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Screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (PCR) reference genes and primers thereof

A technology of Burkholderia Populus and internal reference gene, which is applied in the field of molecular biology, can solve the problems of deviation of experimental results and unstable RNA expression level of internal reference gene, and achieves the effects of high stability and wide adaptability

Active Publication Date: 2020-09-25
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under different experimental conditions, the RNA expression level of internal reference genes is not stable, and the analysis of gene expression levels based on the internal reference genes commonly used in the literature may cause deviations in experimental results

Method used

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  • Screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (PCR) reference genes and primers thereof
  • Screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (PCR) reference genes and primers thereof
  • Screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (PCR) reference genes and primers thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0028] Screening of embodiment 1B.gladioli reference gene

[0029] The strains used were B.gladioli HDXY-02 isolated and preserved in our laboratory and B.gladioli CICC10574 purchased from China Industrial Microbiology Culture Collection Center (CICC). The study found that B.gladioli HDXY-02 can produce obvious yellow pigment when using LB or KMB solid and liquid medium. After separation, extraction and identification, the pigment was determined to be toxin. Further research found that compared with the standard strain B.gladioli CICC 10574 purchased from CICC and B.gladioli HDXY-02, no yellow pigment was observed under either solid or liquid culture conditions. Growth curve and toxin synthesis analysis results (such as figure 1 shown), there is no significant difference in the growth of the two strains, but there is a significant difference in the toxin synthesis ability of the two strains (the bar graph is the toxin production), and B.gladioli HDXY-02 began to produce a la...

Embodiment 2

[0059] The expression stability analysis of the candidate internal reference gene of embodiment 2

[0060] 1. geNorm analysis

[0061] The geNorm software compares the genes in pairs to obtain the relative value of each gene change, and judges the stable value of the internal reference gene according to the M value. The lower the M value, the better the stability. Generally speaking, genes with an M value less than 1.5 indicate Its expression level is relatively stable.

[0062] Input the obtained Ct value into the geNorm program to obtain the expression stability value (M value) of each internal reference gene, and sort the internal reference genes according to the size of the M value. The results are shown in Table 2. At different culture stages, the pyrG and thyA genes of B.gladioli HDXY-02 were relatively stable, and the 16S and gyrB genes of B.gladioli CICC10574 were relatively stable; at different culture temperatures, the thyA genes of B.gladioli HDXY-02 and 16S genes...

Embodiment 3

[0080] Example 3 Internal reference genes are applied to qRT-PCR to detect the expression of each gene in the tox gene cluster

[0081] The results of transcriptome sequencing (sampling point is the late logarithmic phase) showed that compared with B.gladioli CICC10574 as the control, the genes of the toxoflavin synthesis gene cluster toxABCDE and the transport gene cluster toxFGHI were significantly up-regulated in B.gladioli HDXY-02.

[0082] The transcriptome sequencing results were verified by qRT-PCR, and clpX was selected as an internal reference gene for qRT-PCR verification (see Table 6 for the primer sequences of genes related to toxoflavin synthesis). The experimental results were consistent with the results of transcriptome sequencing. Compared with B.gladioliCICC10574, the expression levels of toxoflavin synthesis genes toxA, toxB, toxC, toxD, toxE and transport genes toxF, toxG, toxH, toxI were lower in B.gladioli HDXY- 02 were increased by 5-10 times and 2-5 time...

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Abstract

The invention discloses screening and application of burkholderia gladioli real-time quantitative polymerase chain reaction (qRT-PCR) reference genes and primers thereof. According to obtained transcriptome analysis results and reported reference genes, 12 candidate reference genes with relatively stable expression are preliminarily screened, and gene names are shown as follows: atpD, clpP, clpX,ftsZ, gyrB, lpxC, pyrG, recA, rpoB , rpoD, thyA and 16S; for the candidate genes, amplification primers are designed; reference genes under different culture conditions (temperature, initial pH valueof medium, culture time, treatment with NaCl different in concentration) of burkholderia gladioli are screened; geNorm, NormFinder and Bestkeeper software is employed to analyze qRT-PCR results; and finally, the reference genes with most stable expression under different culture conditions and NaCl treatment condition are obtained by screening. The invention is beneficial to the stability and reliability of analysis and study of gene expression of the burkholderia gladioli under different conditions.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to the screening and application of Burkholderia gladiolus real-time fluorescence quantitative PCR internal reference gene and primers thereof. Background technique [0002] Burkholderia (Burkholderia) is a Gram-negative rod-shaped bacterium that widely exists in water, soil, plants and human bodies. Burkholderia can be used in agriculture for biodegradation, biocontrol and promotion of plant growth. Some Burkholderia bacteria can synthesize small molecule antibacterial substances-toflavin, which has good tumor cell inhibitory activity and antifungal activity (especially the inhibitory activity against azole drug-resistant Aspergillus fumigatus), It is a compound with good application prospects, and can be used as a candidate drug after structural modification. Gladiolus Burkholderia (Burkholderia gladioli) HDXY-02 has the characteristic of high toxin production, and ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/6809G16B40/00G16B25/10G16B25/20G16B30/10G16B20/00G16B20/20C12R1/01
CPCC12Q1/689C12Q1/6851C12Q1/6809G16B40/00G16B25/10G16B25/20G16B30/10G16B20/00G16B20/20C12Q2600/166C12Q2563/107C12Q2561/113C12Q2545/101C12Q2531/113
Inventor 李晓丹汪仁徐晟王蓉江玉梅周正雄李宜奎王松凤
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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