The Construction of Full-length Testis and Ovary cDNA Libraries of Marsupenaeus japonicus

To clone the testis and ovary specific expression genes of Marsupenaeus japonicus, we constructed two full-length libraries using the SMART library construction kit (Clontech). Total RNAs were isolated from tests and ovaries using RDP, a reagent made by us and modified from that used by Chomczynski and Sacchi (1987) for the simultaneous isolation of RNA, DNA, and protein. Oligotex (QIAGEN) was used to separate the mRNA of testes and ovaries from total RNA. The “anchor first-strand cDNA containing asymmetrical SfiⅠ restriction enzyme sites (A B) was synthesized by transcription of mRNA with the SMART technique. The LDPCR was performed using a modified oligo(dT) primer and an anchor primer as the primer set, and anchor first-strand as the template to enrich the population for full-length sequences. After digestion with SfiⅠ and size fractionation using CHROMA SPIN-400 TM Columns, SMART was ligated into the SfiⅠ-digested λ TriplEx2 TM Vector. The ligation mixture was packaged into lambda capsids using the Gigapack Ⅲ Gold packaging extract (Stratagene) to generate full-length testis and ovary libraries of Marsupenaeus japonicus. The titer of un-amplified libraries was 1.04×106 for the testis library and 1.2×106 for the ovary library. The titer of amplified libraries was 7.3×108(Pfu/ml)for the testis library and 9.1×108(Pfu/ml)for the ovary library respectively. The inserts sizes were between 400 bp-3 kb. These results indicate that the quality of these libraries is good enough for further cloning of testis and ovary specific expression genes.