收稿日期: 2008-06-24
修回日期: 2008-12-11
网络出版日期: 2009-12-12
基金资助
国家自然科学基金项目(项目编号：30070597，30571430)
The construction of normalized cDNA library and preliminary EST analysis from the gonad development and sexual differentiation related organs of Scylla serrata
Received date: 2008-06-24
Revised date: 2008-12-11
Online published: 2009-12-12
Supported by
国家自然科学基金项目(项目编号：30070597，30571430)
分别提取锯缘青蟹Scylla serrata卵巢、精巢、眼柄、促雄腺的总RNA，等量混合后经oligotex试剂分离mRNA，利用SMART (Switch mechanisms at the 5’end of RNA transcript)技术及DSN (duplex-specific nuclease) 的特性构建线性化cDNA质粒文库。经检测文库约含有5.3×107重组子，扩增后的文库含有1011以上重组子。文库的插入cDNA长度为0.5-2.5kb，重组率大于98%。从构建的文库中随机挑取5202个克隆进行大规模EST测序，获得3346条ESTs，其中长度大于100bp的高质量ESTs 2697条。经拼接与软件分析，2697 ESTs代表了2522个Unigenes，包括2355个Singlets和167个Contigs，冗余率6.49%。EST序列经生物信息学分析(BLASTX, e<10-6)，发现了Sry-like protein C、Sox14 protein、Sox4b、sex-determining protein fem-1、vitellogenin、vitellogenin receptor、cyclin A、cathepsin C和cyclin-dependent kinase 2等与性别分化和性腺发育相关的基因，以及热休克蛋白家族、泛素系统、抗氧化防御体系和抗病相关的功能基因。该文库具有较高的质量和利用价值，可为更大规模EST分析及采用基因芯片技术开展锯缘青蟹性腺发育及性别分化相关功能基因的研究奠定基础。
关键词： 锯缘青蟹Scylla serrata; 线性化cDNA文库; 性腺发育; 性别分化 EST
邹志华 , 张子平 , 王艺磊 , 陈锦民 , 贾锡伟 , 王淑红 , 林鹏 . 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[J]. 热带海洋学报, 2009 , 28(6) : 88 -94 . DOI: 10.11978/j.issn.1009-5470.2009.06.088
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.
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