The Construction Of Giant Panda's (Ailuropoda Melanoleuca) Hypothalamus CDNA Library

In this study, we used the hypothalamus of giant panda to construct cDNA library. We extracted total RNA from the giant panda's hypothalamus and further purified mRNA from total RNA. Synthesis of first-strand cDNA was catalyzed by Reverse Transcriptase, and the cDNA-mRNA hybrids were synthesized and converted into full-length double-stranded cDNAs. We polished the ends of double-stranded cDNAs, ligated the synthetic linkers or adaptors and digested the attached linkers to create cohesive termini. Fractionation of cDNA was performed by Gel Filtration through Sepharose CL-2B. The final stage of cDNA library construction involved optimizing ligation of the size-fractionated cDNA to bacteriophage arms, followed by packaging and plating of the recombinant bacteriophages. We obtained the primary cDNA library from the recombinant bacteriophages. The recombinant bacteriophages infected host bacteria, then the amplified cDNA library with high quality was obtained.In this study, Total RNA was extracted from the hypothalamus of giant panda using TriZol reagents, we obtain high quality total RNA that three bands are 5S, 18S, 28S from total RNA, while total RNA has high pure, good quality and without degradation.The method of chromatography on oligo(dT) columns is performed to purify without secondary structure poly(A)+ RNA from total RNA. mRNA was used as template and synthesis of first-strand cDNA was catalyzed by Reverse Transcriptase; The cDNA-mRNA hybrids were converted into full-length double-stranded cDNAs (ds-cDNAs). E. co// DNA polymerase I extended the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. ds-cDNAs were fractionated by Gel Filtration through Sepharose CL-2B, and fragments over 10Obp were reclaimed; The produces of cDNAs were purified and ligated to Uni-ZAP XR vector. The produces of ligation were packed in packaging extracts and the primary cDNA library was obtained. The titer of this library is 1.129X106 clones/ug Uni-ZAP XR Vector. Theprimary library was transfected into E.coli and amplified. The titer of this amplified cDNA library is 5.2 X109 pfu/ml. Twelve plaques were cored randomly from the agar plate and incubated. Phagemids were extracted and digested by using both EcoR I and Xho I . The size of released insert fractions was examined by agarose gel electropheresis ranked between 300 bp and 500 bp.In this work, we offered an important resource of gene expression library, which will be helpful to solve these problems of giant panda's low reproduction, difficult pregnancy, difficult judgement of oestrus. Moreover this cDNA library provides powerful means for cloning and studying the genes involved in life activities of giant panda.