Identification Of TaGAPDH8 And Construction Of Transgenic Wheat Of TaGAPDH12

Wheat（Triticum aestivum L.）is one of the most important crop plants in the world,and it supplies rich nutrient for human beings.Whereas most of wheat-growing regions are widespread in the arid and semi-arid areas,production and food quality of wheat are severely impacted by adverse environments such as rising temperature and salinated land during wheat life span.Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）which plays a vital role in the energy supply and balance of redox for cells,is a key enzyme involved in the glycolysis and gluconeogenesis.Recently,mounting researches reveal that the protein serves important roles of stress resistance in plants.To study the function of the TaGAPDH8 and the TaGAPDH12 gene of the TaGAPDH family in stress tolerance,we（1）detected the expression patterns of the TaGAPDH8 gene in the four putative RNA interference（RNAi）T₃ transgenic lines（CW3,CW6,CW12,CW13）at normal growth conditions;the lines which showed lower gene expression levels of the TaGAPDH8 were selected for further study.The expression levels of the TaGAPDH8 and physiological parameters of the lines in heat,PEG and Na Cl conditions were measured,respectively.The growth conditions of the lines which grown in the field at milk stage were measured.To establish the transgenic lines of the TaGAPDH12 and supply materials for further study,（2）the two recombinant vectors（the overexpression vector and RNAi vector）which contained the open reading frame（ORF）of the TaGAPDH12 was transformed into the ZY1（ZY）and CW134（CW）via biolistic particle,respectively.The main results are as follows:1.Identification of the T₃ TaGAPDH8 transgenic plants and measurement of stress-resistance physiological indexesPCR and Southern blot were employed to identify the four putative transgenic wheat lines（CW3,CW6,CW12,CW13）,the results showed that the four lines were stable inheritance of the exogenous gene.Quantitative RT-PCR（q RT-PCR）showed that the expression levels of the TaGAPDH8 gene in these four lines were 0.53,0.75,0.21 and 0.78-fold compared with un-transformed Changwu134（CK）,respectively;the two lines,CW3 and CW12 which expressed lesser TaGAPDH8 were selected for further study.（1）Under severe heat（42℃）conditions,the TaGAPDH8 gene in CK strongly increased to6.4-fold compared with that of 0 h,while the expression levels in CW3 and CW12 changed negligible;the resistance and recovery capability of CK were better than that of the two transgenic lines during severe heat stress and after recovery at room temperature.RWC,SOD activity and cholorophyll content in CK were higher than the transgenic lines.（2）Under PEG treatment,the TaGAPDH8 gene in CK strongly increased to 8.1-fold compared with that of 0 h,while the gene changed little in CW3 and CW12;RWC,SOD and POD activities in CK were higher than that of transgenic lines over time of this stress.（3）Under Na Cl stress,expression levels of the TaGAPDH8 gene in CK or transgenic lines changed insignificant compared with that of 0 h.No significant difference of the physiological parameters could be detected among the three lines.Agronomic traits of T₃ progeny which grown in the field at milk stage were measured,as well as thousand kernel weight and germination rete of the T₃ seeds.Plant height shows little difference among the lines.CW12 has longer spike but slighter thousand kernel weight than CK;CW3 has maximum grain numbers,and transgenic plants have higher germination rete than CK.2.Transformation of the TaGAPDH12 gene in wheatThe overexpression vector that contains the target gene the TaGAPDH12 was transformed into the callus of ZY（800 callus）and CW（200 callus）,and RNAi vector was transformed into the callus of ZY（200 callus）and CW（800 callus）,respectively.Twenty-five young plantlets were differentiated from callus.Four regenerated plantlets of ZY（three overexpression plantlets,one RNAi plantlet）and four regenerated plantlets of CW（RNAi plantlets）were obtained in total after selected using hygromycin.PCR identification indicated that the eight T₀regenerated wheat lines were positive,with the transformation rates of 0.4%.Seeds of T₀generations were harvested and all seeds were planted,nine PCR-positive plants were harvested but no positive T₂ plant could be detected via PCR.