关键词: 毛冠鹿, 睾丸, cDNA 文库, P1 精蛋白

Abstract: 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 5.52×10⁵ independent clones,90.8% of which are recombinant,the titer of the amplified library is 1.05×10⁹ 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.

Key words: cDNA Library, P1 protamine, Testis, Tufted deer (Elaphodus cephalophus)