The Preliminary Study On Genetic Transformation Of CsHIR1 Gene Into Cucumber

Downy mildew,one of the most important diseases caused by the oomycetes in cucumber(Cucumis sativus L.),seriously affected the yield and quality of cucumber.In this study,the role of CsPI and CsHIR1 was preliminarily discussed on cucumber downy mildew resistance.Using cucumber genotypes CCMC(susceptible germplasm)and PI088(highresistant germplasm)as the experimental materials,we analyzed the expression of CsPI and Cs HIR1 at the transcriptional level.The plant expression vector pCAMBIA35S-CsHIR1 was constructed and transfered into cucumber through the optimization of agrobacteriummediated genetic transformation system.The main results were as follows:1.Transcriptional expression analysis of CsPI and CsHIR1 after inoculating P.cubensisWe analyzed the expression of CsPI and CsHIR1 in CCMC and PI088 leaves,0h?3h?6h ? 12 h ? 1d ? 2d ? 4d and 7d after the P.cubensis inoculation.The results showed that 4days after the inoculation,CsPI and CsHIR1 expression was significantly increased in CMCC.However,in PI088 it was up-regulated at the early stage but decreased 12 hours after the inoculation.2.Optimization of regenerationUsing the cotyledon node of CCMC as explant,we found the 3-5d-old explants(semi-sprouting)had higher regeneration rate than the 5-7d-old explants(full sprouting),and the explants broken from seeds had higher regeneration rate than that of cutting from seeds.3.Genetic transformation of cucumberThe plant expression vector p CAMBIA35S-CsHIR1 was constructed by in-fusion technology.Using the cotyledon node of CCMC and PI088 as explant,we transferred CsHIR1 gene into cucumber through the optimization of agrobacterium-mediated genetic transformation system.25 plants were proved to be positive by PCR detection,and 8 of which were harvested self-pollination seeds(T1).The positive rate of the T1 generation was 44.7%detected by PCR.We conducted real time quantitative PCR analysis of T1 positive generation.And the results showed that the relative expression of Cs HIR1 was slightly higher in T1 positive generation than that in non-transformed plants,demonstrating CsHIR1 had been transferred into the cucumber.