中间锦鸡儿种子特异性fad2基因克隆及fad2-1A基因酵母表达载体的构建

Gene Cloning and Eukaryotic Expression Vector Construction of Caragana intermedia fad2

Δ12脂肪酸脱氢酶(Δ12 fatty acid desaturase,Δ12 FAD,也称为fad2)催化油酸生成亚油酸,是植物体内生成多不饱和脂肪酸的关键酶,种子中特异表达的fad2基因负责种子贮脂中多不饱和脂肪酸亚油酸的生成,决定种子油脂的成分和营养价值。采用RT-PCR和RACE技术从中间锦鸡儿未成熟种子中克隆了种子特异表达的fad2-1A、fad2-1B基因,fad2-1A编码的前283个氨基酸与fad2-1B的同源性高达98.9%,前者比后者多编码97个氨基酸,全部氨基酸残基的同源性为70.71%。将fad2-1A克隆到真核表达载体pYES2中,构建了重组质粒pYES2-fad2-1A,经PCR检测、质粒酶切、测序等检测,目的基因确实插入重组质粒,且方向正确,为进一步研究fad2基因的功能和表达调控机理奠定了基础。

Fatty acid desaturase 2 （fad2）are involved in the conversion of oleic acid to linoleic acid in plant. The seed-specific fad2 convert oleic acid to linoleic acid of seed store fat and decide the seed oil composition and nutrition value. Based on the conserved oligo amino acid residues of the published delta-12 desaturase genes from other higher plant species,the seed-specific fad2 genes （fad2-1A ,fad2-1B）were amplified by RT-PCR （reverse transcriptase-polymerase chain reaction）and RACE （rapid amplification of cDNA ends）from the total RNA of immature caragana inermedia seeds. The first 283 deduced amino acid sequences of Fad2-1A and fad2-1B showed high identity which was 98.9%. The fad2- 1A were subcloned into the enkaryotic expression vector pYES2 and the recombinant plasmid was transformed into topl0＇ for identification. The results showed that the recombinant plasmid pYES2-fad2-1A was constructed correctly. All of these were very important to study the fimctions and regnlation of these genes further.