Cloning And Function Analysis Of Phospholipase D Gene In Flat Peach Fruit

Flat Peach fruit (Prums persica L. Batsch) is a typical climacteric fruit and it reaches maturity in short time after harvest and then gets rotten easily. Therefore, how to extend its shelf time becomes a urgent problem that need to be solved. Previous research of Li et al found that phospholipase D (PLD) inhibitor treatment helped quality maintenance of flat peach fruits after harvest. To further investigate the function of PLD in fruit development and postharvest storage of flat peach fruit, we selected and optimized the total RNA extraction method, cloned PLD from flat peach fruit, undertook "Northern Blot of PLD, constructed RNAi expression vector and used for genetic transformation of strawberry. The results are as follows:(1) Flat peach Fruits harvested at100days after fully blossom were used to isolate total RNA and the result showed that modified CTAB method is the most suitable one. Primers were designed based on the known sequence information of peach, a fragment of1097bp was cloned from flat peach fruit using RT-PCR. Multiple sequence alignment showed that flat peach fragment had99.36%similarity to peach PLD and therefore confirmed it was flat peach PLD.(2) A114bp fragment of flat peach fruit PLD was synthesized and used as a probe to analyze PLD expression during fruit development and after different postharvest treatments through northern blot. The results showed that the intensity of PLD expression increased with the development of the fruit, while PLD inhibitor treatment decreased PLD expression to certain extent.(3) A conserved PLD fragment of347bp was amplified by RT-PCR and pSK-PLD-RNAi expression vector was obtained by inserting the fragment into the pSK-int expression vector. The PLD shRNA expression vector pCAMBIA-PLD-RNAi was constructed through inserting pSK-int expression vector into pCAMBIA1301vector. Further confirmation of restriction endonuclease digestion and sequencing showed that PLD shRNA expression vector pCAMBIA-PLD-RNAi was successsfully constructed.(4) In the Agrobacterium tumefaciens mediated strawberry transformation system,5Kmr transgenic strawberry plants were obtained through a succession of Km selecting. By PCR analysis on the resistance plants,4transgenic plants were confirmed positive. The result indicated that the target gene was integrated into strawberry genomes.