Transformation Of Common Wheat By SOS Genes From Arabidopsis Thaliana

Common wheat(Triticum asetivum L.)is one of the most important and widely planted crops in the world.Salt stress is an abiotic stress that affects the production and development of agriculture severely.Extensive researches have now been progressed against this stress,focusing on the breeding of salt tolerant plants. Traditional breeding is slower and less precise because of the barriers between different species.However,gene engineering including transgenic technology have solved the problem.In this article,a novel method of transgenic technique was used to transfer SOS genes from Arabidopsis thaliana to common wheat(Tnticum asetivum L.).According to the SOS pathway,three vectors containing target genes SOS1, SOS2-SOS3,SOS1-SOS2-SOS3 respectively were constructed.They were used separately to transform five genotypes of wheat cultivars by Agrobactenum tumefaciens mediated transformation of shoot apical meristem ceils.Transgenic lines carrying different single or combined SOS gene(s)were identified by PCR and Southern blot analysis.Furthermore,RT-PCR has proved that the SOS genes integrated into the wheat genome could express.1.Shoot apical meristem cells of five types of common wheat were transformed by A.tumefaciens.Compared with other methods,this one has more advantages,such as simpler to operate,fewer copies,higher transformation efficiency,etc.The novel and direct approach of wheat apical mefistems transformation mediated by A.tumefaciens has been proved to obtain transgenic wheat and progenies in this study.2.After selected with solution containing 100mg 1^-1hygromycin or 0.2%Basta,the SOS genes transgemc seedlings were then proved by PCR and Southern blot analvsis.The result indicates that,the transformation efficiency is related to the genotypes of wheat.The highest is Jimai,then Yanmai and the lowest Gaofei.The difference of transformation efficiency depends on the ability of common wheat resistant the intrusion of A.tumefaciens.It was proved that the SOS genes were integrated into the wheat genomes and they could express in the transgenic wheat and progenies.3.Heredity and salt tolerance oftransgenic progenies1)To confirm that the exogenous gene can be inherited to the progenies,the segregation in the T₂ of SOS2- SOS 3 transgenic wheat was detected.The ratio between positive and negative was about 3:1,which is consistent With Mendel rules.2)The analysis of germination rates indicated that under normal condition,no differences were found between transgenic wheat and control.While after salt treatment,transgenic lines of SOS 2-SOS 3 and SOS 1- SOS 2-SOS 3,but not of SOS 1 showed much higher frequencies than control.3)After treatment with different concentrations of NaCl for two weeks, SOS2-SOS3 and SOS1-SOS2-SOS3 transformed plants showed more tolerant than control,while there was no obvious difference between SOS 1 transformed plants and control.Ion detection showed that Na⁺ contents of SOS2-SOS3 and SOS1-SOS2-SOS3 transgenic plants were totally lower and the K⁺ / Na⁺ were higher than control,while no obvious differences of the indexes were found between SOS1 transgenic lines and the control.4)MDA content and the activity,of CAT were analyzed and the results were as follows:Treat with salt for two weeks,MDA content increased in both transformed and control wheat,but the increasing degree in control and SOS 1 transgenic wheat were greater than the two other types of transgenic wheat. Treat with salt solution of 200mM NaCl for 24h,no significant increase of CAT activity was found,while after 48h,CAT activity of SOS2-SOS3 transformed plants increased much greatly than the control,but that of SOS1 transformed wheat only increased a little greatly than the control.5)The result of RT-PCR of TaSOS1 suggested that compared with TaSOS1 in SOS1 transformed wheat and control,the expression of TaSOS1 gene in SOS2-SOS3 transformed plants was upregulated.We suggest that protein kinase complex SOS2/SOS3 can phosphorylate TaSOS1 and stimulate its activity in wheat,which enhanced the salt tolerance of transgenic wheat.The results above proved that the SOS(salt overly sensitive)pathway in Arabidopsis thaliana can operate in common wheat and that the expression of plasma membrane Na⁺/H⁺ antiporter SOS1 depends on the regulation of the kinase complex SOS2/SOS3 in the pathway.The result of RT-PCR analysis of TaSOS1 indicated that the complex SOS2/SOS3 may be concerned with other pathway by regulating the expression of TaSOS1.