Cloning And Transformation Of PEPC And DGAT Gene Related To Oil Content Of Rapeseed

Rapeseed (Brassica napus L.) is one of the most important oilseed crop planted widely in theword and as a major source of edible vegetable oil and protein for human. Vegetable oils are mainproduction of rapeseed, they are not only the main sources of necessary fatty acid for humanbeing and animals, but also the industrial materials of plastics, textile dyes, printing ink, leather,detergents and leather and lubricants. Compared with other countries, most of rapeseed cultivarsin China contain lower amounts of oil content. Therefore, it has significant meaning to improve oilcontent of rapeseed.Vegetable oil content of seed shows closely correlation with the activities ofPhosphoenolpyruvate carboxylase (PEPC) and Acyl CoA: diacylglycerol acyltransferase (DGAT;EC 2.3.1.20). PEPC controls the direction of the flow of phosphoenopyruvate which is the commonsubstrate of protein and lipid biosynthesis and determines the ratio of the protein/lipid content ofplant seed; Acyl CoA:diacylglycerol acyltransferase catalyzes the last and only committed stepwhich was regard as a rate-limiting reaction in triacylglycerol biosynthesis, by using adiacylglycerol and a fatty acyl CoA as its substrates, DGAT may regulate the flow of carbon intoTAG and plays an important role in regulating lipid content of plant seed.In the present research, the DGAT gene and PEP gene fragment were obtained from Arabidopsisthaliana and Rapeseed, respectively. Seed-specific plant expression vectors are constructed, thesense DTAT and antisence PEPC gene are transformed into rapeseed by Agrobacteriumturaefacience-mediated cotyledons node co-transformation system. The purpose is to enhancethe oil content of rapeseed through expressing antisence PEPC gene to inhibit PEPC geneexpression of rapeseed so as to facilitate the flow of phosphoenolpyruvate into lipid at times oflipid accumulation and over expressing DGAT gene so as to improve lipid biosynthesis rates andreduce feedback inhibition of fatty acid synthesis occurs in seed of rapeseed. The main resultswere as followings:1.Cloning of cDNA and DNA encoding DGAT from Arabidopsis and construction of theirtransformation for seed-specific expression vectorUsing PCR and RT-PCR methods the cDNA and DNA encoding DGAT were obtained from Arabidopsis and subsequently ligated into pGE M- T Easy vector, The sequence analysis wasindicated that the ligated fragment length of cDNA and DNA encoding DGAT was 1563 bp and3020 bp, respectively. Comparison of their sequence indicated that genomic DNA encoding DGATfrom Arabidopsis consists of 16 extrons and 15 introns with start codon and termination codon.,which shared 100% homology with the corresponding sequence of the reported DGAT. The DGATgene cloned from Arabidopsis thaliana, encodes a 520 amino acid protein with a predictedmolecular mass of 59.0 kDa and an iso-electric point at pH 8.53.The deduced amino acid sequence showed high identities with other plant DGAT fromBrassica napus, soybean, N. tabacum and Oryza sativa, which were 89%,73%,69%and 66%,respectively. But it had low identities with house mouse, which was only 38%. Comparison of theplant glycerol-3-phosphate acyltransferase and lysophosphatidatic acid acyltransferase sequenceswith the plant DGAT did not show any significant sequence homology, suggesting they areunrelated.The gene of the recombinant pGEM- T Easy vector was digested by BamHI (the newrestriction sites were introduced by PCR amplification primers) and ligated into thecorresponding BamH I sites of the pBI121.N and constructed the expression vector of DGAT.The recombinant pBI121.N plasmid which contain sense seed-specific expression vector of DGATwas selected out by PCR and restriction enzyme digestion and named pBI121.ND₁ andpBI121.ND₂. Transformation of Agrobacterium strains LBA4404 with pBI121.ND₁ andpBI121. ND₂ was carried out and identified by plaque-PCR2.Cloning of a sequence encoding PEP from Bassica napus and constructing of antisenceseed-specific pep expression vectorA PEP gene fragment was amplified from Xiangyou 15' s genomic DNA by polymerase chainreaction and subsequently ligated into pGE M- T Easy vector, The DNA sequence analysis wasindicated that the ligated fragment was 576 bp in size and shared 95% homology with thecorresponding sequence of the reported PEP gene. The 576 bp fragment of the recombinant pGEM-T Easy vector was digested by BamHI/Sacl (the new restriction sites were introduced by PCRamplification primers) and ligated into the corresponding sites of the pBI121.N in the antisenseorientation and constructed PEP transformation for seed-specific expression vector (pBI121.NP).Transformation of Agrobacterium strains LBA4404 with pBI121.ND was carried out and identifiedby plaque-PCR 3. The genetic transformation and obtaining the transgenic B. napusBy optimizing the parameters affecting the regeneration of rapeseed cotyledons node andtransformation mediated by Agrobactenium tumefaciens, we have established a high-efficiencyAgrobacterium-mediated co-transformation system.. The cDNA encoding DGAT fromArabidopsis and the antisense pep gene encoring Phosphoenolpyruvate carboxylase from XiangyouNO.15. were introduced into a Brassica napus variety, Xiangyou NO.15. The regenerated seedlingswith Kanamycin resistant was obtained. PCR and Southern hybridization analysis of transgenicplants showed that the DGAT or PEP gene integrated into genome of transgenic Brassica napus and11 transgenic rapeseeds were obtained. Among of 11 transgenic rapeseeds, there are 2 transgenicrapeseeds with both DGAT cDNA and antisense pep gene, 4 transgenic rapeseeds with single DGATcDNA, and 5 transgenic rapeseeds with single antisense pep gene.4. The effects of PEPC and DGAT gene expression on lipid and protein content in rapeseedDTAT gene can express in all transgenic rapeseed, Northern blots revealed that it was present athigh concentration in developing embryos and at a low concentration in leaf. The PEPC activitiesof transgenic lines were decreased, the most reduction could be up 11%. Analysis of tdacylglyceroland protein content in seed of the 11 transgenic lines showed that the expression of DGAT andantisenece PEPC gene resulted in the increase of oil content and the average 1000-seed weightby anywhere. The off content increased ranging 0.7 to 6.9 percentage, representing net overallincreases of 1.7 to 17.4%, and exhibited that transgenic lines with both DGAT and PEPC werebetter in increase oil content than transgenic lines with single DG,4T or single PEPC. At the sametime, the protein content in seeds decreased in a degree. The correlation analysis indicated that therewere an significant negative correlation between protein content and oil content, The correlationcoefficient between of them is-08801.