Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Zhou, Lifeng; Chen, Fengmao; Ye, Jian‐Ren; Pan, Hongyang

doi:10.3389/fgene.2018.00269

Cited by 24 publications

(22 citation statements)

References 62 publications

(82 reference statements)

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“…Quantitative analysis was performed with pyrG2 as the internal reference gene primer, and the qPCR-specific primers in Table 3 were the suhB and ropN gene primers. Quantitative PCRs were performed on an ABI 7500 real-time PCR instrument (Zhou et al 2018).…”

Section: Burkholderia Pyrrocinia Jk-sh007 Biofilm-related Gene Cloninmentioning

confidence: 99%

Adding nutrients to the biocontrol strain JK-SH007 promotes biofilm formation and improves resistance to stress

Chen

Liu

et al. 2020

AMB Expr

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Burkholderia pyrrocinia JK-SH007 is an important biocontrol strain for the prevention and treatment of poplar canker disease. Its powerful biocontrol function is inseparable from its successful colonization of poplar trees. Bacterial biofilms can ensure the long-term colonization of a host. To explore the mechanism of action of biofilms in the biocontrol process, we manipulated various exogenous factors to explore the morphology of the JK-SH007 biofilm in vitro. The addition of glycerol and MgSO 4 to TSB medium stimulated biofilm production, increased the resistance of JK-SH007 to disease, enhanced the survival of JK-SH007 in nutrient-poor environments and maintained the antagonistic ability of JK-SH007 against the poplar canker pathogen. Therefore, we constructed and optimized a biofilm-forming system to produce a large number of stable JK-SH007 biofilms. The optimized system showed that the optimal incubation time for JK-SH007 biofilm formation was 14 h, the optimal temperature of the static culture was 25 °C, and the optimal pH was 5. The optimal medium for biofilm formation was TSB medium, 1% glycerol and 50 mM MgSO 4 . RT-qPCR experiments showed that an increase in the expression of the suhB gene promoted JK-SH007 biofilm formation, while an increase in the expression level of the ropN gene inhibited JK-SH007 biofilm formation. The possible mechanism by which JK-SH007 was inhibited by biofilm formation under natural culture was revealed. These results indicate the importance of adding nutrients to JK-SH007 biocides produced on a commercial scale. This is the first report of JK-SH007 producing a long-lasting biofilm that guarantees antagonism.

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Section: Burkholderia Pyrrocinia Jk-sh007 Biofilm-related Gene Cloninmentioning

confidence: 99%

Adding nutrients to the biocontrol strain JK-SH007 promotes biofilm formation and improves resistance to stress

Chen

Liu

et al. 2020

AMB Expr

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“…1 a, b). According to the literature data, at least two reference genes should be used for a given organism in every experiment [ 11 ]. Zhang et al .…”

Section: Resultsmentioning

confidence: 99%

“…DNA quantification, in relative quantification PCR method, is based on the determination of differences in expression between the target and reference genes. The reason for using one or more reference genes is to reduce the influence of a number of variables during the real-time PCR reaction, such as RNA integrity and quantity, efficiency in cDNA synthesis, and PCR amplification [ 11 ]. However, the most important aspect of the real-time PCR experiment planning is the selection of the reference gene, whose expression must be stable for that specific organism we work with [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

The Selection of Reliable Reference Genes for RT-qPCR Analysis of Anisakis simplex Sensu Stricto Gene Expression from Different Developmental Stages

et al. 2020

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Background Anisakis simplex s. s. is a parasitic nematode with a complex life cycle in which humans can become accidental hosts by consuming raw or not fully cooked fish containing L3 larvae. The growing popularity of raw fish dishes has contributed to an increase in the incidence of anisakiasis, which has spurred scientific efforts to develop new methods for diagnosing and treating the disease and also to investigate the gene expression at different developmental stages of this parasite. The identification of reference genes suitable for the normalization of RT-qPCR data has not been studied with respect to A. simplex s. s. Methods In the present study, eight candidate reference genes were analyzed in A. simplex s. s. at two different developmental stages: L3 and L4. The expression stability of these genes was assessed by geNorm and NormFinder softwares. Results In general, our results identified translation elongation factor 1 α ( ef-1α ) and peptidyl-prolyl isomerase 12 ( ppi12) as the most stable genes in L3 and L4 developmental stages of A. simplex s. s. Validation of the selected reference genes was performed by profiling the expression of the nuclear hormone receptor gene ( nhr 48 ) in different developmental stages. Conclusions This first analysis selecting suitable reference genes for RT-qPCR in A. simplex s. s. will facilitate future functional analyses and deep mining of genetic resources in this parasitic nematode. Electronic Supplementary Material The online version of this article (10.2478/s11686-020-00220-3) contains supplementary material, which is available to authorized users.

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“…UBQ was also used as a reference gene in cotyledons of Cunninghamia lanceolata [11]. However, this gene was unsuitable for data normalization in Bursaphelenchus mucronatus and lettuce (Lactuca sativa) [49,50]. Compared with Ts, HIS had higher expression stability in tissue samples and UBQ had higher expression stability in coldtreated samples.…”

Section: Discussionmentioning

confidence: 99%

“…5, Table 2) [8,23,51]. However, 18S could be used for data normalization in Bursaphelenchus mucronatus and C. sinensis under metal stress [45,49].…”

Section: Discussionmentioning

confidence: 99%

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

Tu¹,

Fan²,

Li³

et al. 2020

Preprint

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Background: Passiflora edulis is a tropical fruit with high edible and medicinal values that is widely cultivated in southern China. The development of new P. edulis variety with cold resistance and research on the mechanism of cold resistance are the basis for further promoting the cultivation of this species. In a previous study, our laboratory discovered a cold-resistant variety Pingtang 1, which is used as a material for the study of cold resistance mechanism in P. edulis. However, functional genes of P. edulis have not been well studied, especially via relative quantitative analysis. In addition, research on reference genes in P. edulis has not been reported.Results: We used three tools to test the expression stability of 10 candidate reference genes in multiple tissue samples and cold-treated samples of P. edulis. We found that Ts and EF1 showed the highest expression stability in all samples. Further analysis showed that HIS and EF1 were stably expressed in tissue samples, whereas UBQ and EF1 were stably expressed in cold-treated samples. Interestingly, EF1 was stably expressed in not only multiple tissue samples but also cold-treated samples. To validate the selected reference genes, we used the target gene ICE1 and investigated its expression level in cold-treated leaves. Interestingly, the expression of ICE1 increased with increasing cold treatment times.Conclusions: In this study, we successfully selected reference genes from among 10 candidates for P. edulis RT-qPCR data normalization. We selected Ts and EF1 as reference genes for normalization in all samples. HIS and EF1 were ideal for data normalization in tissue samples, whereas UBQ and EF1 were ideal for cold-treated samples. Our work substantially benefits the study of functional genes in P. edulis.

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Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Cited by 24 publications

References 62 publications

Adding nutrients to the biocontrol strain JK-SH007 promotes biofilm formation and improves resistance to stress

Adding nutrients to the biocontrol strain JK-SH007 promotes biofilm formation and improves resistance to stress

The Selection of Reliable Reference Genes for RT-qPCR Analysis of Anisakis simplex Sensu Stricto Gene Expression from Different Developmental Stages

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

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