Construction Of Expression Vector Of LeEIL2 And Transformation Into Tomato

Ethylene, as the most simple plant hormone, plays major roles in regulation of plant growth and development. In 1970s, ethylene biosynthesis pathway had been elucidated. But the research of ethylene signal transduction pathway was started from 1990s. In Arabidopsis, a model of the pathway as following has been proposed: C2H4 -ETR - CTR- EIN2- EIN3- Ethylene responses. Ein3 located at the most downstream position of the ethylene signal transduction pathway and Ein3 gene encodes a novel nuclear-localized protein. Ein3 gene was cloned From Arabidopsis at first, and was cloned from tobacco, melon and banana subsequently. LeEIL 1/2/3 were EIN3-like in tomato. LeEIL2 has the close correlation of the tomato' s ripening and senescence. The full- length of LeEIL2 gene is 1845bp, LeEIL2 encodes transcriptional factor .In this study, In order to investigate the role of LeEIL2 in tomato fruit ripening, three expression vectors had been constructed and mobilized into Agrobacterium twnefacious EHA105. Transgenic tomato was developed using Agrobacterium-mediated transformation. The main results are as follows:1. Excised LeEIL2 fragment from pGEM-Teasy-LeEIL2 fragment with EcoRI, ligased to pGr2935S and antisense expression vector pGr2935S -LeEIL2 fragment was constructed.2. Excised a fragment from pGr2935S -LeEIL2 fragment with BamHI, ligased to pBI121 and antisense expression vector pBI121-LeEIL2 fragment was constructed.3. Excised LeEIL2 from pGEM-Teasy-LeEIL2 with SacI and Xbal, ligased to pBI121 and antisense expression vector pBI121-LeEIL2 was constructed.4. Construction of pGreen0029-LeEIL2 and pGA643-HindIII5. Digested pGreen0029-LeEIL2 and pGA643-HindIII plasmid with HindIII and Xbal, purified the small fragment and large fragment respectively. Ligation of the two fragments and construction ofsense expression vector pGA643-LeEIL2.6. The expression vector pBI121-LeEIL2 fragment, pBI121-LeEIL2 and pGA643-LeEIL2 was mobilized into Agrobacterium tumefacious EHA105 by chemical-based DNA direct transformation respectively.7. Transgenic Tomato was developed using Agrobacterium-mediated transformation with kanamycin resistance selection. The presence of antisense LeEIL2 and sense LeEIL2 in transformants were confirmed by PCR and positive plants has been obtained...