The Clone Of FAD3Gene In Flax And Construction And Genetic Transformation Of The FAD3Gene Vector

Flax (Linum usitatissimum L.) is the panicum miliaceum, and it belongs to theflax linen genera. Flax has a long cultural history in our country. Oil seed Flax isChina’s important oil crops. With the development of economy and improvement ofliving standards, people’s demand for oil seed Flax is already by the edible type turninto the health protection gradually, especially the linseed oil is widely attention. Theoil content of oil seed Flax is high, and ALA of necessary for human body is rich inoil seed Flax, and it is important for the prevention and treatment of cardiovascularand cerebrovascular diseases, it can also reduce the human body cholesterol levels.The gene of FAD3codes endoplasmic reticulum ω-3fatty acid desaturase, it maincatalyses the linoleic acid into the linolenic acid, and its over-expression can make thecontent of linolenic acid increase. Therefore, the research of ω-3fatty acid desaturase(FAD3) gene expression and function has the vital significance. This research hascloned the flax FAD3gene and constructed the vectors, and has studied the flaxhypocotyl high-frequency adventitious shoot induction in different genotypes. As thisbasis of tissue culture for flax genetic transformation, the study has usedAgrobacterium-mediated method to introduce the genetically engineered bacteria ofthe FAD3gene into the flax. The main results as follows:(1) The clone of FAD3gene in flax and construction of expression vectorsIn this paper, using the cDNA of Flax fruit of15to20days after flowering toclone the FAD3gene, and constructed two expression vectors pMDC139-FAD3andpEarleyGate100-FAD, and one interference vector pBIB-BASTA-35S-FAD3, and putthese three recombinant plasmid into the agrobacterium LBA4404, and had thefollow-up study of genetic transformation.(2) The flax hypocotyl adventitious shoot induction in different genotypesIn this paper, the5flax varieties which have different prominent features wereused as experimental material. To study the flax hypocotyl adventitious shootinduction in different genotypes, and different seed disinfection method of these5 flax varieties and select the culture medium of adventitious shoot, and compareanalysis the differences of flax hypocotyl adventitious shoot induction andadventitious shoot rooting in different genotypes. The study was aimed at setting updifferent adventitious shoot induction system of the different flax varieties, andprovided the research foundation of flax tissue culture for flax genetic transformation.The results showed that the optimal disinfection treatments of five flax varieties wereto put seeds in70％alcohol solution for0.5min, and then in1％sodiumhypochlorite solution for10min. Selected that the optimum culture medium ofadventitious shoot was MS+0.5mg/l6-BA+0.02mg/l NAA. The conclusion was thatLongya No.10has the highest adventitious shoot induction rate and adventitious shootrooting rate, and it’s easy to do the follow-up research work.(3) The genetic transformation with the flax FAD3gene by Agrobacterium mediatedThe study has used Agrobacterium-mediated method to introduce the geneticallyengineered bacteria of the FAD3gene into Longya No.10. In the process of genetictransformation, based on the sensitivity of the selection agent test, this paperdetermined that the best concentration of hygromycin as a selection agent was25mg/l,and the best concentration of PPT as a selection agent was10mg/l. And thetransgenic flax plants were identified by PCR and stable GUS expression analysis.Obtained three positive plants which contained the plasmid of pMDC139-FAD3, andfour positive plants which contained the plasmid of pEarleyGate100-FAD3, and threepositive plants which contained the plasmid of pBIB-BASTA-35S-FAD3.