Computational protocol: TRPV4 associates environmental temperature and sex determination in the American alligator

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Protocol publication

[…] Total RNA was extracted from female neonatal gonad, using RNeasy kit (Qiagen, Valencia, CA, USA). Full coding region of the AmTRPV4 was determined by standard procedure using SmartRACE kit (Takara, Ohtsu, Japan), and finally full-length AmTRPV4 was cloned with KOD + polymerase (Toyobo, Osaka, Japan). The amplified full-length AmTRPV4 cDNA product was then subcloned into pOX + vector for electrophysiological analysis. Primer information is reported in .Multiple sequence alignment for vertebrate TRPV4 homologues was performed using the CLUSTAL W. Phylogenetic relationships of TRPV4 were then examined using TRPV4 amino acid sequences derived from GenBank/EMBL database. The TRPV4 genes and species used are summarized in . Based on TRPV4 conserved sites, which include the ankyrin repeat domains and transmembrane domains (652 residues in AmTRPV4), with all the alignment gap sites were eliminated, minimum-evolution methods was applied to construct an evolutionary tree, using MEGA 5 software. The statistical confidence was then computed by bootstrap method with 1000 replications. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Alligator mississippiensis
Diseases Substance-Related Disorders