Functional Analysis And Overexpression Of LeEIL1 In Arabidopisis Ein2 Mutant

As a phytohormone, ethylene is a very important regulator in plant growth and development. The researches on ethylene signal transduction pathway and the regulation mechanism have received considerable attention. In Arabidopsis, a model of the ethylene signal transduction pathway has been proposed as followed: C2H4→ETR→CTR→EIN2→EIN3/EILs→ERFs→ethylene responses. The EIN3/EILs, located at the downstream position of the ethylene signal transduction pathway, is a novel nuclear protein. Furthermore, EIN3/EILs may also mediate other phytohormones signaling pathways and stress factors signaling pathways. So it is important to reveal the function of EIN3/EILs in signal transduction pathway. So far, four genes of EIN3/EILs are isolated from tomato. They are LeEIL1, LeEIL2, LeEIL3 and LeEIL4. The LeEIL1, which expressed in the whole stage of the tomato fruit, is one of the very important components in the ethylene transduction pathway in tomato. In this research, the LeEIL1 was over-expressed in ein2 mutant of Arabidopsis. The main results are as follows:①The fragment of LeEIL1, which digested from the plasmid pBluescript SK-LeEIL1 by the BamHⅠ, was inserted into the plasmid pDH51 that digested by BamHⅠto construct the plasmid pDH51-LeEIL1. The fragment, CaMV 35S Promoter-LeEIL1-CaMV 35S Terminator, was excised from pDH51-LeEIL1 by EcoRⅠ, and was then inserted into the plasmid pBIN19 that digested by EcoRⅠto construct the over-expression vector pBIN19-LeEIL1.②The pBIN19-LeEIL1 was transformed into Agrobacterium tumefacious strain LBA4404 by the freeze-thaw method. The LeEIL1 was then transferred into ein2 mutant plants of Arabidopsis by Agrobacterium tumefacious through the floral dip method. Two transgenic Arabidopsis plants were selected by kanamycin resistance, and named LeEIL1-ein2①and LeEIL1-ein2②respectively.③The results of southern blotting indicated that there were multiple copies of the transgene LeEIL1 in LeEIL1-ein2①a nd one copy in LeEIL1-ein2②.④The results of northern blotting with LeEIL1 suggested that the transgene LeEIL1 was over-expressed in LeEIL1-ein2①and LeEIL1-ein2②. The expression level of transgene LeEIL1 in LeEIL1-ein2②is higher than that in LeEIL1-ein2①.⑤The triple response of transgenic plant indicated that the transgenic plants partially restored ethylene response. There occurred a slight constitute triple response in the LeEIL1-ein2①and LeEIL1-ein2②.⑥The florescence of LeEIL1-ein2①and LeEIL1-ein2②was earlier than ein2 mutant but still later than WT Arabidopsis.⑦The fragments of four ethylene-regulated genes AtPR3, AtPR4, AtPDF1.2 and AtGST2 were cloned as probes for northern blotting respectively. The northern analysis showed that the expression level of AtPR3 and AtPR4 was enhanced in the transgenic Arabidopsis. The expression level of AtPDF1.2 was not distinct different among transgenic Arabidopsis, ein2 mutant and WT Arabidopsis. In addition, the expression level of AtGST2 in transgenic plants was slight lower than that of ein2 mutant and WT Arabidopsis.