Cloning And Expression Vector Construction Of KCS Genes In Cardamine Hirsuta And Thlaspi Arvense And The Transformation Of Brassica Napus And Arabidopsis Thaliana

Nervonic acid (C24:1) is a kind of very long chain monounsaturated fatty acids. Nervonic acid is an essential fatty acid which helps to maintain brain health by assisting in the biosynthesis and maintenance of nerve cell myelin. Nervonic acid is recognized as the most promising brain health products in21st Century. At present, nervonic acid oils is widely used for pharmaceutical, nutraceutical and industrial applications. But the human can hardly synthesize nervonic acid and just depend on eating. Natural plants rich in nervonic acid differ in fruit oil and nervonic acid development cost too much, so finding high nervonic acid plant with low cost becomes an urgent problem and. This is an effective means of achieving this goal by plant gene-engineering. The biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes:β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxacyl-CoA dehydratase (HCD) and trans-2,3-enoyl-CoA reductase (ECR). KCS catalysis the rate-limiting step in the biosynthesis of very long chain fatty acids (VLCFAs) including nervonic acid.In this study, the full-length CDS of β-ketoacyl-CoA synthase (KCS) gene from Cardamine hirsute and Thlaspi arvense was cloned. The two cloned sequences were ligated into pFGC-5941.nap, then the two over-expression vector was transformed into Brassica napus GX-29and Arabidopsis thaliana (Col) by Agrobacterium-mediated. Whether can improve nervonic acid composition during seed development of Brassica napus GX-29and Arabidopsis thaliana (Col). The study paved the way for further obtaining natural nevonic acid resources with low cost in the future. The results are as follows:1. Sequence analysis shows that CarKCS sequence and Cardamine graeca KCS reported in Genbank homology up to88%, Lunaria annua sequence up to86%and Arabidopsis thaliana KCS18sequence up to87%. ThlKCS respectively up to85%、87%、86%.2. Bioinformatic analysis shows that the CarKCS and ThlKCS respectively encodes505and506amino acid. There are two highly hydrophobic transmembrane domains in the N-terminal and include all amino-acid residues Ser282, Cys223, His302, His387, His391and His420of the enzyme active site as Cardamine graeca, Lunaria annua and FAE1KCS18. In the phylogenetic tree, CarKCS and ThlKCS like KCS18belongs to FAE1-like subfamily of KCS gene family.3. Two over express vector was constructed.4. The two over-expression vector was transformed into Arabidopsis thaliana (Col) and respectively got4CarKCS resistant plants and2ThlKCS resistant plants.5. The two over-expression vector was transformed into Brassica napus GX-29and respectively got31CarKCS resistant plants and27ThlKCS resistant plants.