南极冰藻Chlamydomonas sp.ICE-L的cDNA文库构建及品质检测  

Construction and Quality Detection of cDNA Library From Antarctic Ice Alga Chlamydomonas sp.ICE-L

作　　者：王能飞[1] 沈继红[1] 金治平[2] 赵爱云[3] 林学政[1] 

机构地区：[1]国家海洋局第一海洋研究所 [2]清华大学生物系,北京100084 [3]青岛大学天然色素研究所,山东青岛266071

出　　处：《海洋科学进展》2006年第4期520-525,共6页Advances in Marine Science

基　　金：国家自然科学基金项目--南极冰藻抗冻蛋白的研究(40306027);青岛市自然科学基金项目--极端环境下南极冰藻抗逆基因的研究(05-2-JC-56);国家海洋局青年基金项目--南极冰藻抗逆藻种的筛选和cDNA文库构建(2006103);中国极地科学战略研究基金--南极海冰区藻类对气温升高的适应性研究

摘　　要：为构建南极冰藻Chlamydomonas sp．ICE-L的cDNA文库，提取对数生长期南极冰藻ICE-L的总RNA，以此为模板，通过PowerScript逆转录酶逆转录合成第一链cDNA；再以第一链cDNA产物为模板，用LD-PCR合成第二链cDNA。该cDNA产物经分级分离转入大肠杆菌中，即获得南极冰藻IcBL的cDNA原始文库，其滴度为1．6×10^6 cfu／mL。扩增后的cDNA文库的滴度为1．0×10^10 cfu／mL。用PCR方法测得文库的重组率大干97％，插入cDNA的长度为0．5～1．8kb，0．9kb以上插入片段占50％以上。取适量扩增文库稀释并铺平板，挑取72个独立菌落，对其中22个独立菌落所插入的cDNA进行测序，克隆到了一个具有5’和3’非编码区的40S ribosomal protein S5全长基因序列，GenBank收录号为AM167929。In order to construct the cDNA library from Antarctic ice alga Chlamydomonas sp. ICE-L, the total RNA from the ice alga was taken as template to synthesize the first strand cDNA by using PowerScript reverse transcriptase, and the LD-PCR technique was used to synthesize the second strand cDNA. The cDNA products were transported into colon bacilli to obtain the unamplified cDNA library with a titer of 1.6× 10^6 cfu/mL, and the amplified cDNA library has a titer of 1.0× 10^10 cfu/mL. It is shown from the PCR detection results that more than 97 % of clones in the primary cDNA library are recombinant and the insert cDNA sizes are in the range 0.5 kb to 1.8 kb. It is shown from the sequencing analysis results that a full-length 40s ribosomal protein S5 cDNA with 5＇ and 3＇ unnumbered regions was isolated, and its accession number of GenBank is AM167929.

关 键 词：PCR 南极冰藻 RIBOSOMAL protein S5 CDNA文库 

分 类 号：Q949.2[生物学—植物学]