Lu MX et al. (2018), Identification and Functional Analysis of the F...

XB-ART-54622

Front Physiol 2018 Jan 01;9:57. doi: 10.3389/fphys.2018.00057.

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Identification and Functional Analysis of the First Aquaporin from Striped Stem Borer, Chilo suppressalis.

Lu MX , Pan DD , Xu J , Liu Y , Wang GR , Du YZ .

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Aquaporins are integral membrane proteins some of which form high capacity water-selective channels, promoting water permeation across cell membranes. In this study, we isolated the aquaporin transcript (CsDrip1) of Chilo suppressalis, one of the important rice pests. CsDrip1 included two variants, CsDrip1_v1 and CsDrip1_v2. Although CsDrip1_v2 sequence (>409 bp) was longer than CsDrip1_v1, they possessed the same open reading frame (ORF). Protein structure and topology of CsDrip1 was analyzed using a predicted model, and the results demonstrated the conserved properties of insect water-specific aquaporins, including 6 transmembrane domains, 2 NPA motifs, ar/R constriction region (Phe69, His194, Ser203, and Arg209) and the C-terminal peptide sequence ending in "SYDF." Our data revealed that the Xenopus oocytes expressing CsDrip1 indicated CsDrip1 could transport water instead of glycerol, trehalose and urea. Further, the transcript of CsDrip1 expressed ubiquitously but differentially in different tissues or organs and developmental stages of C. suppressalis. CsDrip1 mRNA exhibited the highest level of expression within hindgut and the third instar larvae. Regardless of pupae and adults, there were significantly different expression levels of CsDrip1 gene between male and female. Different from at low temperature, the transcript of CsDrip1 in larvae exposed to high temperature was increased significantly. Moreover, the mRNA levels of CsDrip1 in the third instar larvae, the fifth instar larvae, pupae (male and female), and adults (male and female) under different humidities were investigated. However, the mRNA levels of CsDrip1 of only female and male adults were changed remarkably. In conclusions, CsDrip1 plays important roles in maintaining water homeostasis in this important rice pest.

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Genes referenced: ncoa6 tpm3

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	Figure 1. Sequence analysis, predicted topology and structure of CsDrip1. (A,B) Nucleotide and deduced amino acid sequence of CsDrip1 variant A (JQ011314) and variant B (JQ011315). Six transmembrane domains (TMs) are also shown by the red underline and C-terminal “SYDF” sequence found in insect AQPs with dark underline. The NPA boxes are shown with gray highlight. Orange indicates amino acids residues constituting the ar/R constriction region. Blue dots and green dots, respectively are potential phosphorylation sites and protein kinase C-specific sites. (C) Homology modeling of the CsDrip1 is compared with bovine aquaporin-1 (PDB ID: 1J4N) as template using Phyre2. Intracellular (B,D) and extracellular (A,C,E) loops and transmembrane helices are shown (TM1 in blue, TM2 in red, TM3 in yellow, TM4 in green, TM5 in purple, TM6 in orange). (D) The structure depicted is from the extracellular side of the membrane. The classical NPA motifs are shown in gray sphere, and Ar/R selectivity residues regions (Phe-69, His-194, Ser-203, and Arg-209) are shown in gray stick.
	Figure 2. Neighbour-joining phylogenetic tree showing the Chilo suppressalis Drip1 (JQ011314 and JQ011315) with respect to other insect AQPs. BmDrip1 (Bombyx mori, AB178640), CmDrip1 (Cydia molesta, AB469882), AaDrip1 (Aedes aegypti, AF218314), PhDrip1 (Pediculus humanus, DS235154), TcDrip1 (Tribolium castaneum, CM000285), LlDrip1 (Lutzomyia longipalpis, EU124628), AgDrip1 (Anopheles gambiae, AAAB01008984), Cf Drip1 (Coptotermes formosanus, AB433197), HieDrip1 (Haematobia irritans exigua, U51638), CvDrip1 (Cicadella viridis, X97159), DpDrip1 (Drosophila pseudoobscura, CM000071), BtDrip1 (Bemisia tabaci, EU127479), GmDrip1 (Glossina morsitans, EZ422826), CqDrip1 (Culex quinquefasciatus, DS232465), ApDrip1 (Acyrthosiphon pisum, EU925136), D. plexippusDrip1 (Danaus plexippus, EHJ75085), EoDrip1 (Ectropis oblique, AMO02270.1), SlDrip1 (Spodoptera litura, AGT50942.1), ObDrip1 (Operophtera brumata, KOB73271.1), PrDrip1 (Phormia regina, BAM26200.1), MdDrip1 (Musca domestica, AIB09141.1), LhDrip1 (Lygus hesperus, AHI85743.1), EsPrip (Eurosta solidaginis, FJ489680), BgPrip (Blattella germanica, FR744897), Cf Prip (Camponotus floridanus, GL435707), BaPrip (Belgica Antarctica, AB602341), PvPrip (Polypedilum vanderplanki, AB281619), and BmPrip (Bombyx mori, AB458833). Numbers on the branches are the bootstrap values obtained from 1,000 replicates (only bootstrap values >50 are shown).
	Figure 3. Functional assay of Chilo suppressalis aquaporin (CsDrip1). (A) Water permeability of CsDrip1. (B) Glycerol permeability of CsDrip1. (C) Trehalose permeability of CsDrip1. (D) Urea permeability of CsDrip1. The graphs of oocytes assayed in the experiments were indicated above each bar. Error bars represented SEM (n ≥ 9). Student's t-test was performed to find means that were significantly (p < 0.05) different from the water-injected control oocytes (indicated with asterisk).
	Figure 4. Relative mRNA expression levels of the CsDrip1 gene in different tissues (organs) of the Chilo suppressalis. HG, hindgut; MG, midgut; FG, foregut; HC, haemocytes; MT, Malpighian tubules; EP, epidermis; FB, fat body and HE, Heads. Values are denoted as the mean ± SE. Significant differences are indicated as P < 0.05.
	Figure 5. Relative mRNA expression levels of the CsDrip1 gene in different developmental stages of the Chilo suppressalis. E, eggs; L1, the first instar larvae; L2, the second instar larvae; L3, the third instar larvae; L4, the fourth instar larvae; L5, the fifth instar larvae; MP, male pupae; FP, female pupae; MA, male adults; and FA, female adults. Values are denoted as the mean ± SE. Significant differences are indicated as P < 0.05.
	Figure 6. Relative expression levels of CsDrip1 gene in the Chilo suppressalis under temperature stress. The larvae were exposed for −11°C, −9°C, −3°C, 0°C, 27°C, 36°C, 39°C, or 42°C for 2 h. Values are denoted as the mean ± SE. Significant differences are indicated as P < 0.05.
	Figure 7. Relative expression levels of CsDrip1 gene in the Chilo suppressalis under humidity treatments. (A) The third instar larvae treated by 25, 50, 75, and 95% for 12 h, respectively. (B) The fifth instar larvae treated by 25, 50, 75, and 95% for 24 h, respectively. (C) The female pupae treated by 25, 50, 75, and 95% for 24 h, respectively. (D) The male pupae treated by 25, 50, 75, and 95% for 24 h, respectively. (E) The female adults treated by 25, 50, 75, and 95% for 24 h, respectively. (F) The male adults treated by 25, 50, 75, and 95% for 24 h, respectively. Values are denoted as the mean ± SE. Significant differences are indicated as P < 0.05.

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