CDNA Library Construction And Library Screening Of Hainan Characteristic Animals

A cDNA library from the peripheral blood leucocytes in Cervus eldi hainanaus, Bos taurus hainanaus, Wuzhishan Miniature Pig was constructed with the SMART (switching mechanism at 5'end of RNA transcript) technique. The total RNA was extracted from the peripheral blood leucocytes and reversely transcripted into the full-length cDNA by using Powerscript^TM reverse transcriptase, the double strand cDNA was synthesized and amplified by long-distance PCR (LD-PCR). The PCR products were digested by proteinase K. After digestion with SfiI and size fractionation using CHROMA SPIN-400^TM columns, the cDNAs (＞500bp) were ligated to the SfiI digested, dephosphorylated pDNR-LIB vectors. The ligation mixture was transformed into E.coli. DH5αby electroporation. Then, the library was amplified, and the clones were picked randomly from the library for screening the size of cDNA inserts. The primary constructed cDNA libraries contained 1.9×10⁶, 1.2×10⁶, 1.5×10⁶ independent clones. The titers of the cDNA libraries were estimated as 1.59×10¹⁰cfu/mL, 3×10⁹cfu/mL, 9.7×10⁹ cfu/mL with almost 100% the percentage of the recombinant clones, respectively, and those of the amplified libraries were 7.4×10¹²cfu/mL, 4.3×10¹⁰cfu/mL, 6.4×10¹⁰cfu/mL, respectively. The results showed that the inserts varied from 0.5 to 2.0kb with the average size was larger than 1100bp, which indicated that the libraries built set up a strong basis for screening the full-length cDNAs. These data showed that the libraries meet the requirement of a standard cDNA library. The Cervus eldi hainanaus, Bos taurus hainanaus, Wuzhishan Miniature Pig cDNA libraries from the peripheral blood leucocytes constructed could lay a basis for subsequent work of cloning and selecting of some kinds of fine functional genes.A full-length PMSA6 gene cDNA was cloned from the cDNA library of the peripheral blood leucocytes in Wuzhishan Miniature Pig by Colony PCR. The bioinformatic analysis showed that the PMSA6 cDNA sequence is 1029 bp length, containing an open reading frame of 741 bp, flanked by 97 bp 5'UTR and 192 bp 3'UTR, encoding 246 amino acids. The relative molecular weight is 37.680 kDa, pI 8.76. Further analysis indicated that the expression sequence of PMSA6 and the protein encoded are highly homologous to some mammals reported, showing that the PMSA6 gene is evolutionaring conservatively in animal kingdom, and the structure and function of PMSA6 gene is highly conservative.