Cloning, Expression Of A Eukaryotes Elongation Factor1A Gene From Ziziphus Jujuba Mill.(ZJeEF-1α) And The Functional Analysis Associated With Jujube Witches’ Broom (JWB)

Eukaryotes elongation factor1A (eEFIA) is an abundant and a multifunctional protein. It binds and leads aminoacyl tRNAs to the acceptor site of the ribosome during the peptide chain elongation phase of protein synthesis. It appears to be involved in several cellular processes. Recently, some interesting data had reported that eEFIA was not only a house-keeping gene and it is also very important to plant development and stress conditions. A Eukaryotes elongation factor1A of Ziziphus jujuba Mill.(ZJeEF-la, JF488063) was firstly isolated by SON-PCR method and3’ RACE.The related function was also studied by semi-RT-PCR and western-blotting in this paper.The main results were as follows:1The eukaryotes elongation factor1A of Ziziphus jujuba Mill.(ZJeEF-la, JF488063) was firstly isolated by SON-PCR and3’RACE.The2343bp included a complete open reading frame of1344bp which encoding a polypeptide of477amino acids, a5’ non-coding region of502bp, a3’non-coding region of497bp.2Sequence alignment showed that ZJeEF-1α was highly homologous to the eEFIA from Malus domestica (AJ223969.1),Populus trichocarpa (EF147714.1), Glycine max (AK285610.1), Litchi chinensis (DQ471426.1) and other homologous gene, and their similarity were above85%. The predict-protein is characterized by the molecular weight of49.47kDa, the isoelectric point of9.56, the electricity of14.23in neutral condition (pH=7), presumably as an acidic protein.3. Through the full length genomic amplification and western blotting analysis, the result showed that genome length of ZJeEF-1α was1486bp, including a section of115bp intron sequences. And there was alternative splicing and two transcript of ZJeEF-1α.4Prokaryotic expression of ZJeEF-1α was carried out, and a large quantity of the fusion protein of60kDa was detected in bacterium precipitation.The NCBI analysis predicted that ZJeEF-1α encode a protein of49kDa. And the protein of6×His Tag of pET-32a was about19.3kDa. So the expressed protein in bacterium precipitation was the expressed protein of ZJeEF-la and a large quantity of the fusion protein was obtained.The anti-rabbit serums of the ZJeEF-1α multi-clones were used to identify the specificity and the ideal experimental result was obtained.5The results of semi-quantitative RT-PCR and the western-blotting showed that there was a close relationship between the ZJeEF-1α and the disease resistance to Jujube Witches-Broom (JWB).’Xingguang’, a resistant cultivar to jujube witches’broom (JWB), was also used as material to study the expressing of ZJeEF-1α. After infecting by JWB, the expression of ZJeEF-1α were consistent in resistant scions of’Xingguang’and healthy controls, but its expression were obvious differences in the susceptible scion of ’Xingguang’ and ’Fupingdazao’ and healthy controls. Those results showed that the transcription and expression of ZJeEF-1α were significantly affected by JWB pathogen. The high expression of ZJeEF-1α may enhance the resistance to JWB.6. The eukaryotic expression vector of pCAMBIA3301-35S::ZJeEF-1α was constructed and then transformed into Arabidopsis theliana by agrobacterium. Then two postive transgenic lines of ZJeEF-1α were obtained.