Quantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔC t method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.
Galeruca daurica (Joannis) is a new outbreak pest in the Inner Mongolia grasslands in northern China. Heat shock protein 10 and 60 (Hsp10 and Hsp60) genes of G. daurica, designated as GdHsp10 and GdHsp60, were cloned by rapid amplification of cDNA ends techniques. Sequence analysis showed that GdHsp10 and GdHsp60 encoded polypeptides of 104 and 573 amino acids, respectively. Sequence alignment and phylogenetic analysis clearly revealed that the amino acids of GdHsp10 and GdHsp60 had high homology and were clustered with other Hsp10 and Hsp60 genes in insects which are highly relative with G. daurica based on morphologic taxonomy. The mRNA expression analysis by real-time PCR revealed that GdHsp10 and GdHsp60 were expressed at all development stages and in all tissues examined, but expressed highest in eggs and in adults' abdomen; both heat and cold stresses could induce mRNA expression of GdHsp10 and GdHsp60 in the 2nd instar larvae; the two Hsp genes were expressed from high to low with the extension of treatment time in G. daurica eggs exposed to freezing point. Overall, our study provides useful information to understand temperature stress responses of Hsp60 and Hsp10 in G. daurica, and provides a basis to further study functions of Hsp60/Hsp10 relative to thermotolerance and cold hardiness mechanism.
Image haze removal is critical for autonomous driving. However, it is a challenging task for the existing image dehazing algorithms to eliminate the block effect completely and handle objects similar to light (such as snowy objects and white buildings). To address this problem, we propose a novel singleimage dehazing method based on superpixels and Markov random field. We obtain the transmission map in the superpixel domain to eliminate the block/halo effect and introduce Markov random field to revise the transmission map in the superpixel domain. The key idea is that the sparsely distributed, incorrectly estimated transmittances can be corrected by properly characterizing the spatial dependencies between the incorrectly estimated superpixels and the neighbouring well-estimated superpixels. The experimental results demonstrate that the proposed method outperforms state-of-the-art image dehazing methods. INDEX TERMS Superpixel, Markov random field, haze removal, edge preservation, dark channel prior.
The evolutionary mechanisms underlying the biogeochemical niche conservatism in forests remain incompletely understood. Here we aimed to determine how the strengths of biogeochemical niche conservatism vary among elements and between life forms. We measured leaf concentrations of basal elements (C, N, P, K, Ca, and Mg) in a wide range of life forms in a subtropical montane evergreen broad‐leaved forest. We found that differences in life forms such as evergreen/deciduous woody species and herbaceous/woody species significantly affected leaf elemental composition. The significant phylogenetic signal was present in leaf C, N, K, and Mg concentrations but absent in leaf P and Ca concentrations in all species. These contrasting strengths of biogeochemical niche conservatism were best generated by Ornstein–Uhlenbeck processes toward optima. Woody species were evolutionarily selected to show lower optimal leaf N, P, and K concentrations and higher optimal leaf C, Ca, and Mg concentrations than herbaceous species. The number of optima varied from the least in leaf C concentration to the most in leaf Ca concentration, suggesting the stronger convergent evolution of leaf Ca concentration. The positions of optima toward the tips were more selected in woody species, suggesting the more frequency of species‐specific adaptations in woody species. The positions of optima were also selected at the nodes towards the species groupings from certain life forms (e.g., the group of 12 Polypodiales ferns in leaf Ca evolution and the group of three evergreen Theaceae species in leaf P evolution) that were converged to present similar leaf elemental composition. During the evolution of biogeochemical niche, strong correlations were found among leaf C, N, P, and K concentrations and between leaf Ca and Mg concentrations. In conclusion, the strengths of biogeochemical niche conservatism can vary among elements and between life forms due to the different tempo and mode of Ornstein–Uhlenbeck processes.
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