| Molecular Biological Studies of Ethylene Receptor Gene LeETRl and LeETR2 inTomatoesZheng Tie-song (Tutors: He Guo-qing, Ying Tie-jin) AbstractThe phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. It's very import to study the biosynthesis of endogenesis ethylene and ethylene signaling pathway. Although the regulation of ethylene biosynthesis has been studies extensively, much less is known about the regulation of ethylene perception and transduction.In this paper for the first time the sense and antisense expressions of ethylene receptor gene LeETRl and LeETR2 have been studied simultaneously after the partial special DNA sequences of LeETRl and LeETR2 had been cloned. The studies will be helpful for realizing roundly about the functions of ethylene receptor LeETRl and LeETR2 in tomato's ethylene signaling pathway. The main results in this paper are as follows:1. The 2 pairs of special primers have been designed according to the reported DNA sequences of LeETRl and LeETR2. Then partial 3'-terminus cDNA sequences 313bp of LeETRl, and 265bp of LeETR2 have been cloned from tomato tissues by RT-PCR. The cloned cDNA sequences were proved the same as those reported with DNA sequencing. Only single special band produced respectively when using the cloned DNA sequence to study the expressions of LeETRl mRNA or of LeETR2 mRNA with RPA (RNase protect analysis) method, which indicated that the cloned cDNA sequences have high specificity.2. The expression characteristics of LeETRl mRNA in tomato mutant Epi and its wild type VFN8 have been studied with RPA. The results showed that LeETRl mRNA expressed constitutionally in VFN8. And its expression was not affected by the endogenous ethylene. However, the high expression of LeETRl mRNA in some tissues in Epi suggested that LeETRl gene may have some important roles in the morphogenesis of leaves and fruit ripening in tomatoes.3. The expressive characteristics of LeETR2 mRNA in tomato mutant Epi and its wild type VFN8 have been studied with RPA technique. The results showed that LeETR2 mRNA expressed in all the tissues examined, and it's expression was unaffected by the endogenous ethylene in leaves. However, LeETE2 mRNA was developmentally regulated, but partially induced by ethylene in fruits. The results indicated that LeETR2 mRNA exhibits different expression pattern from other ethylene receptor genes in tomato. LeETl, LeETRl and Nr may belong to different ethylene receptor systems. They have different roles in ethylene perception and transduction.4. Integrated in vitro tomato regeneration systems with high efficient shoots and roots regeneration have been established using cotyledons of tomato species 9551, T9253, Estrella, 9209 and Bl. The basal medium used was modified medium MS (i.e. inorganic components of medium MS and organic components of medium B5) . The media for shoot regeneration are the basal media with different97concentrations of BA and IAA according to different tomato species. The optimal phytohormones concentrations for tomato 9551, T9253, Estrella, 9209 and Bl are BA 2.5 mg/L + IAA 1.0 mg/L, BA 2.0 mg/L + IAA 0.2 mg/L, BA 2.5 mg/L + IAA 0.4 mg/L, BA 2.0 mg/L + IAA 0.4 mg/L, and BA 2.5 mg/L + IAA 0.4 mg/L respectively. The optimal rooting media for all the tomatoes are the basal media with no phytohormone. Shoos regeneration frequencies of tomato 9551, T9253, Estrella, 9209 and Bl on their optimal media are 92A%* 88.5?cK 91.6%> 89.2% and 94.1% respectively.5. The lay mode of tomato explants especially that of hypocoryls affected evidently the regeneration frequency. Soots regeneration will descend greatly if hypocotyls are laid upside down.6. Tomato 9551, T9253, Estrella, 9209 and Bl have been proved have different sensitivities to antibiotics Km. Cotyledon calli and shoots regeneration of tomato 9551, T9253, Estrella and 9209 can be retarded completely when 25 mg/L Km added into their media. But Cotyledon calli and shoots regeneration of tomato Bl can be retarde... |
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