AlA Accumulation Pattern And Cloning Of ω-3Fatty Acid Desaturase CDNA In Perilla, And Its Prokaryotical Expression

The content and composition of fat in food was closely related to human health.Fattyacids can be divided into Unsaturated fatty acids (monounsaturated fatty acids andpolyunsaturated fatty acids) and saturated fatty acids.Polyunsaturated fattyacids(PUFAs)separated as ω–3,ω–6series. ω–3series contains ALA,DHA and EPA,etc. Ofthem. ALA is not only the precursor of DHA and EPA,But also necessary for human healthand evolution.It is the basic object in lives evolution and has physiological function inimproving intelligence and memory,as well as in protecting eyesight. Preventing myocardialinfarction and hyperlipemia.In normal conditions,the fatty acid Contains rich linoleic acid,andthe content of ALA is poor.The main source of ALA is deep-sea fish oil,but the deep–sea fishoil too low to fulfill our market in china.Plant linolenic acid from linoleic acid by omega-3fatty acid desaturase catalytic.Fattyacid desaturase is a membrane-bound protein exists as gene family. plants fatty aciddesaturase affects growth and development,and participate in the defense response of thebiotic and abiotic. Fatty acid desaturase, which is the enzyme in the fatty acid synthesis andmodification pathways. Is one of the target enzyme in the genetic engineering of plant oilsand fats.Generally,There are three genes fad3,fad7,fad8encoding fatty acid desaturase. Plantfatty acid desaturase is conservative in the evolution.Has the highly conserved domain. Theresearch of omega-3fatty acid desaturase gene has broad application prospect in the Plantlipid improvement,.We could controlled Fatty acid expression by means of molecularbiology.Thus its genetic manipulation has been applied in a wider area.Perilla frutescens belong to perilla Labiatae. Is the special fuel oil crops,Strong adaptab-ility,Suitable for large area planted in the west. Originating in China,Wide distribution andcultivated application about nearly2000years history. Perilla frutescens Is the first issued bythe Ministry of health as both food and medicine which is one of the60kinds of goods. Rich in ALA activity.It is a broad development prospects plant ALA resources. and a goodapplication prospect of the oil engineering research object. But the perilla culture area is notbig,and output is low,and we could not get many ALA only rely on perilla culture.So,weshould research the physiological mechanism in the accumulation of ALA and cloning the keygene from it.Analysis it is structure and function,we use prokaryotic expression to validate theexact express of ω–3fatty acid desaturase, it has important theory and practice purport inimprove the plant ALA output by transgenes.In our research,we abstract all the fatty acid from perilla(perilla frute-scens (L.) Britt.var. arguta (Benth.)Hand. Mazz.).The highest extraction ratio is20.08%.We studied the fattyacid's components and ratio by gas chromatography.The main components of fatty acid inperilla is unsaturated fatty acid.The content of saturated fatty acid is gradually decrease fromblooming to maturate,but the content of unsaturated fatty acid is gradually increase,and thelinolenic acid is the highest.At the same time,we construct the accumulation of a–linolenicacid's dynamic model in perilla's seeds development process.Full length ω–3fatty acid desaturase cDNA was cloned from perilla (perilla frute-scens (L.) Britt. var. arguta (Benth.)Hand. Mazz.) by PCR using specific primers and wasligated into PUCm–T easy vector. Positive clones were selected and the recombinantplasmid was isolated from E. coli DH5a and identified by sequencing.The results indicatedthat the target cDNA was found to be very hight conservative from the published. Accordingto the sequence information to infer the components of amino acid,Molecular Weight andprot-ein structure.Compared with other six plant's construct,functions and homology ofprotein which coden by ω–3fatty acid desaturase gene.Digested by Xhol and SacI.The targetgene was constructed Pires–hrGFP–1a vector digested with the same enzymes.Transform thevector to E.coli BL21(DE3).Cultured in the LB with0.5%ampicillin.We screening the positi-ve cloning with PCR.Induce the target protein(zs047039–1,zs59477.1) expression byrevulsive IPTG.12%SDS–PAGE detected two express protein.Their Molecular Weight are45KDa,49KDa respectly.