Studies On Tranformation Of Anti-sense Gene Of AGP Small Subunit Into Amorphophallus

Amorphophallus has been cultivated for a long time. It is not only the traditional food and medicine, but also the raw and processed materials for papermaking, building, mining and food additive in modern time. A main standard of Amorphophallus is the content of konjac glucomannan. One important way for Amorphophallus processing is to exclude impurity, such as starch, and extract refined powder, which is mainly composed of crystallization of konjac glucomannan. The higher content of konjac glucomannan and the lower content of starch, the better of the quality of the refined powder.AGP is the first and key ferment hi the process from metabolizing to starch-composing. In plant, this ferment is composed of two large subunits and two small subunits. In different species, small subunit is of still more conservation. By means of genetic operation on the ferment, we can increase or decrease the starch content.In this paper, AGPSl antisense expression vector was constructed. The Gene-gun and Agrobacterium tumefacions were used for transformation to Amorphophallus. At last, the genetic transformation plants were identified, the results were showed as follows:1. Construction of expression vector pBIN-AGPSlPlasmid pCR-AGPSI and p35S-GUS were digested with SacI + Xbal and electrophoresi-sed on 1% agarose gel. Targeting fragments were extracted from corresponding gel fragments and antisense working vector p35S-AGPSl was obtained through ligation. In this vector, 3' end of AGPSl cDNA was closely attached to CaMV35S promoter and 5' end to Nos terminator. In the same, plasmid p35S-AGPSl, and pBINPLUS were digested with EcoRI+Hindlll, then through electrophoresis, extraction and ligation. Antisense expression vector pBIN-AGPSl was constructed. Consequently, pBIN-AGPSl was transformed to Agrobacterium tumefacions by freezing melting, then which can be used for transformation to Amorphophallus.2. Construction of expression vector pPAT-AGPSlPlasmic pBSKS+ and pG5 were digested with EcoRI + HindIII, then through electrophoresis, extraction and ligation,plasmid p35S-PAT with herbicide pat was obtained.In the same,after the digestation of plasmid p35S-PAT and p35S-AGPSl with EcoRI+HindIII,antisense expression vector pPAT- AGPSl was constructed through electrophoresis,extracction and ligation. pPAT-AGPS1 was transformed to Ecoll before it was transformed to Amorphophallus with gene-gun.3. Genetic transformation to Amorphophallus by Agrobacterium tumefacionsThe influence of Kan and Carb on differentiation of shoot was studied respectively. The effect of factors, such as precultured time and cultured time, on transformation was studied as well. The results showed that 75mg/L Kan, 300mg/L Carb and preculturing two days, culturing two days and postponing six or seven days in selection are the best condition in genetic transformation by Agrobacterium tumefactions.4. Genetic transformation to Amorphophallus by Gene-gunThe influence of Basta concentration, ratio between nitrogen pressure and bombardment distance on genetic transformation was studied in this paper. In the condition of four hours before bombardment and sixteen hours after bombardment, treating the Amorphophallus with high pressure. The best treatment in genetic transformation by Gene-gun was: Inig/L Basta, 1100Psi/9cm ( ratio between nitrogen pressure and bombardment distance ) and culturing six or seven days for resume after bombardment.5. Identification of the genetic transformation plantExtracting the DNA from genetic transformation plants, amplifying it by PCR and analysing it with PCR-Southern, we can prove that the target gene was found inAmorphop- hallus.