Cloning Important Genes Involved In Fatty Acid Metabolic Pathway And Construction Of Expression Vectors With Them

Peanut(Arachis hypogaea L.)is the world's fourth largest oil seed crop grown worldwide in subtropical and tropical regions.China is the largest nation of peanut production and comsumption in the world.Obtaining higher quantity and quality of peant oil is great importance in the contry.It has been shown that the major quality determinant in edible plant oils is the fatty acid composition at present.For a long time,our breeding aim in peanut focus on high yield and did not take serious the quality.Althougth there is about 50%of peanut output was used to oil production in the country and a few peanut varieties contain as high as 61%of rough fat. Many breeders didn't take the question of breeding high oil peanut as an important thing.As a result,the varieties used in production so far show poor oil output.Enhancing the oil content in peanut verieties has becomed an main issue of peanut quality improvement in the world.To increase both oil quantity and quality in peanut quickly,biology technology was employed in the study.We have cloned intact or partial coding region of PEPCase gene,ω-3 fatty acid desaturase gene and DGAT gene for construction of anti-RNA expression and over expression vectors,and then make use of the vectors for transformation mediated by Agrobacterim..The main results are as follow:Primers were synthesized according to the published sequence on internet,ω-3 fatty acid desaturase gene in soybean,PEPCase gene and DGAT gene in peanut were isolated,respectively, by RT-PCR.The PEPCase gene and DGAT gene were constructed on cloning vector pMD18-T, sequenced,and then tranfered to plant expression vector pCAMBIA 1300a and pSC1300.ω-3 fatty acid desaturase gene was constructed on plant expressing vector pCAMBIA 1300 directly, then identified by sequencing too.Sequencing results showed that PEPCase gene is 1950 bps with an identity of 88%to the sequence on GenBank,DGAT gene is 1038bps,98%identity with that on GenBank,andω-3 fatty acid desaturase gene is 1173bps in length,99%identity with that on GenBank.To construct antiRNA expression vectors of PEP,redesigning three pairs of primers according to the sequenced result of PEP gene,we obtained three different fragments,named PEP-1;PEP-2 and PEP-3,by PCR.To have the gene fragments specifically express in peanut embryo,these fragments were inserted inversely after a soybean oleosin gene promoter which already existed in a derived vector of pCAMBIA 1300a by digestion and ligation and three anti-RNA expressing vectors,named pCAMBIA1300 a-antiPEP1,pCAMBIA1300 a-antiPEP2 and pCAMBIA 1300 a-antiPEP3 were got afterwards.Sequencing results showed that all PEP gene fragments have been correctly constructed on the plant expression vectors,the length of the segments PEP-1,PEP-2,and PEP-3 was 643 bps,1319 bps,1925 bps,with 90%,84%,87% indentity to the sequence on GenBank,respectively.These expressing vectors have been transformed into Agrobacterium tumefaciens EHA105.ω-3 fatty acid desaturase gene and DGAT gene fragments with length of 1173bps and 1038bps,obtained from soybean and peanut respectively,were constructed on available plant expressing vectors pCAMBIA1300-7s and pCAMBIA1300-2s,with seed specific 7S promoter (from soybean)and 2S promoter(from B.nabulus),and we got two over expression vectors, pCAMBIA1300-FAD3-7s and pCAMBIA1300-DGAT-2s.Above all,we obtain three anti-RNA plant expressing vectors with PEPCase gene,and two over expressing vector withω-3 fatty acid desaturase gene and DGAT gene.The work done founded a good basis for improving fatty acid composition in peanuts in the near future.