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A Novel Aquaporin 12-like Protein from Chilo suppressalis: Characterization and Functional Analysis.
Lu MX
,
Song J
,
Xu J
,
Wang G
,
Liu Y
,
Du YZ
.
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Aquaporins (AQPs), which are members of the major intrinsic protein (MIP) family, play an important role in the transport of water and other small, uncharged solutes across membranes. In this study, we identified gene encoding two aquaporin 12-like (AQP12L) proteins, CsAqp12L_v1 and CsAqp12L_v2, from Chilo suppressalis, a serious rice pest in Asia. Phylogenetic analysis indicated that CsAQP12L_V1 and CsAQP12L_V2 were grouped in a well-supported cluster that included other members of Lepidoptera. The two proteins are almost identical, except that CsAQP12L_V1 lacks 34 amino acids that are present in CsAQP12L_V2 at site 217. The qRT-PCR indicated that both CsAqp12L and CsAqp12L_v2 were expressed in heads, epidermis, foregut, midgut, and hindguts, with the highest level of expression in hindguts, heads, and epidermis. Expression of CsAqp12L and CsAqp12L_v2 was detected in all life stages and both sexes and was highest in first instar larvae and lowest in eggs. Expression of CsAqp12L and CsAqp12L_v2 was not significantly altered by exposure to brief changes in temperature. There were no significant differences in the third instar larvae, male and female pupae, and female adults in response to adverse humidity. However, the mRNA level of CsAqp12L in the fifth instar larvae and CsAqp12L_v2 in male adults was induced significantly by low humidity, respectively. Moreover, Xenopus oocytes injected with cRNAs of CsAQP12L_V1 and CsAQP12L_V2 showed no significant changes in permeability to water, glycerol, trehalose, or urea. The two CsAQP12L variants likely localize to an intracellular location in C. suppressalis and may respond to novel stimuli.
Figure 1. Comparative analysis of Chilo suppressalis AQP12L (CsAQP12L) with AQP12L proteins in other insect species. (A) Multiple sequence alignment of CsAQP12L_V1 and CsAQP12L_V2 from C. suppressalis (CsAQP12L_V1, MF033357; CsAQP12L_V2, MF033358), Papilio xuthus (PxAQP12L_V1, XM_013311304; PxAQP12L_V2, XM_013311305), Papilio machaon (PmAQP12L_V1, XM_014505979; PmAQP12L_V2, XM_014505980), Amyelois transitella (AtAQP12L_V1, XM_013345423; AtAQP12L_V2, XM_013345424), Plutella xylostella (PxyAQP12L_V1, XM_011560529; PxyAQP12L_V2, XM_011560536). Conserved motifs are bordered with a rectangle and the ar/R selectivity site is indicated by black solid dots. (B) Phylogenetic analysis of AQP12L proteins using the Bayesian inference (BI) method. The number associated with each internal branch represents Bayesian posterior probabilities. The amino acid sequences used for construction of the phylogenetic tree were obtained from GenBank and were deposited under the following accession numbers: CsAQP12L_V1 and CsAQP12L_V2 (C. suppressalis see above), PxAQP12L_V1 and PxAQP12L_V2 (P. xuthus, see above), PmAQP12L_V1 and PmAQP12L_V2, (P. machaon, see above), AtAQP12L_V1 and AtAQP12L_V2 (A. transitella, see above), PxyAQP12L_V1 and PxyAQP12L_V2 (P. xylostella, see above), LsAQP12L_V1 and LsAQP12L_V2 (Lepeophtheirus salmonis, KR005665, KR005666), H NlAQP12L (Neodiprion lecontei, XM_015664627), and DaAQP12L (Diachasma alloeum, XM_015259511).
Figure 2. Expression of CsAqp12L in different tissues and organs of Chilo suppressalis. (A) CsAqp12L; (B) CsAqp12L_v2. Abbreviations: FG, foreguts; MG, midguts; HG, hindguts; FB, fat bodies; MT, Malpighian tubules; HE, head; EP, epidermis; and HC, hemocytes. Data represent mean ± SE for four replications. Means labeled with different letters are significantly different (P < 0.05, one-way ANOVA, Tukey).
Figure 3. Expression of CsAqp12L genes in different developmental stages of Chilo suppressalis. (A) CsAqp12L; (B) CsAqp12L_v2. Data represent mean ± SE for four replications. Means labeled with different letters were significantly different (P < 0.05, one-way, Anova, Tukey).
Figure 4. Expression of CsAqp12L genes from Chilo suppressalis in response to different levels of relative humidity. Third instar larvae were exposed to 25%, 50%, 75%, and 95% RH for 12 h. Fifth instar larvae, pupae, and adults were exposed to 25%, 50%, 75%, and 95% for 24 h. (A) Expression of CsAqp12L gene. (B) Expression of CsAqp12L_v2 gene. Histograms indicate relative expression levels. Data represent mean ± SE for four replications. Means labeled with asterisks were significantly different (P < 0.05, one-way ANOVA, Tukey).
Figure 5. Expression of CsAqp12L and CsAqp12L_v2 in larvae of Chilo suppressalis exposed to temperature stress. Larvae were exposed to −9 °C, −8 °C, −6 °C, −3 °C, 0 °C, 27 °C, 30 °C, 33 °C, 36 °C, 39 °C, 42 °C, and 43 °C for 2 h. Histograms indicate relative expression levels. Data represent mean ± SE for four replications. Means labeled with asterisks were significantly different (P < 0.05, one-way ANOVA, Tukey).
Figure 6. Functional assays of Chilo suppressalis aquaporin (CsAQP12L). (A) Water permeability in Xenopus oocytes microinjected with CsAQP12L_V1 cRNA, CsAQP12L_V2 cRNA, and nuclease-free H2O (control). (B) Solute permeability assays in Xenopus oocytes exposed to 140 mM of glycerol, trehalose, and urea. Values represent the mean ± SE of oocytes.
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