The construction of normalized cDNA library and preliminary EST analysis from the gonad development and sexual differentiation related organs of Scylla serrata

To study the molecular mechanism in gonad development and sexual differentiation of Scylla serrata,a normalized cDNA library was constructed using the combination of SMART(Switch mechanisms at the 5′end of RNA transcript) and DSN(duplex-specific nuclease) technique.Total RNA was isolated from testis,ovaries,eyestalks and androgenic gland,and then mixed equal amount of RNA from each tissue to make a total RNA pool.Oligotex(QIAGEN) was used to isolate the mRNA from the total RNA pool.The first strand cDNA was synthesized by transcription of mRNA with the SMART technique.The LD-PCR was performed using a modified SMART primer as the primer set,and first-strand cDNA as the template to amplify the cDNA population.After treatment with DSN,the normalized cDNAs were digested with Sfi I enzyme and size fractionated to remove the small products(500bp).These normalized SMART cDNAs were ligated into the Sfi I-digested pDNR-LIB Vector.E.coli(TOP10) were transformed with the ligation mixture to generate a normalized cDNA plasmid library.The titer of unamplified cDNA libraries was 5.3×106 cfu.ml-1.The titer of amplified cDNA libraries was above 1011 cfu.ml-1.The cDNA inserts sizes ranged between 0.5-2.5 kb.Recombination rate was more than 98%.A large-scale expressed sequence taq(EST) sequencing project was undertaken for the purpose of differentially expressed genes between male and female crabs.A total of 5 202 clones were randomly analyzed by single-pass sequencing from the 5′end.Clustering and assembling of these ESTs resulted in a total of 2 697 high quality sequences with 167 overlapping contigs and 2 355 singletons.The redundancy of the cDNA library was 6.49%.Twenty-seven ESTs showed significant homology(BLASTX e-value10-10) to known genes such as genes related to gonad development and sex differentiation,heat shock protein family,ubiquitin system,disease resistant,antioxidant defense system.Nine ESTs named Sry-like protein C、 Sox14 protein、 Sox4b、 sex-determining protein fem-1、 vitellogenin、vitellogenin receptor、 cyclin A、 cathepsin C、 cyclin-dependent kinase 2 were proven to be associated with gonad development and sex differentiation by other investigators.This normalized cDNA library provides a useful resource for gene identification and functional genomic studies of Scylla serrata.