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Construction and Preliminary Identification of cDNA Library for Velvet in Sika Deer (Cervus Nippon Hortulorum)

Author: YanLi
Tutor: LiHePing
School: Northeast Forestry University
Course: Animal Genetic Breeding and Reproduction
Keywords: Deer Deer antler cDNA library
CLC: S865.42
Type: Master's thesis
Year: 2010
Downloads: 66
Quote: 1
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Abstract


The test with deer antler growth and development of related genes cloned the full-length sequence , using SMART technology to build the full - length cDNA library of the Northeast sika deer antler tip organizations . Adopt healthy 4 -year-old northeast captivity deer as experimental animals , whichever antler velvet antler tip organization as experimental material , the SV Total RNA isolation and purification of total RNA extraction kit ; reverse-transcribed into single-stranded cDNA , LD - PCR was used to synthesize double-stranded cDNA ; the PCR product was purified by hydrolysis of proteinase K , purified, digested with Sfil ; digested product was passed through a CHROMA SPIN-400TM column fractionation , recycling the components of the fragment length of more than 500bp cDNA , and pDNR -LIB plasmid vector was transformed into E.coli.DH5a efficient competent cells , and built the original library ; titers and recombination efficiency of identification library after library amplification ; single colonies were randomly picked plaques with carrier both ends of the cloning site of the universal primers for PCR amplification, the quality of the cDNA library to detect . The test results show that : total RNA electrophoresis , 18S, 28S bands clearly obvious no degradation of total RNA quality , high purity , in line with the requirements of building a database . Upon identification, the library obtained about 2.56x106 recombinants , recombination efficiency is close to 100% , the length of the inserted fragment , the average length of between 0.5-2.0kb 1.1kb . This test library indicators meet the requirements of the standard cDNA library screening of the full-length cDNA , can be used to build a deer velvet antler cDNA library . Deer antler growth tissue cDNA library was constructed successfully , not only save precious the sika genetic resources , but also for the subsequent EST sequencing analysis , gene expression profiling to build , laid the foundation for early analysis of functional gene expression in the of Velvet Antler growth and development period .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Hunting,domestication of wild animals > A variety of wild animal domestication > Medicinal animals > Deer
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