Construction And Transformation Of The Antisense PBI121-NR-LEACS2 Binary Expression Vector Into Tomato

Ethylene, which plays an very important regulatory role in various physiological and developmental process of plant, such as fruit ripening, has been studied as a major content in the postharvest physiology. In the past ten years, the regulation of ethylene reception and signal transduction pathway has been studied extensively in the model plant Arabidopsis, but much less is known about the same research in tomato-the model plant material of ripening and senescence research of fruits and vegetables.Six ethylene receptor genes have been cloned from tomato so far, there are LeETRl-6,and their functions are far from being understood. With ethylene receptor genes isolated from tomato, referring to the ethylene signal transduction pathway of Arabidopsis, our research is focused on the functions of ethylene receptor genes in tomato. NR (never ripe) gene (i.e. LeETR3) is a very special one in the six tomato ETR1 homolgues. Its mRNA expression is restrictly regulated by the fruit development, and its expression mode is highly compatible with that of the ethylene production of the climactric fruits. Judged from the time of its mRNA expression, NR might be the pivotal factor which startups System-2 Ethylene synthesis in tomato.In this study, the antisense pBI121-NR-LEACS2 binary expression vector is constructed and transformed into tomato through Agrobacterium-mediated method. This study will help to make the function of NR protein and its role in the feedback regulation of ethylene biosynthesis clear, which will enrich the ethylene theory and the mechanism of ripening and senescence of fruits and vegetables. The main results obtained in this study are as follows:(1) Excised 35S-NR(reverse)-nos fragment from pBI121-NR with EcoRI and HindIII, ligased to pGreen0029 and the first vector pGreen0029-NR was constructed(2) Excised 35S-NR(reverse)-nos fragment from pGreen0029-NR with BamHI and HindIII, ligased to pLP100 and the second vector pLP100-NR was constructed(3) Excised 35S-NR(reverse)-nos fragment from pLPlOO-NR with EcoRI, ligased to pBI121-LEACS2 and the Antisense pBI121-NR-LEACS2 Binary Expression Vector was constructed eventually(4) The Antisense pBI121-NR-LEACS2 Binary Expression Vector was mobilized into Agrobacterium tumefacious EHA105 by chemical-based DNA direct transformation(5) Transgenic tomato plants were gained through Agrobacterium-mediated method. Seven PCR positive trans-gene plants have been obtained by PCR testing...