陈曦,周艳菊,夏明钰,刘东春,崔征,王东
CHEN Xi1,ZHOU Yan-ju1,XIA Ming-yu2,LIU Dong-chun1,CUI Zheng1,WANG Dong1(1.Laboratory of China-Korea Molecular Pharmacognosy

• 1:沈阳药科大学中韩分子生药学研究室
• 2:沈阳药科大学生命科学与生物制药学院

摘要(Abstract)：

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。
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 SMARTerTM 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 which were obtained by EST analysis.Conclusions The constructed full length cDNA library can be used for further screening and cloning functional genes from the venom gland of B.bufo gargarizans.

关键词(KeyWords)： 中华大蟾蜍;耳后腺;全长cDNA文库;EST分析
Bufo bufo gargarizans;venom gland;full length cDNA library;EST analysis

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 陈曦,周艳菊,夏明钰,刘东春,崔征,王东
CHEN Xi1,ZHOU Yan-ju1,XIA Ming-yu2,LIU Dong-chun1,CUI Zheng1,WANG Dong1(1.Laboratory of China-Korea Molecular Pharmacognosy

DOI: 10.14066/j.cnki.cn21-1349/r.2012.12.002

参考文献(References)：

• [1]国家药典委员会.中华人民共和国药典:一部[M].北京:中国医药科技出版社,2010:360.
• [2]楼之岑,秦波.常用中药材品种整理和质量研究(北方编):第2册[M].北京:北京医科大学中国协和医科大学联合出版社,1995:1134-1200.
• [3]高艳荣,张莉,张磊,等.蟾酥及其有效成分的药理作用及机制研究进展[J].武警医学院学报,2003,12(5):406-408.
• [4]戴维克.药用天然产物的生物合成[M].北京:化学工业出版社,2007:199-207.
• [5]DMITRIEVA R I,BAGROV A Y,LALLI E,et al.Mam-malian bufadienolide is synthesized from cholesterol inthe adrenal cortex by a pathway that is independent ofcholesterol side-chain cleavage[J].Hypertension,2000,36(3):442-448.
• [6]DMITRIEVA R I,LALLI E,DORIS P A.Regulation ofadrenocortical cardiotonic steroid production by dopa-mine and PKA signaling[J].Front Biosci,2005,10:2489-2495.
• [7]GARRAFFO H M,GRAS E G.Biosynthesis of bufadi-enolides in toads.VI.Experiments with[1,2-3H]cho-lesterol,[21-14 C]coprostanol,and 5 beta-[21-14 C]pregnanolone in the toad Bufo arenarum[J].Steroids,1986,48(3/4):251-257.
• [8]PORTO A M,GRAS E G.Biosynthesis of the Bufadien-olide Marinobufagin in Toads Bufo paracnemis fromCholesterol-20-14C[J].Experientia,1971,27(5):506-506.
• [9]SANTA COLOMA T A,GARRAFFO H M,PIGNATA-RO O P,et al.Biosynthesis of bufadienolides in toads.V.The origin of the cholesterolused by toad parotidglands for biosynthesis of bufadienolides[J].Steroids,1984,44(1):11-22.
• [10]D'ALESSIO J M,GERARD G F.Second-strand cDNAsynthesis with E.coli DNA polymerase I and RNase H:the fate of information at the mRNA 5'terminus and theeffect of E.coli DNA ligase[J].Nucleic Acids Res,1988,16:1999-2014.
• [11]ZHU Y Y,MACHLEDER E M,CHENCHIK A,et al.Reverse transcriptase template switching:A SMARTTMapproach for full-length cDNA library construction[J].BioTechniques,2001,30:892-897.