毛冠鹿睾丸组织cDNA文库的构建及P1精蛋白基因的克隆

Construction of cDNA library from testis of Elaphodus cephalophus and clonin g of P1 protamine gene

为构建毛冠鹿睾丸组织cDNA文库,用TRIzol试剂提取总RNA,利用SMART(switchingmechanismat5’endofRNAtranscript)技术合成cDNA第1链,双链cDNA经LDPCR(LongdistancePCR)扩增并经sfiⅠ酶切和过柱分级分离后,克隆入经sfiI酶切的λTrip1EX2载体,经体外包装而成cDNA原始文库。该毛冠鹿睾丸组织cDNA原始文库含有独立克隆数为5.52×105,重组率达90.8%。文库扩增后,滴度达1.05×109pfu/ml,插入cDNA平均长度为1.01kb。通过随机挑取噬菌斑,并克隆测序,从该文库中克隆了毛冠鹿睾丸组织特异表达基因:P1精蛋白基因(GenBank序列号:DQ299383),该cDNA具有5’和3’非编码区,从第95至250个核苷酸为一完整阅读框(ORF),此阅读框编码一个51Aa的P1精蛋白。以上结果表明本文构建的毛冠鹿睾丸组织cDNA文库符合建库要求,可作为进一步克隆毛冠鹿睾丸组织特异表达基因的可靠材料。

To construct a cDNA library of tufted deer （Elaphodus cephalophus） testis, the total RNA was extracted using TRIzol reagent and the first-strand cDNA was synthesized by using the SMART （ switching mechanism at 5＇ end of RNA transcript） technique. The double-strand cDNA was amplified by LD-PCR （Long-distance-PCR） and then digested by sfi I restriction enzyme. Following cDNA fractionation through CHROMASPIN-400 column, fragments over 500 bp were collected and ligated with λTriplEx2 vector. The recombinants were packaged in vitro using Packagene Lambda DNA Packaging extract. The primary cDNA library contains 5.52 × 10^5 independent clones, 90. 8% of which are recombinant, the titer of the amplified library is 1.05 × 10^9 pfu/ml and the average length of exogenous inserts is 1.01 kb. Testis-specific genes of tufted deer were isolated after the phage clones were randomly selected and sequenced, the full-length cDNA with both 5＇ and 3＇ untranslated regions of a testis-specific gene, P1 protamine, was obtained from the cDNA library （ GenBank accession number： DQ299383 ）. Sequence analysis showed that this 431 bp long cDNA spans an open reading frame from nucleotide 95 to 250, encoding a 51-aminoacid long protein. It is the first time that the P1 protamine gene of tufted deer has been cloned. These results show that the cDNA library is eligible and is useful for screening and cloning other testis-specific genes of tufted deer.