African Journal of
Agricultural Research

  • Abbreviation: Afr. J. Agric. Res.
  • Language: English
  • ISSN: 1991-637X
  • DOI: 10.5897/AJAR
  • Start Year: 2006
  • Published Articles: 6574

Full Length Research Paper

Efficient construction of a normalized cDNA library of the high oleic acid rapeseed seed

Zhang Zhenqian1, Xiao Gang1,2, Liu Ruiyang1, Tan Tailong1, Guan Chunyun1,2* and Wang Guohuai1,2
1Oil Crops Institute/National Oil Crops Improvement Center, Hunau Agricultural University, China. 2Pre-State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops, Changsha 410128, China.
Email: [email protected]

  •  Accepted: 22 August 2011
  •  Published: 12 September 2011

Abstract

The cDNA library normalized by reassociation is a newly developed, effective platform for gene discovery and function analysis. In order to screen the genes related to high oleic synthesis, a normalized cDNA library was constructed. Total RNAs were prepared from high oleic acid rapeseed seeds using TRIzol reagent. CDS III/3´ primer was used to synthesize the first-strand cDNA with SMART technique; and the double strand (ds) cDNA fraction formed by abundant transcripts is degraded by 1/2 duplex specific nuclease (DSN) dilution. Then amplified with twice the PCR cycles, which was 11 and 12 respectively. Finally, the cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector. The titer of the unamplified cDNA library was 2.61×106 cfu/ml with a recombinant rate of 100%, and the titer of the amplified library was 9.24×109cfu/ml. Electrophoresis gel results fragments ranged from 800 bp to 2 kb, with an average size of 1000 bp. These results indicated that the normalized full length cDNA library has been constructed successfully, which is convenient for further studying the oleic synthesis mechanism in rapeseed.

 

Key words: Normalization, cDNA library, rapeseed, high oleic acid.