锯缘青蟹性腺发育与性别分化相关组织混合线性化cDNA文库构建及EST初步分析

doi:10.11978/j.issn.1009-5470.2009.06.088

Abstract

Abstract:

To study the molecular mechanism in gonad development and sexual differentiation of Scylla serrata, a normalized cDNA library was constructed by 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 5.3×106cfu/ml．The titer of amplified cDNA libraries was above 1011cfu/ml．The cDNA inserts sizes ranged between 0.5-2.5kb．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 5202 clones were randomly analyzed by single-pass sequencing from the 5` end. Clustering and assembling of these ESTs resulted in a total of 2697 high quality sequences with 167 overlapping contigs and 2355 singletons. The redundancy of the cDNA library was 6.49%. Twenty-seven ESTs showed significant homology (BLASTX e-value<10-10)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 proved 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.

Key words: Scylla serrata, gonad development, sexual differentiation, normalized cDNA library, EST

ZOU Zhi-hua,ZHANG Zi-ping,WANG Yi-lei,CHEN Jin-min,JIA Xi-wei,WANG Shu-hong,LIN Peng
. The construction of normalized cDNA library and preliminary EST analysis from the gonad development and sexual differentiation related organs of Scylla serrata[J].Journal of Tropical Oceanography, 2009, 28(6): 88-94.

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

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