Cloning Of The PEPC Gene And Construction Of A Seed-specific IhpRNA Expression Vector In Brassica Napus L

Rapeseed(Brassica napus L.)is one of the most important oilseed crop planted widely in the world and as a major source of edible vegetable oil and protein for human.It also has a important role in the international agriculture enconomy.Vegetable oils are main production of rapeseed.They are not only the main sources of necessary fatty acid for human being and animals,but also the industrial materials of plastics,textile dyes,printing ink,leather,detergenis and leather and lubricants.Rape cultivated area of our country exceeds 100 million must(mainly in Yangtse Valley),accounts for 1/3 of cultivated area the world,and has been provided exceeding 50%edible oil giving our country people..However,compared with other countries,such as Canada,most of rapeseed cultivars in China contain lower amounts of oil content::before 2005,international rapeseed average oil content approximately 42%,but our country rapeseed average oil content is 38%only.At present our country rapeseed annual yield is about 13 million tons,and can provide rapeseed oil 4.7 million tons,which has accounted for half of the total yield of edible oil(annual vegetable oil yield is about 8.5million tons in China).And the gap between the edible oil production and consumption is very large(the total consumption of vegetable oil is about 12million tons).Our country importation of edible vegetable oil was 1.79million tons in 2000 and was 6.211million tons in 2005,the annual growth rate has reached 28.2%,so to relive edible oil consumption,it must be done that we should try our best to increase rapeseed yield,expand rapeseed planting area and improve oil content of rapeseed.Therefore,it has significant meaning to improve oil content of rapeseed.Phosphoenolpyruvate carboxylase(PEPC) show closely correlation with vegetable oil content.It controls indirectly the direction of the flow of phosphoe nopyruvate which is the common substrate of protein and lipid biosynthesis and determines the ratio of the protein lipid content of plant seed.We make use of the RNAi technology to restrain PEPC,make solubility sugar be so as to flow inio Metabolic Pathway on Fatty Acid,and gain Genetically—modified rape with high oil content.The main results were as followings: 1.The confirming of a conserved region of PEPC genePEPC gene(GenBank accession D13987) is used to do nucleotide blast with vegetable genes in GenBank.The result is that the extron has high conservatism, which is at the site 2637-2859bp of PEPC gene,named "s".2.The clone of"s" gene and napin promoterA full-length napin promoter sequence(1138bp)and a conserved region(181bp)of PEPC gene were amplified by PCR,from the genomic DNA of Brassica napus L.and recombined into the pMD 18-T vector respectively.3.Construction of inverted repeat cassetteTo develop an ihpRNA vector for seed-specific expression targeting the PEPC gene in Brassica napus L.,an inverted repeat unit of the 181bp segment of PEPC gene was first cloned into a mediating vector,pBS-NEI vector,with a spliceable PEPC intron sequence in between.4.Construction of the seed-specific expression vector pCAMNapinAn ihpRNA expression vector pCAMNapin was constructed through inserting the seed-specific napin promoter into the multiple clonal sites in the binary vector pCAMBIA1391.5.Construction of PEPC gene's seed-specific expression ihpRNA vectorthe whole inverted repeat cassette was digested from pBS-B2-NEI-B1 vector,and inserted into the 3-end of the napin promoter in the vector pCAMNapin.It was finally confirmed by the digestion of restriction enzymes,that a seed-specific expression ihpRNA vector,pCAMNapin-B2-NEI-B1,targeting the PEPC gene in Brassica napus L was constructed.