CAJ | 학술논문

分别提取锯缘青蟹Scylla serrcg"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 inechanisms 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5.3×10~7重组子.扩增后的文库含有10~(11)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5 202个克隆进行大规模EST测序,获得3 346条ESTs,其中长度大于100bp的高质量ESTs 2 697条.经拼接与软件分析,2 697 ESTs代表了2 522个Unigenes,包括2 355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Sty-like protein C、Sox14 protein、Sox4b、sex-determining protein fem-1、vitellogenin、vitellogenin receptor、cvcl.n 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.该义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冈的研究奠定基础.
분별제취거연청해Scylla serrcg"란소、정소、안병、촉웅선적총RNA,등량혼합후경oligotex시제분리mRNA,이용SMART(Switch inechanisms at the 5'end of RNA transcript)기술급DSN(duplex-specific nuclease)적특성구건선성화cDNA질립문고.,경검측문고약함유5.3×10~7중조자.확증후적문고함유10~(11)이상중조자.문고적삽입cDNA장도위0.5-2.5kb.중조솔대우98%.종구건적문고중수궤도취5 202개극륭진행대규모EST측서,획득3 346조ESTs,기중장도대우100bp적고질량ESTs 2 697조.경병접여연건분석,2 697 ESTs대표료2 522개Unigenes,포괄2 355개Singlets화167개Contigs,용여솔6.49%.EST서렬경생물신식학분석(BLASTX,e<10~(-6)),발현료Sty-like protein C、Sox14 protein、Sox4b、sex-determining protein fem-1、vitellogenin、vitellogenin receptor、cvcl.n A、cathepsin C화cyclin-dependent kinase 2등여성별분화화성선발육상관적기인,이급열휴극단백가족、범소계통、항양화방어체계화항병상관적공능기인.해의고구유교고적질량화이용개치,가위경대규모EST분석급채용기인심편기술개전거연청해성선발육급성별분화상관공능기강적연구전정기출.
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 nuelease) technique. Total RNA was isolated from testis, ovaries, eyestalks and androgenic gland, and then mixed equal amount of RNA from eacch 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 10~(11) cfu·ml~(-1). The cDNA inserts sizes ranged between 0.5-2.5 kb. Recombination rate was more than 98%. A largescale 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-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 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.