Cloning Of PLDα CDNA In Strawberry And Studies Of Antisense Gene Genetic Transformation

Strawberry (Fragaria ananassa Duch.) is a kind of perennial herb, which was cultivated widely in the world. Strawberry fruit is a typical non-climateric ftuit and get rotten easily after picked, which was accelerated by the catabolic breakdown of cellular structure such as the cellwall and the cytomembrane. Therefore keeping integrity of membrane structure and compartmentalization plays an important role in the process of ripening. PLDαis related to maturity of strawberry fruit and increasing activity of PLDαis the first step to damage membrane in cell senescence. Effectively controlled the activity of PLDαwill conduced to study the effect of PLDαgene on storage period.Total RNA was extracted from the mature fruit of Darselect strawberry by Plant RNA Reagent Kit, then HKD2 domain gene order was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and cloned into pMD19-T simple vector. The positive clones were sent to sequence. After confirming its validity cloning vector was digested with double digestion, the PLDαgene fragment was inverted inversely into plant expression vector pBI121 to construct antisense expression vector pBI121-PLD. Then the recombinant plasmid was transferred into Agrobacterium tumerfaciens LBA4404 by freeze-thaw method and transformed into strawberry to obtain the PLDαdeficiency plant. This would be helpful to study the function of the gene in strawberry fruit ripening and senescence. In the experiment we construct plus sense expression vector pBI121-PLD also and transformed into strawberry, the aim is to compare with each other in the later fruit storage period.In the Agrobacterium tumefaciens mediated strawberry transformation system, the Agrobacterium tumefaciens in log phase was diluted to OD600 between 0.3 and 0.6 with MS liquid medium without hormone and preincubated strawberry leaves were dipped in them for 10 minutes, then the explants were cocultured for 3 days. After that, they were washed by MS liquid medium and blotted with filter paper then cultured on different medium with Cef 300mg·L^-1 for 7 to 10 days. Finally, the explants were cultivated on the selective medium with Cef 300mg·L^-1 and Km 20mg·L^-1.6 Kmr transgenic strawberry plants were obtained through a succession of Km selecting. By PCR and Southern hybridization analysis on the resistance plants, the results showed that 4 are positive and indicated that target gene was integrated into strawberry genomes.