中华大蟾蜍耳后腺全长cDNA文库的构建

目的构建中华大蟾蜍(Bufo bufo gargarizans)耳后腺全长cDNA文库。方法采用TRIzol法提取耳后腺总RNA,经亲和色谱纯化获得总mRNA。以总mRNA为模板,利用In-FusionSMART-erTMDirectional cDNA Library Construction Kit获得初始文库,测定文库滴度、重组率及插入片段长度。随机选取211个克隆进行测序。结果初始文库的滴度为1.3×109cfu.L-1,重组率90%,插入片段大小分布为0.4～4.0 kbp,平均片段大小约为1.1 kbp。共获得180个表达序列标签(ex-pressed sequence tag,EST)。结论此方法所构建文库各项指标均符合要求,为从功能基因组水平研究中华大蟾蜍耳后腺的基因表达及其功能奠定基础。

Construction of full length cDNA library from venom gland of Bufo bufo gargarizans

Objective To construct a full length cDNA library from venom gland of Bufo bufo gargarizans. Methods Total RNA was extracted from the venom gland of B. bufo gargarizsas by TRIzol method. The mRNA was purified with mRNA Purification Kit. In-Fusion^® SMARTer^TM Directional cDNA Library Construction Kit was used to obtain the primary library, and the titer, recombinant rate and the length of insert fragments of the library were checked. A total of 211 clones were randomly selected for sequencing. Results The titer of primary library was 1.3×10⁹ cfu·L^-1with a recombinant rate of 90%. The length of insert fragments ranged from 0.4 kbp to 4.0 kbp, and the average length was about 1.1 kb.180 unigenes were obtained by EST analysis. Conclusion The constructed full length cDNA library can be used for further screening and cloning functional genes from the venom gland of B. bufo gargarizans.