Kosteletzkya Virginica Shoot Tip Regeneration System Of The Establishment And Construction Of Plant Transformation Vector Of GPD1 And DGAT

In order to improve the oil content of Kosteletzkya virginica, promoting biodiesel industry to ease the energy crisis to the global pressure, this experiment Kosteletzkya virginica shoot tips as explants for tissue culture. Kosteletzkya virginica is optimized to establish a good high frequency plant regeneration system. And build an efficient high oil content seed-specific promoter napin gene driven GPD1 and DGAT plant expression vector. For a future high-frequency, highly efficient regeneration system of Kosteletzkya virginica, using transgenic methods to cultivate a good amount of high oil Kosteletzkya virginica new line to provide some reference data.The following is the study carried out by the main conclusions of the work and to obtain:1. With 98% of concentrated sulfuric acid soaking the seeds of Kosteletzkya virginica 1h or so, sterile water will clean the surface of concentrated sulfuric acid, sterile water to soak the seeds for more than 10h , stripped kinds of skin inoculated into the culture medium to obtain the free vaccine. Seedling age on the 3rd to take the non-vaccine, tweezers and a scalpel to remove the stem and the top growing point, were cut 0.5 ~ 1.0mm shoot-tip meristem inoculation into the culture medium. Culture temperature (26±1)℃; relative humidity of 70% ~ 80%; illumination time of 14h / d; light intensity 2000lx. With MS (sucrose 3%, agar 0.8%, pH 5.8) culture medium as the basic medium. Kosteletzkya virginica best medium for shoot tip germination MS + IAA0.3mg / L + ZT0.3mg / L, germination rate was 91.11%; the proliferation of the optimal medium for the MS + IAA0.1mg / L + KT0.5mg /L and proliferation factor of 4.67; rooting medium was MS (sucrose 3%, agar 0.6%, pH 5.8), rooting rate was 90%. Lianmiao after transplanting, the survival rate of up to 60%.2. The use of PCR amplification technology from yeast INVSc1 (Saccharomyces cerevisiae), Brassica napus (Brassica napus L.) genomic DNA and Arabidopsis (Arabidopsis thaliana) cDNA was amplified, respectively, 3 - phosphoglycerate dehydrogenase (GPD1) , seed-specific promoter napin, and diacylglycerol acyltransferase (DGAT) gene fragments were cloned them into the pMD18-T (cloning vector) into. The use of pBluescriptKS (intermediate cloning vector) will be napin and GPD1 gene fragment was inserted into pCAMBIA1390 (plant expression vector) of the multiple cloning site points to construct the seed-specific promoter napin-driven GPD1 gene plant expression vector p1390NG. The napin and DGAT gene fragment in turn inserted into pCAMBIA1390 (plant expression vector) of the multiple cloning site points to construct the seed-specific promoter napin-driven DGAT gene plant expression vector p1390ND, respectively, using freeze-thaw method will p1390NG and p1390ND two plant expression vector into Agrobacterium tumefaciens LBA4404, EHA105, AGL1 in the ongoing resilience of the laboratory energy plants Kosteletzkya virginica, and Datura (Datura candida) seed oil content and oil The genetic improvement research, this experiment the carrier to build for the future use of genetic manipulation technology to improve fuel energy plants the seed oil content provides the foundation and materials, and can be directly applied to Mandala and GM Kosteletzkya virginica molecular breeding. Oil energy plants can be applied to genetic improvement of seed oil content.