Cloning and chaeacterization of cDNA involved in sex determination of the giat tiger shrimp Penaeus monodon

Abstract

Sex-specific cDNA markers of the giant tiger shrimp (Penaeus mondon) were identified by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed from conserved regions (DM domain) of DMRT1, dsx and mab-3 homologues and peritrophin, zinc finger protein gene homologues and from other sex-related transcripts. Nevertheless, they did not reveal sex-specific expression profiles in this species. Five non-specific RT-PCR fragments (PMO720, PMO880, PMO920, PMT700 and PMT1700) exhibiting sex-specific expression patterns were cloned and sequenced. Primers were designed from PMO720, PMO920 and PMT1700. Expression of these transcripts was examined and displayed differential expression levels between males and females P. monodon. Single nucleotide polymorphism (SNP) fixed in each gender of P. monodon was examined at the genomic level by comparing DNA sequences of the amplified zinc finger gene segments (450 bp from Zincfinger-F and Finger-R). Polymorphism was found at both within and between genders but did not fix in each sex. Therefore, sex determination SNP markers were still not found in this species. Additionally, subtractive cDNA libraries of ovaries and testes of P. monodon were constructed. A total of 216 clones (155 clones from subtractive cDNAs of ovaries and 61 clones from those of testes) were unidirectionally sequenced. Most of the expressed genes in ovaries encoded thrombospondin (TSP 45 clones, accounting for 28.7%), peritrophin (17 clones, 10.8%) and unknown transcripts (76 clones, 49.7%). Conversely, almost all of the ESTs in P. monodon testes were unknown transcripts (59 clones, 96.7%). Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) was carried out for further characterization of TSP. Results indicated successful amplification of 5{167} RACE-PCR but not 3{167} RACE-PCR. Gender-specific expression of various candidate sex-linked gene homologues was examined by RT-PCR. While XNP-1 and peritrophin were expressed in both ovaries and testes, VCP7, PM233 and AGESTX revealed sex-specific expression in female P. monodon. Expression of TSP was found in both male and female of 4-month-old P. monodon but temporal sex-specific expression was found in P. monodon adults. Expression of TSP and AGESTX homologues was semi-quantitatively estimated. The relative expression level of AGESTX in ovaries of 4-month-old and adults of P. monodon was not significantly different (P>0.05) but that of TSP was significantly different statistically (P<0.05).