Transformation Of Maize With Trehalose Synthase Gene (TPS1) Cloned From Saccharomyces Cerevisiae

Drought is the main limiting factor in the production of maize.Maize breeding is use to improve the ability of drought tolerance.It is the most economical and efficient way to deduce the loss of drought.But the drought ability of maize is controlled by microeffect multiplegene.Good result is not received because it's difficult to improve the drought ability by breeding.Transgenic technology can break the species isolation and utilize the gene of other species.Trying to improve the ability of drought tolerance,a large number of genes such as SOD and DREB are transferred into maize.But the transgenic plants can not fit the damand of maize production,because the ait-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself.Technically it is very important to make use of hight ait-drought ability of the gene which is more consistant with maize.Trehalose has the trait of adversity tolerance.So it's very significant to transfer the related gene to maize and breed now maize lines which have the strong ability of drought tolerance.Genomic DNA of Saccharomyces cerevisiae was extracted from strain AS.1416 by the method introduced by Adams et al.(1998).According to the sequence of gene TPS1,a pair of specific PCR primers was designed using primer 5.0.At the 5'ends of the forward and reverse primers,recognition sites of restriction endonucleases BamH I and Apa I.The specific amplified fragment was separated by agarose gel electrophoresis and inserted into cloning vector pMD19-T and named pT-TPS1.Plasmid pT-TPS1 containing gene TPS1 and plasmid pC1300UbiDREB containing promoter ubiquitin,were digested by restriction endonucleases BamH I and Apa I, respectively.Fragments of gene TPS1 and plasmid pC1300UbiDREB without gene DREB were separated by low melting-point agarose gel eledtrophoresis and ligated by T₄-DNA ligase to form plasmid pC1300UbiTPS1.According to its multiple cloning sites,the expression structure of pC1300UbiTPS1 was confirmed by restriction digestion of BamH I and Apa I.Two specific fragments of 1603 and 11700 bp were separated by agarose gel electrophoresis.They were the same long as gene TPS1 and the backbone of plasmid pC1300UbiTPS1.This result showed that gene TPS1 had been inserted into vector pC1300UbiTPS1 and the plasmid pC1300UbiTPS1 had the expression structure as designed.Expression vectors with TPS1 gene promoted by maize ubiquitin promoter and stress inducible promoter(mwcs120)of monocotyledon respectively were used to transfer embyronic calli of "18-599R" and "18-599W" mediated by Agrobacterium.After two times of screening on gradient hygromycin medium,340 and 607 pieces of positive calli were screend from inbred lines "18-599R" and "18-599W",respectively.The rates of positive calli were 6.4 and 10.2%.No significant difference was found between these two rates.However,the regeneration ability of" 18-599R" was higher than "18-599W".The regeneration rate of "18-599R" was more than three times of "18-599W".54 and 31 regeneration plants were obtained.1 positive plant was obtained by PCR amplification.Reseearch found that the percentage of positive clallus regenerated was the highest when callus subculture for 7～9 days.Supplementary the cysteine(400 mg/L)in co-culture medium was essential to efficient transfer foreign gene to gramineous plants.In addition,the infecting A.tumefaciens concentration was OD₆₀₀=0.5,10 minutes infection time and co-culture 3 days were the optimal conditions for Agrobacterium tumefaciens mediated maize transformation.The positive plant which was just tested by PCR need further study such as molecular hybridization,expression test,molecular mark selection and drought tolerance identification until it is breeding to be steady transgenic breed.It's exploration to improve the ait-drought ability on method of transgene and it is also helpful to study the mechanism of trehalose metabolism.