

Under the NaCl treatment condition, gyrB and rpoD genes of B.gladioli HDXY-02 are relatively stable, and gyrB and pyrG genes of B.gladioli CICC10574 are relatively stable; in different culture stages, the pyrG and 16S genes of B.gladioli HDXY-02 are more stable, and the 16S and gyrB genes of B.gladioli CICC10574 are more stable; the thyA and 16S genes of B.gladioliHDXY-02 are more stable, and the lpxC and pyrG genes of B.gladioli CICC10574 are more stable at different culture temperatures; the thyA and pyrG genes of B.gladioli HDXY-02 were more stable, and the pyrG and thyA genes of B.gladioli CICC10574 were more stable, depending on the initial pH. The results of the analysis of the NormFinder are similar to the results of the geNorm in terms of ranking. The ftsZ gene has a high M value under various conditions and is relatively unstable.

TABLE 3 analysis results (stability values) by the NormFinder software

3. Bestkeeper analysis

The stability of each reference gene was mainly judged by bestkoeper by calculating the standard deviation (SD value) and the coefficient of variation (CV value). The smaller the Standard Deviation (SD) and coefficient of variation (Cv) calculated by the BestKeeper program, the more stable the reference gene.

From the results of the BestKeeper program analysis (Table 4), the more stable genes of B.gladioli HDXY-02 were thyA and pyrG, and the more stable genes of B.gladioli CICC10574 were gyrB and clpP at different culture stages; the more stable genes of B.gladioli HDXY-02 are lpxC and 16S, and the more stable genes of B.gladioli CICC10574 are thyA and pyrG at different culture temperatures; under different pH conditions, the most stable gene in both strains is thyA.

TABLE 4 Bestkeeper software analysis of the expression stability of each reference gene

4. Comprehensive analysis

Since two strains were analyzed simultaneously, the expression stability of the reference genes of two strains of Burkholderia gladioli HDXY-02 and CICC10574 was analyzed using the 3 software GeNorm, NormFinder and BestKeeper, and the stability of each reference gene was comprehensively ranked according to the calculated geometric mean (Table 5).

The result shows that the stability of the lpxC gene under all conditions is highest in comprehensive ranking, the lpxC gene can be used as an internal reference gene applicable to Burkholderia gladioli HDXY-02 and the standard strain CICC10574 under different experimental conditions, and the expression of the lpxC gene is most stable under different initial pH values and culture temperatures except different culture times. The most stable internal reference gene was expressed at different culture times as 16S. The comprehensive analysis of the ftsZ gene is most unstable under all conditions, and the ftsZ gene is not suitable for being used as an internal reference gene in the analysis of the gene expression difference in B.gladioli HDXY-02 and B.gladioli CICC 10574.

TABLE 5 stability ranking of various reference genes in different strains

Example 3 application of reference genes to qRT-PCR for detecting expression level of each gene of tox gene cluster

Transcriptome sequencing results (sampling points are late log phase) show that the genes of the toxoflavin synthesis gene cluster toxABCDE and the transporter gene cluster toxFGHI are significantly up-regulated in b.gladioli HDXY-02, using b.gladioli CICC10574 as a control.

The sequencing result of the transcriptome was verified by qRT-PCR, and qRT-PCR verification was performed using clpX as an internal reference gene (the sequence of the toxoflavin synthesis related gene primer is shown in Table 6). The experimental results are consistent with the sequencing results of transcriptome, compared with B.gladiolicICCC10574, the expression levels of toxoflavin synthesis genes toxA, toxB, toxC, toxD, toxE and transport genes toxF, toxG, toxH and toxI are respectively up-regulated by 5-10 times and 2-5 times in B.gladioli HDXY-02 (figure 3), the results of qRT-PCR also prove the credibility of transcriptome data to a certain extent, and simultaneously show that the clpX gene has feasibility in the application of gene expression level analysis of two strains.

TABLE 6 Duoxigenin Synthesis related Gene primer sequences

Experiments show that the synthetic amount of B.gladioli CICC10574B.gladioli CICC10574 toxoflavin can reach more than 10 times of that of untreated after NaCl with the final concentration of 10g/L is added into a KMB culture medium. The lpxC is selected as an internal reference gene, and the expression quantity of the toxoflavin synthesis related gene in B.gladioli CICC10574 is improved by 4.72-14.93 times after NaCl treatment (shown in figure 4), which indicates that the synthesis capacity of toxoflavin in B.gladioli CICC10574 is improved by activating the transcription of the toxoflavin synthesis related gene in B.gladioli CICC10574 with NaCl.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

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<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>primer_bind

<222>(1)..(20)

<223>thyA-F

<400>21

ctctaccagt tcctgcccaa 20

<210>22

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>primer_bind

<222>(1)..(20)

<223>thyA-R

<400>22

gccgatgaag taggtgaacc 20

<210>23

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>primer_bind

<222>(1)..(20)

<223>16S-F

<400>23

aaagcctgat ccagcaatgc 20

<210>24

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>primer_bind

<222>(1)..(20)

<223>16S-R

<400>24

gattaacgct cgcaccctac 20

Claims

1. Burkholderia gladioli (B.)Burkholderia gladioli) The method for screening the reference gene is characterized in that: after stress treatment is carried out on different culture medium initial pH values, culture time and culture temperature or NaCl with different concentrations, total RNA is extracted, reverse transcription is carried out to synthesize cDNA, real-time fluorescent quantitative PCR (qRT-PCR) verification is carried out, and geNorm and BestKeeper software are adopted to carry out numerical controlAccording to the analysis, thereby screening outB. gladioliThe most stably expressed internal reference gene after different culture conditions and NaCl treatment specifically comprises the following steps:

(1) adopting an on-line design to synthesize specific qRT-PCR primers of candidate reference genes, determining the amplification specificity of the primers by utilizing fluorescent quantitative PCR, diluting cDNA according to 10 times of gradient, respectively carrying out quantitative PCR, establishing a standard curve and calculating the amplification efficiency of the primers;

(2) selecting different treatments separatelyB. gladioliExtracting total RNA as experimental material, reverse transcribing to synthesize cDNA and qRT-PCR analysis;

(3) and (3) inputting the Ct value obtained by qRT-PCR into Normfinder, geNorm and BestKeeper software to perform internal reference gene stability analysis, and performing comprehensive analysis by combining the results of the three software.

2. The candidate reference gene of step 1 of claim 1 (c) ((atpD、clpP、clpX、ftsZ、gyrB、lpxC、pyrG、recA、rpoB、rpoD、thyAAnd16S) The qRT-PCR primers are respectively the following base sequences:

atpDthe forward primer atpD-F of (1) is shown as SEQ ID NO.1, and the reverse primer atpD-R is shown as SEQ ID NO. 2;

clpPthe forward primer clpP-F is shown as SEQ ID NO. 3, and the reverse primer clpP-R is shown as SEQ ID NO. 4;

clpXthe forward primer clpX-F is shown as SEQ ID NO. 5, and the reverse primer clpX-R is shown as SEQ ID NO. 6;

ftsZthe forward primer ftsZ-F is shown as SEQ ID NO. 7, and the reverse primer ftsZ-R is shown as SEQ ID NO. 8;

gyrBthe forward primer gyrB-F is shown as SEQ ID NO. 9, and the reverse primer gyrB-R is shown as SEQ ID NO. 10;

lpxCthe forward primer lpxC-F is shown as SEQ ID NO. 11, and the reverse primer lpxC-R is shown as SEQ ID NO. 12;

pyrGthe forward primer pyrG-F is shown as SEQ ID NO. 13, and the reverse primer pyrG-R is shown as SEQ ID NO. 14;

recAthe forward primer recA-F is shown as SEQ ID NO. 15, and the reverse primer recA-R is shown as SEQ ID NO. 16;

rpoBthe forward primer rpoB-F is shown as SEQ ID NO. 17, and the reverse primer rpoB-R is shown as SEQ ID NO. 18;

rpoDthe forward primer rpoD-F is shown as SEQ ID NO. 19, and the reverse primer rpoD-R is shown as SEQ ID NO. 20;

thyAthe forward primer thyA-F is shown as SEQ ID NO. 21, and the reverse primer thyA-R is shown as SEQ ID NO. 22;

16Sthe forward primer 16S-F is shown as SEQ ID NO. 23, and the reverse primer 16S-R is shown as SEQ ID NO. 24.

3. The method of claim 1B. gladioliThe strains are HDXY-02 and CICC 10574.

4. The method of claim 1, wherein the reference gene is selected by fluorescence quantitative PCRB. gladioliThe application of the expression and function of toxoflavin synthesis related transport gene is characterized in that the toxoflavin synthesis related transport gene is screened outclpXThe gene is the most stable internal reference gene under the general culture condition,lpxCthe gene is the most stable reference gene under NaCl treatment.

5. The reference gene of claim 4B. gladioliThe application of the expression and function research of toxoflavin synthesis related transport gene is characterized in thatB. gladioliHDXY-02 andB. gladioliCICC10574 is used as a sample, and toxoflavin is used for synthesizing a gene clustertoxA、toxB、toxC、toxD、toxEGenes and Transporter Gene clusterstoxF、toxG、toxHAndtoxIselected as a target geneclpXThe gene is a reference gene and can be used for discussing different genesB. gladioliAnalyzing the expression rule of the flavin synthesis related gene in the strain.

6. The reference gene according to claim 4B. gladioliToxoflavin synthesis related transport genesThe expression and function research aspect of (1), characterized in that the most stable reference gene under NaCl treatment conditionlpxCCan be used as an assayB. gladioliAnd (3) an internal reference gene for analyzing the expression of a toxoflavin synthesis related gene after NaCl treatment in the CICC 10574.