Cloning Of A Cucumber CDNA Encoding Phospholipase D(PLD) And Construction Of The Sense And Antisense Expression Vectors

Phospholipase D (PLD) is widly distributed in the plant tissues. PLD and its product, phosphatidic acid, have been implicated in multiple plant stress responses by functioning in signal transduction cascades and by influencing the stablity of lipid membranes.Cucumber is one of the main vegetables in China.In the cultivation of it, salt, drought and other stresses are often encountered. In this study, the full-length cDNA of a phospholipase D (PLD) was amplified by RT-PCR and RACE methods from young leaves of cucumber (Cucumis sativus L.). Furthermore, we constructed the sense and the antisense expression vectors of cuPLDα.The expression of cuPLDαunder NO3- treatment was also studied using RealTime-PCR methods. The main results were as follows:1. With RT-PCR and RACE methods, the full-length cDNA of a phospholipase D (PLD),named cuPLDα,was amplified from cucumber (Cucumis sativus L.). It's accession number in GenBank is EF363796.2. Sequencing analysis showed that the full-length of cuPLDαcDNA consisted of 2,756 bps which contains an open reading frame of 2,427 nucleotides coding for a 808-amino acid protein of 91.8 kD.The predicted PI was 5.37.Like in most plant PLDs, two HKD motives, IYIENQFF motif and one C2 domain are present in cuPLDα.The gene can be assigned to the [alpha]-type of plant PLDs.There is no meaningful transmembrane helices in cuPLDα.3. A new method of site-directed mutagenesis was introduced: splicing the non-mutant regions of two or more positive clones which have mutations. 4. RealTime-PCR showed that cuPLDαmRNA increased after half an hour treatment with NO3- and cuPLDαincreased in a concentration-dependent manner. The cuPLDαexpression of the 182 mmol/L NO3- treatment increased nearly 4.66 fold over the 14mmol/L NO3- treatment,which was the greatest increase.5. In order to determine the function of PLD in cucumber, a sense and an antisese vector were constructed and introduced into tobacco and cucumber using Agrobacterium-mediated plant transformation and ovary-injection, respectively.