Identifying And Characterizing Of Homozygote From Antisense LeETR1 And LeETR2 Transgenic Tomato Plants

Ethylene is a simple, readily diffusible gaseous hormone and it regulates multiple developmental processes. Such ethylene-dependent processes have long been presumed to be dependent on the synthesis or activation of ethylene receptors. Although the regulation of ethylene biosynthesis is especially well characterized, much less is known about the regulation of ethylene perception. Based on the results that were investigated on the mutants ETR1, a member of ethylene receptor genes, ETR1, ERS1, ETR2, EIN4, ERS2, ethylene receptor genes might play a negative role in regulating response to ethylene. However, so far only the results obtained by Whitelaw et al (2002) concerning LeETRl gene were partly consistent with the negative model and partly against it. Obviously, the action models for these ethylene receptors are far from being clearly understood.The purpose of this study is to verify the possible functions of tomato ethylene receptor genes LeETR1 and LeETR2 in regulating ethylene response. By introducing antisense LeETR1 or LeETR2 genes into tomato, the following aspects were investigated.Methods: (1) The segregation of foreign target gene in the T1 by histochmical GUS assays; (2) Identification of pure line from transgenic tomatoes (Tl) through examining GUS expression in pollens in conjunction with PCR analysis of marker gene (npt ); (3) The transcript levels of LeETRl or LeETR2 in anti-sense transgenic plants; (4) The phenotypes of the transgenic plants in tomato during whole life cycle under ethylene-treated and non-treated conditions.Results showed that: (1) In general, the segregation ratios of target gene in some Tl lines are conformed to 3:1, however, some are not possibly due to gene silence or missing in the self-pollinated progeny; (2) Two homozygous plants were identified from 10 putative transgenic plants. Their frequencies of GUS expression in pollens were 97.755% 0.800% and 96.556% 0.600% respectively. To verify these results, the self-pollinated progenies of these two putative homozygotes were examined in T2 by histochenmical GUS staining of vegetative tissues and PCR analysis using specific primers. These results demonstrated that merely examining the segregation of reporter gene in pollens is a reliable method that could avoid the laborious testing process of the pure line from transformed plants; (3) The transcript levels of LeETRl and LeETRl were checked by Northern blotting analysis. Expression of target genes was suppressed in LeETR1 or LeETR2 antisense transgenic plants. Meanwhile, the signal intensity ofLeETR2 transcript was also affected in LeETRl antisense plants; (4) In addition, the phenotypes observed most consistently in transgenic lines were delayed senescence and abscission both in antisense (LeETRl and LeETR2 plants. Interestingly, a great reduction in plant size and outgrowth of axillary buds as well were only found in LeETR2 antisense plants. While the fruits ripening process was delayed in LeETRl antisense fruits. Epinastic of leaves in LeETRl antisense plants was only occasionally observed, which was not observed in LeETR2 antisense plants; (5) When treated by exogenous ethylene (25ul/L), the abscission of antisense LeETR2 petiole explants was delayed. Moreover, leaf epinasty failed to be induced by ethylene treatment c, which suggested that these tissues were insensitive to ethylene.The shorter internodes and out-growth of anxillary buds found in antisense LeETR2 and leaf epinasty observed in antisense LeETRl plants are phenotypes consistent with the negative regulation model of ethylene. LeETR2 gene might play a more important role in regulating the morph-genesis in tomato. However, based on the negative regulation model, delayed abscission is perhaps against to what might be expected in plants with reduced ethylene receptor. To explain the paradoxes, it is essential to characterize the receptors gene and their interaction with other hormones, such as auxin, abscisic acid and cytokinin in regulating plant developments through signal pathways.