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Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (St å l)

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ABSTRACT

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies.

No MeSH data available.


Stability of the reference gene expression in N. lugens reared on rice under different temperatures.Candidate genes (ylsActin, ylsEF1α, ylsGADPH, ylsRPS11, ylsRPS15e, ylsRPS15p, ylsTub, ylsUBQ) were evaluated using the comparative ΔCt method (A), NormFinder (B), geNorm (C) and BestKeeper (D).
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f5: Stability of the reference gene expression in N. lugens reared on rice under different temperatures.Candidate genes (ylsActin, ylsEF1α, ylsGADPH, ylsRPS11, ylsRPS15e, ylsRPS15p, ylsTub, ylsUBQ) were evaluated using the comparative ΔCt method (A), NormFinder (B), geNorm (C) and BestKeeper (D).

Mentions: With the samples exposed to different temperature conditions, the comparative ΔCt method, NormFinder and BestKeeper analyses indicated that ylsRPS15e and ylsGAPDH were the most stable genes, while ylsACT and ylsTUB were the most stable genes found by geNorm (Fig. 5). According to RefFinder, the overall order from most to least stable is ylsRPS15e > ylsGADPH > ylsACT > ylsTUB > ylsEF1α > ylsUBQ > ylsRPS15p > ylsRPS11 (Table 2). The geNorm analysis revealed that almost of all pairwise (Vn/Vn + 1) variations were below 0.15 (Fig. 3). Thus, the combination of reference genes recommended for this subset is ylsRPS15e and ylsGADPH.


Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (St å l)
Stability of the reference gene expression in N. lugens reared on rice under different temperatures.Candidate genes (ylsActin, ylsEF1α, ylsGADPH, ylsRPS11, ylsRPS15e, ylsRPS15p, ylsTub, ylsUBQ) were evaluated using the comparative ΔCt method (A), NormFinder (B), geNorm (C) and BestKeeper (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304208&req=5

f5: Stability of the reference gene expression in N. lugens reared on rice under different temperatures.Candidate genes (ylsActin, ylsEF1α, ylsGADPH, ylsRPS11, ylsRPS15e, ylsRPS15p, ylsTub, ylsUBQ) were evaluated using the comparative ΔCt method (A), NormFinder (B), geNorm (C) and BestKeeper (D).
Mentions: With the samples exposed to different temperature conditions, the comparative ΔCt method, NormFinder and BestKeeper analyses indicated that ylsRPS15e and ylsGAPDH were the most stable genes, while ylsACT and ylsTUB were the most stable genes found by geNorm (Fig. 5). According to RefFinder, the overall order from most to least stable is ylsRPS15e > ylsGADPH > ylsACT > ylsTUB > ylsEF1α > ylsUBQ > ylsRPS15p > ylsRPS11 (Table 2). The geNorm analysis revealed that almost of all pairwise (Vn/Vn + 1) variations were below 0.15 (Fig. 3). Thus, the combination of reference genes recommended for this subset is ylsRPS15e and ylsGADPH.

View Article: PubMed Central - PubMed

ABSTRACT

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies.

No MeSH data available.