| To construct a cDNA library of tufted deer (Elaphodus cephalophus) testis, the total RNA was extracted by 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 sfl I . Following cDNA fractionation through CHROMASPIN- 400 column, fragments over 500bp were collected and ligated with λTriplEx2 vectors. 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 deerThe genomic DNA was extracted from tissues of tufted deer and was digested by EcoR I, EcoR V and Pst I,respectively. The digested DNAs were transferred on the nylon membrance and hybridized with the PRM1 probe, which was labelled by using PCR DIG labelling Mix.The results of southern blotting showed that the PRM1 gene has two types of copy in the genome of tufted deer.In order to study the expression of the PRM1 in tufted deer, total RNA was extracted from 8 types of tissues from tufted deer,and the first-strand cDNA were synthesized.The RT-PCR was...
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