Offsrping Detection Of Transgenic Maize With Trehalose Synthase Gene (TPS1) Cloned From Saccharomyces Cerevisiae

Drought is one of the main limiting factors in maize production.It is the most economical and effective way to improve maize drought-tolerance ability by varity improving.However,maize drought-tolerance is controlled by multi-genes,so genetic improvement had more difficulties over the years.Using other species' useful genes, transgenic technology can break the reproductive isolation between species.Goddijn, Holmstrom,Pilon-Smits,Romero,et al.transformed TPS1 which came from E.coli and yeast in tobacco and potato successfully.And the obtained transgenic plants had higher drought tolerance than non-transgenic plants.Wu rui transformed trehalose synthase gene which came from E.coli,otsA and otsB,into India rice.At last,not only rice tillering and yield had improved,but also cold hardiness,drought and salt tolerance of the new rice variety all had improved.Owing to strong drought tolerance of TPS1,it is significant to transfer the related gene that had strong drought tolerance ability in maize.In this study,for amplifying TPS1 of Sacchromyces cerevisiae,we extracted DNA from strain AS.1416 of Saccharomyces cerevisiae and the extracted method described by Adams.Sequencing analysis showed that eleven single nucleotide mutations were found and ten of them were included in encoding region.But nine of tem were samesene mutaion which did not change the amino acid sequence encode protein.Only mutation at 1135 site that G changed to A,so that the encoding protein of glycine at 355 changed to asparamide.Two new restriction sites Speâ… and Apaâ… had introduced in both ends of the TPS1 amplified fragment.The fragment was separated by agarose gel electrophoresis and inserted into cloning vector pMD19-T named pT-TPS1.Using the sequence construct monocotyledon expression vector pCWTPS1300 that was include trehalose synthase gene TPS1 cloned from Saccharomyces cerevisiae which promoted by stress induced promoter wmcs120 cloned from wheat,selectable marker gene Hyg which promoted by plant constitutive 35S promoter and selactalbe marker gene of E.coli.The expression vectors mediated by Agrobacterium were used to transfer embyronic calli of "18-599R"and "18-599W".After three rounds,with selection gradient of hygromycin medium,obtained 340 and 607 pieces of resistant calli from "18-599R"and "18-599W",respectively.The conversion rate of callus were 6.4%and 10.2%,both of them had no significant differences.However,the regeneration ability of "18-599R" was significantly higher than that of "18-599W".54 and 31 regeneration plants were obtained by tissue culture,1 positive plant obtained by PCR amplification.Owing to absence pollen of positive plant that PCR detected,we used the non-transgenlc materials'pollen to pollinate the female flower of T₀ generation and then obtained T₁ generation.By PCR detection,the T₁ generation were all positive plants.Then, taken the purpose fragment as probe,pCWTPS1300 as positive comparison,we carried on Sourthern hybridization by using DIG High Prime DNA Labeling and Detection Starter Kitâ…¡Kit purched from Roche company.Finally,the result showed that T₁ generation is a single copy.The analysis of high performance liquid chromatorgraphy showed that the trehalose content in leaves of T₁ generation transgenic plants was not higher than non-transgenic control,but the content of other saccharides in transgenic plants were significantly lower than that of control.10 offspring obtained from the T₁ self-generation,each offspring randomly selected 10 seeds to germinate and then obtained T₂ generation.Chosing the positive plants from T₂ generation by PCR detection,carried on the cold treatment that in 12℃sustained 3days and salt treatment that in 2%NaCl sustained 3days,repectively.Using RT-PCR to detection,we obtained 1544 bp in cold treatment.The result explained that only in the low-temperature-induced TPS1 can expression at mRNA level.On the basis of non-homozygous in T₁ generation,we need to identify and bred homozygote,and identification drought-tolerance of the tansgenic plants.Exploring the trans-exogenous trehalose synthase gene in maize can improve possibility of maize drought-tolerance and also can contribute to research on the mechanism of trehoalose.