Strawberry Ethylene Etr1 Gene Cloning And Its Plant Expression Vector Construction

Cultivated strawberry (Fragaria×ananassa Duch.) is a fruit crop species with high values, but its fleshy fruits, which are getting less firm during progressive ripening, are liable to a post-harvest deterioration that impairs their preservation and causes great economic losses. Further research on gene function of Etr1 may demonstrate a relation between ethylene and the ripening of non-climacteric fruits and pay the way for improving storage performance of strawberry fruit through biological techniques. Using genomic DNA as template, a Etr1 gene fragment was amplified by Polymerase Chain Reaction (PCR). FaEtr1gene antisense and RNAi expression vectors were constructed. Using All-Star in vitro plantlet leaf as explants, the effects of basic medium, hormones, physiological state of the explants, seedling ages and dark-culture time on strawberry leaf regeneration were studied to optimize the regeneration conditions. Efficient regeneration system of All-Star strawberry was obtained. Using All-Star strawberry leaf discs as explants, genetic transformation system mediated by Agrobacterium tumefacien was established. Kanamycin resistant plantlets were obtained. Gus chemical staining and PCR detection showed that Etr1-RNAi gene was transformed into All-Star strawberry plantlets. The main results were as follows:1. A pair of specific primers were designed according to the published Chandlar strawberry ethylene receptors Etr1 gene conservative amino acid sequence. A617 bp fragment was amplified. Sequence analysis showed that the nucleotide sequence of the cloned fragment was 98% identity with Chandler-Etr1 in GenBank, and no intron sequence, which demonstrated that the fragment was All-Star strawberry Etr1 fragment.2. Double digested by XbaⅠand BamHⅠ, All-Star strawberry Etr1 nucleotide sequence was subcloned into XbaⅠ- BamHⅠsite of pBI221 vector in reverse orientation, which resulted in plasmid pBI221-anti-Etr1. A fragment including CaMV 35S promoter and Etr1 antisense sequence was cut from pBI221-anti-Etr1 vector by SmaⅠand HindⅢ. The fragment was directionally inserted into the same restriction enzymes digested pBI121 plasmid, which resulted in pBI121-anti-Etr1.3. Double digested by BamHⅠand SmaⅠ, All-Star strawberry Etr1 nucleotide sequence was subcloned into BamHⅠ-SmaⅠsite of pBI221 in sense orientation, which resulted in plasmid pBI221-sense-Etr1 The plasmid pBI121-anti-Etr1 and pBI221-sense-Etr1 were double digested by BamHⅠand EcoRⅠ, respectively. The sequence with Etr1sense fragment was inserted into the double digested pBI121-anti-Etr1 vector, which result in pBI121-Etr1-RNAi plasmid.4. The vector of pBI121-anti-Etr1 and pBI121-Etr1-RNAi was transferred into A. fumefeciens strain LBA4404 by the freezing-thaw method. The transformation was confirmed by PCR amplification with specific primers.