Cloning And Analysis Of Genes Of Fatty Acid Biosynthesis-related Enzymes (KASI,FatB) And Genetic Transformation Of AhFAD2B In Arachis Hypogaea L.

The fatty acid biosynthesis is catalyzed by three main enzymes which are acetyl-CoA carboxylase and fatty acid synthase and acyl-ACP thioesterase. The first step reaction with acetyl-CoA to form malonyl-CoA is catalyzed by acetyl-CoA carboxylase. The stretching of the growing acyl chain is catalyzed by the followed fatty acid synthase which mainly consisted ofβ-ketoacyl-ACP synthase(KAS).The condensation of malonyl-ACP with acyl-ACP to formβ-ketoacyl-ACP is catalysed by smallβ-ketoacyl-ACP synthase (KAS) family enzymes. KASIII condenses acetyl-CoA with malonyl-ACP to form 4:0-ACP, KASâ… is responsible for the elongation of 4:0-ACP to 16:0-ACP, KAS II mediates the elongation of 16:0-ACP to 18:0-ACP. The last step to form acyl-carrier protein and dissociative fatty acid is catalysed by Acyl-ACP Thioesterase. Further, the fatty acid carbon chain with different lengths is generated under the role of a series of desaturase. Thereinto , Oleic acid and Linoleic Acid which are the highest content of peanuts fatty acids are catalyzed byâ–³¹²fatty acid dehydrogenase(FAD2) which is the key enzyme with Oleic acid to form Linoleic Acid.This study was to clone the key gene KASI encoding fatty acid synthesis and to construct the expression vector of the key gene FAT ,also using the expression vector with gene anti-ahFAD2B for genetic transformation, to make the foundation of improving the peanut quality through the biotechnology methods. The main research results are as followeds:1.Cloning and genetic analysis of gene KASIAccording to the published sequence of KASI gene on GeneBank in Arabidopsis thaliana ,Zea mays and Oryza. Using homology cloning technology, KASI gene fragments from cultivated species and seven wild species. The sequence homology among different cultivated species and bewteen cultivated species and wild species is analysed and compared.Two different copies sequence of gene KASI named respectively dKASI-1 and dKASI-2 was obtained, the rate of homology is 95.91%, the genetic evolution analysis of two copies sequence between cultivated species and two wild groups with A and B shows that the two copies sequences are respectively from two wild groups, the rate of homology with dKASI-1 and group A of wild species is 99.69%, the rate of homology with dKASI-2 and group B of wild species is 100%. 2. Cloning and analysis of the gene FatB in peanut and construction of its expression vectorThe primers were designed according to the published sequence of gene AhfatB on GeneBank(EF117305). The RNA extracted from leaves of cultivar Shanhua7 was used for RT-PCR,the full-length cDNA fragment of AhfatB was amplified. The aimed fragment was recycled after Agarose Gel Electrophoresis. The recombinant plasmid pEASY-AhfatB through connecting the aimed fragment and the cloning vector pEASY-T1. Sequencing analysis indicated that the full-length of the fragment was 1617bp, the inserted fragment of nucleotide and amino acid sequence was showing 99.51% and 100% homology to the reported sequence AhfatB. The recombinat plasmid pEASY-AhfatB were diges- ted by restricton enzyme BamHI and SacI, recycling the aimed fragment , the recombimant was named PBI121-AhfatB.We connect the aimed fragment and the 35S promoter meanwhile resecting GUS gene to construct the expressing vector PBI121-AhfatB.3. Genetic transformation and detection of gene anti-ahFAD2BUsing agrobacterium-mediated technique, we pick the little germinal layer for an explant with fenghua2 , using the expression vector with gene anti-ahFAD2B and marker gene nptâ…¡for genetic transformation. To sift the transgenosis seedings with the Kanamycin, the eighty-seven resistant seedings were transplanted to the field. The detection through the resistance gene nptâ…¡shows that the aimed gene has been transformed into the genome of the peanut, and the seeds has been harvested.