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Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development
ZHENG Lu-ying, ZHANG Zhi-jun, ZHANG Jin-ming, LI Xiao-wei, HUANG Jun, LIN Wen-cai, LI Wei-di, LI Chuan-ren, LU Yao-bin
2019, 18 (4): 854-864.   DOI: 10.1016/S2095-3119(18)61973-2
Abstract238)      PDF (2568KB)(549)      
Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic.  Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species.  A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR.  However, the accuracy and consistency of this technique depend on the reference gene(s) selected.  In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder.  The results showed that EF1-β, RP-L32 and RP-18S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm.  RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis.  Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase (JHAMT) gene.  Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages.  The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.
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Overexpression of vacuolar proton pump ATPase (V-H+-ATPase) subunits B, C and H confers tolerance to salt and saline-alkali stresses in transgenic alfalfa (Medicago sativa L.)
WANG Fa-wei, WANG Chao, SUN Yao, WANG Nan, LI Xiao-wei, DONG Yuan-yuan, Yao Na, Liu Xiu-ming, CHEN Huan, CHEN Xi-feng, WANG Zhen-min, LI Hai-yan
2016, 15 (10): 2279-2289.   DOI: 10.1016/S2095-3119(16)61399-0
Abstract1601)      PDF in ScienceDirect      
    The vacuolar proton pump ATPase (V-H+-ATPase), which is a multi-subunit membrane protein complex, plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. In this study, three genes encoding V-H+-ATPase subunits B (ScVHA-B, GenBank: JF826506), C (ScVHA-C, GenBank: JF826507) and H (ScVHA-H, GenBank: JF826508) were isolated from the halophyte Suaeda corniculata. The transcript levels of ScVHA-B, ScVHA-C and ScVHA-H were increased by salt, drought and saline-alkali treatments. V-H+-ATPase activity was also examined under salt, drought and saline-alkali stresses. The results showed that V-H+-ATPase activity was correlated with salt, drought and saline-alkali stress. Furthermore, V-H+-ATPase subunits B, C and H (ScVHA-B, ScVHA-C and ScVHA-H) from S. corniculata were introduced separately into the alfalfa genome. The transgenic alfalfa was verified by Southern and Northern blot analysis. During salt and saline-alkali stresses, transgenic linevacuolar proton pump, salt tolerance, saline-alkali tolerance, alfalfa
s carrying the B, C and H subunits had higher germination rates than the wild type (WT). More free proline, higher superoxide dismutase (SOD) activity and lower malondialdehyde (MDA) levels were detected in the transgenic plants under salt and saline-alkali treatments. Moreover, the ScVHA-B transgenic lines showed greater tolerance to salt and saline-alkali stresses than the WT. These results suggest that overexpression of ScVHA-B, ScVHA-C and ScVHA-H improves tolerance to salt and saline-alkali stresses in transgenic alfalfa.
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