The Construction Of CDNA Library From Cerebrum Of Elaphodus Cephalophus And Study Of Neurotransmitter Transporter Gene

The Cerebrum cDNA Library from tufted deer (Elaphodus cephalophus) was constructed by using SMART technique. Total RNA was extracted from cerebrum tissue of tufted deer, and poly (A)~+ RNA was purified further by Oligotex mRNA Kit. CDS III/ 3'PCR Primer primed the reverse transcription. The double-strand cDNA was amplified by LD- PCR(long distance PCR) and digested by Sfi I. After cDNA size fractioned by CHROMA SPIN- 400, fragments over 500 bp were collected and ligated with λTripIEx2 vector. After packaged in vitro , the full- long cDNA library of cerebrum tissue was constructed successfully. The library contains 5.1×10~5 clones and the amplified library has a titer of 1.5×10~9 pfu/ml, in which 85% clones are recombinant and the average sizes of inserted cDNAs are about 1.0 kb. These results show that the Cerebrum cDNA Library is qualified for screeening genes. A full- length rig (rat insulinoma gene) gene including 5' and 3'untranslated regions was isolated from the cDNA library . Sequence analysis shows that this 541 bp cDNA spans an open reading frame from nucleotide 43 to 477, encoding a 51- aminoacid rig protein.In order to get the sex-linked gene, we sequenced and obtained 82 ESTs from testis cDNA Library of tufted deer, 27 genes are 3'-sequence and 55 sequences are 5'-sequence. After homologous comparison in the GenBank with the sequence analysing software BLAST, 4 genes are new and 8 genes are repeated, 3 sequences are located on the X chromosome.The genomic DNAs were extracted from tufted deer and were digested by Hind III respectively. The digested DNAs were transferred on the nylon membrance and hybridization was done between the nylon membrance and the Neurotransmitter transporter gene probe, which was labelled by using PCR DIG labeling Mix. The results of southern blotting showed that the Neurotransmitter transporter gene has two obvious bands in the genome of tufted deer. In order to study the expression of the Neurotransmitter transporter in tufted deer, total RNA was extracted from 8 tissues of...