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

Drought is the main limiting factor in the production of maize. Maize breeding is use toimprove the ability of drought tolerance. That's the most economical and efficient way todeduce the loss of drought. But the drought ability of maize is controlled by microeffectmultiplegene. Good result is not received because it's difficult to improve the droughtability by breeding. Transgenic technology can break the species isolation and utilize thegene of other species. Trying to improve the ability of drought tolerance, a large number ofgenes such as SOD and DREB are transferred into maize. But the transgenic plants can notfit the damand of maize production, because the ait-drought ability of the gene is notenough and it is not consistant with the metabolization of maize itself. Technically it isvery important to make use of hight ait-drought ability of the gene which is moreconsistant with maize. Trehalose has the trait of adversity tolerance. So it's very significantto transfer the related gene to maize and breed now maize lines which have the strongability of drought tolerance.Trehalose synthase gene (TPS1) was cloned from stein AS.1416 of Saccharomycescerevisiae by specific PCR. Sequence analysis showed 11 single nucleotide mutationscompared to the previously reported sequence. Ten of the mutations were located at theencoding domain, while 9 of them were synonymous mutations, which had no influence tothe amino acid sequence of the encoding protein. Only the mutation from G to A at site1135 changed glycin of the 355th amino acid to asparagine. Expression vector with TPS1gene promoted by stress inducible promoter (mwcs120) of monocotyledon and used totransfer embyronic calli of maize by microprojectile bombardment. Positive plants wereassayed PCR amplification. The average transformation rate was 0.56%. The transgenicplants and seeds had been obtained.The positive plants which were just tested by PCR need further study such asmolecular hybridization, expression test, molecular mark selection and drought toleranceidentification 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 mechanismof trehalose metabolism.We searched NCBI database to acquire many protain sequences of TPS1 of higherplants and prokaryote. Using sequence analysis software, several conservative segments isfound. Based on the segment, we desgined premiers to ampligy the special band in maize.After the sequcece analysis, it is found that the fragment have 59.87% similarity withSelaginella lepidophylla and 98.07% similarity with yeast. But none band received whenwe used another pair of premiers in amplified reaction. This result proves that in totalmaize genome there is homologous fragment with TPS gene. And maize doesn't have theability to synthesize trehalose, bossibly because maize doesn's have the complete genesequence. But in order to clone the full sequence of TPS or find its function, more study isneed to do in the further research.