玉米叶片cDNA文库构建及Cah墓因克隆

CONSTRUCTION OF MAIZE LEAF CDNA LIBRARY AND CLONING OF CAB GENE

从玉米叶片中提取总ＲＮＡ后，再利用ｏｌｉｇｏ（ｄ７）－纤维素亲合层析往，从中分离出ｍＲＮＡ，然后以ｏｌｉｇｏ（ｄ７）－ＸｂａＩａｄａｐｔｏｒ作为引物，ｍＲＮＡ作为模板，合成相应的ｃＤＮＡ．并定向克隆进λＧＥＭ－４载体，构建出滴度为２×１０５ｐｆｕ的玉米叶片ＣＤＮＡ文库、最后，利用寡核苷酸探针，从ｃＤＮＡ文库中筛选到８个阳性克隆。

Poly (A)+ RNA was obtained by running the total RNA. extracted from freshleaves of maize. through a column of oligo(dT)-cellulose affanity chromatography. Complementary DNA(cDNA) was synthesized by reverse transcription according to theRibodone Systems'procedures using oligo(dT)-XbaI adaptor as primer.In order to cloneit into vector.the cDNA was ligated with EcoR Ⅰ adaptor and digested with XbaI. Finally.a maize leaf cDNA library with a title of 2×105 pfu was established. Eight positive cloneswere identified through three rounds of in situ hybridization using an oligonucleotideprobe.