棉 花 学 报     Cotton Science    2009，21(5)：351-355

The Construction of cDNA Library from Cotton Seed
WANG De-long, YU Ji-wen, YU Shu-xun^*, ZHAI Hong-hong, FAN Shu-li, SONG Mei-zhen, ZHANG Jin-fa
( Cotton Research Institute , Chinese Academy of Agriculture Science , Key Laboratory of Cotton Genetic Improvement,Ministry of Agriculture , Anyang , Henan 455000, China )

Abstract：SMART technique was used to construct the full length cDNA library of cotton seed. Total RNA was isolated from sea-island cotton embryo about 25-35 days. Full length ds-cDNA (Double-strained complement DNA) was amplified by using 21 round LD-PCR（Long-distance PCR）. After Sfi Ⅰ digestion and Chroma spin-400 fractionation, cDNAs (>500 bp) were ligated to pDNR-Lib vector and transformed to DH10B afterward. The total clones of the library was measured to be 5×10⁶ with a titer of 1.5×10⁸cfu·mL^-1. The inserted fragments ranged from 0.5 kb to 2.5 kb, and the recombinant rate of the library was about 100％. 180 monoclones were selected to sequence, and 13 EST concerned with lipid metabolism were identified. These results show that the cDNA library can be used for screening genes of fatty acid metabolism.
Ke words：cotton; embryo; lipid; cDNA library; EST      [Full Text, 2832KB]