为克隆筛选棉花腺体形成相关的基因,运用SMART技术构建cDNA文库.首先抽提棉花腺体形成时期的mRNA,mRNA逆转录后合成cDNA,经均一化处理后,SfiⅠ酶切,连接质粒载体,电转化,成功构建了棉花腺体形成时期的cDNA文库.经鉴定原始文库滴度为5.8×105cfu/mL,其重组率高达94%,插入片段的平均长度约为1.4 kb.
A pair of PCR primers was designed based on the conserved sequences of WD40 family gene of different varieties.The normolization library was screened by PCR methods,the cDNA insert size of positive clones were analyzed by PCR method.A full-length cDNA of WD40(GenBank accession number:EU219610)in cotton was obtained from sequencing.This gene is 1 796 bp in length,containing an open reading frame encoding 274 amino acids and a stop codon from 35th to 860th position.The bioinformatics characterization indicates that the protein is encoded by WD40 domain.pI and molecular weight of the protein encoded were predicted to be 4.24 and 29 kDa,respectively.The yielded gene was accondingly named as GDRP1.RT-PCR analysis showed that the expression of GDRP1 varied during the gland formation process.These results will be helpful for our further study on the gland formation of cotton.
