Studies On Desaturation-related Genes Of Brassica Napus

1,Increased levels of oleic acid may enhance the nutritional and functional value of rapeseed,rapeseed oil is primarily composed of palmitic,stearic,oleie,linoleic and linolenic fatty acids.Delta-12 desaturase(FAD2) in plants converts oleic acid(18:1) to linoleic acid(18:2) by inserting a double bond at the delta-12 position.Fatty acid desaturase-2 gene(fad2) encodes delta-12 desaturase that functions in the endoplasmic reticulum.Fifty-six fad2 DNA clones and nine fad2 seed cDNA clones of B.napus cv. Xiangyou 15 were randomly selected and sequenced in this study.These fifty-six DNA sequences share 91%～99.9%identity in nucleotides.Through combination of identical sequences,eleven different sequences were found,of which each represented various copy of fad2 gene.Deduced amino acid sequences revealed that many stop codons occurred in the coding region of six copies,the other five copies shared 90.6%～99.74%amino-acid identity.Two differential cDNA were found from nine fad2 seed cDNA sequence,no stop codons in the coding region,revealing that more than one fad2 gene express in developing seeds,and oleic acid content is controlled by multigene.The eleven copies in genome could be divided into two groups based on their homology,and designated as fad2Ⅰand fad2Ⅱ,respectively.RT-PCR analysis in seeds at twenty-seven days after pollination showed that fad2Ⅰexpressed strongly in seeds,and no expression of fad2Ⅱwas found,but both of them expressed in leaves.2,Fatty acid composition of two Brassica napus EMS mutants(HO and LO)in different developmental periods were examined by using gas chromatography.The results revealed that oleic acid percentage in HO and LO is low at early developmental time(the first 20 days after pollination,DAF ),while it increased rapidly during 20 to 30 DAF,from less than 5%increased to 52%(in LO) and 65% (in HO).3,According to HO and LO seed fatty acid composition accumulation patterns, selecting 27 DAP seeds of HO and LO for suppression subtractive hybridization,and a differential expression cDNA library was constructed which including four hundred and eighty clones.All SSH clones were arrayed and screened by dot blot hybridization,one hundred and six positive clones was obtained,accounts for 22.1% of the total clones.All these one hundred and six clones were sequenced,and eighty-eight uniESTs were obtained.The results of BLASTN showed that seventy-three uniESTs had homologous sequences in the nr database.The BLASTX results indicated that nineteen uniESYs had significant protein homology,majority are regulating factors.Other sixty-nine uniESTs had not protein homology which is expected to be novel genes.4,Six ESTs were selected for RT-PCR analysis,including four known function ESTS(stearyl-ACP desaturase gene,TCP family transcription factor,growth hormone induced protein,transcription factor) and two unknown EST,B57 and B315.