Cloning And Specific Expressing Vector Construction Of Key Gene FAD2 On Improving Fatty Acid Quality In Peanut

Peanut (Arachis hypogaea L.) is the world's fourth largest oilseed crop, and is grown worldwide in many subtropical and tropical regions. China is to one of the largest nations of peanut production in the world. A major determinant of quality in edible plant oils is the fatty acid composition. The peanut oil comsists mainly of oleic and linoleic acid. Oleic acid can reduce the content of low-density lipoproteincholesterol (LDL) in human body, but can't reduce the content of high-density lipoproteincholesterol (HDL). However, polyunsaturated fatty acid such as linoleic acid and linolenic acid can reduce LDL and HDL synchronously. The ratio of Oleic acid and linoleic acid is on the lower side in the available peanut varieties, so enhancing the oleic acid content in peanut oil has been mainly problem of peanut quality improvement in the world. To improve the composition of oleic acid in peanut, We makes a research on improving peanut oleic acid content employing the methods of molecular biology, bio-informatics technology and gene engineering. We obtained the coding region and partial cds of oleate desaturase gene, and embryo-specific expression promoters. Then we construct the anti-RNA expressing vector and RNAi mediated expressing vector by digestion and ligation. Main results as follow::1. To make interested genes specific express in peanut embryo, primers were synthesized according to the published sequence on internet, then the promoters of oleosin gene and a' subunit of β-conglycinin gene in soybean, the promoter of 1.7S napin seed storage protein gene in Brassica napus and the promoter of Lea gene DC3 in carrot were isolated, respectively, by PCR. The 7S, 2S, DC3 and oleosin promoter are constructed on cloning vector pGM-T and mediated expressing vector pSPROK, respectively, and then were identified by sequencing. Sequencing results showed: the 7S promoter is 878 bps with identity of 99.8% to the sequence on GenBank; the 2S promoter is 1145bps, 100% identity with the sequence on GenBank; the DC3 promoter is 153bps, 100% similarity with the sequence on GenBank; the oleosin-a and oleosin-b promoters are 595bps and 319bps, respectively, 97% and 96% with the sequence on GenBank, respectively.2. Extracting genomic DNA from peanut leaves and designing the primers according to the published sequence of A12 fatty acid desaturase gene on GenBank, we obtained two different fragments of FAD2 gene, named FAD2-2 and FAD2-1, by PCR. The fragments were constructed on mediated expressing vector pSPROK with soybean oleosin protein gene promoter before them by digestion and ligation, then they were transferred into plant expressing vector pCAMBIA1300. Three anti-RNA expressing vector, named pCAMBIA1300-1a, pCAMBIA1300-2a and pCAMBIA1300-2b were got afterwards. Sequencing results showed that FAD2-2 has been inserted in plant expressing vector pCAMBIA1300, the segment was 593bps, containing a coding region of 558bps, encoding 186 amino acid, and 97% indentity with the sequence on GenBank; FAD2-1 has also been inserted in plant expressing vector pCAMBIA1300, containing a coding region of 1153bps, encoding 384 amino acid, with 99% indentity with the sequence on GenBank. At present, three plant expressing vectors have been...