Cloning Two Novel Phospholipase D Genes And Researching The Response Of Low Temperature In Chorispora Bungeana

Two novel phospholipase D genes, CbPLDα and CbPLDδ were isolated fromChorispora bungeana, a rare alpine subnival plant species that is highly tolerant of lowtemperature stress. We investigated the patterns of tissue-spceific expression and lowtemperature stress-related expression of CbPLDα and CbPLDδ by real time quantitative PCR.we investigated the response of PLD to low temperature stress and the role of PLD inmetabolism pathway of rare alpine subnival plant. We introduce CbPLDα and CbPLDβ withthe CaMV35S promoter, into tobacco. The main results are as follows:(1) Through PCR and RACE techniques, a whole sequence of PLDα cDNA was isolatedfrom Chorispora bungeana (GenBank accession No. KF248008). The cloned full-lengthcDNAof Chorispora bungeana (CbPLDα) was2804bp. The cDNAcontained a2430bp ORFencoding a protein of810amino acids with a calculated molecular weight of about91.89kDaand with a PI of5.71. A5’ untranslated region (UTR) of72bp was found upstream of the firstATG codon, and a3’ untranslated region (UTR) of302bp was found downstream from thestop codon. Secondary structure analysis showed that CbPLDα had "FIYIENQYF" region andtwo HKD motifs belonging to PLD family as their activity region. The putative CbPLDαpeptide contained46.11%random coil,28.31%alpha helix,19.04%extended strand and6.55%beta turn. The alpha helix and random coil constituted interlaced domination of themain part of the secondary structure. A comparison of the predicted protein sequences of theCbPLDα with PLDs of other plants shows that CbPLDα is highly identical to PLD fromArabidopsis thaliana PLDα (94.6%).(2) Through PCR and RACE techniques, a whole sequence of PLDδ cDNA was isolatedfrom Chorispora bungeana (GenBank accession No. KF248008). The cloned full-lengthcDNAof Chorispora bungeana (CbPLDδ) was2872bp. The cDNAcontained a2592bp ORFencoding a protein of864amino acids with a calculated molecular weight of about98.01kDaand with a PI of6.8. A5’ untranslated region (UTR) of126bp was found upstream of the firstATG codon, and a3’ untranslated region (UTR) of155bp was found downstream from thestop codon. Secondary structure analysis showed that CbPLDδ had "FIYIENQYF" region andtwo HKD motifs belonging to PLD family as their activity region. The putative CbPLDδpeptide contained47.97%random coil,27.11%alpha helix,19.24%extended strand and 5.68%beta turn. The alpha helix and random coil constituted interlaced domination of themain part of the secondary structure. A comparison of the predicted protein sequences of theCbPLDδ with PLDs of other plants shows that CbPLDδ is highly identical to PLD fromThellungiella halophila (92.1%).(3) CbPLDα and CbPLDδ expression patterns under low temperature stress (4℃、0℃、﹣4℃) were analyzed by real time quantitative PCR and the influence of freezing treatmenton PLD activities was studied in Chorispora bungeana. The results indicated that CbPLDαand CbPLDδ is ubiquitously expressed in Chorispora bungeana, with almost no tissuespecificity. Following exposure to4°C, the microsome-associated PLD activities weresignificantly induced. PLD activities were fluctuated around the comparison at the early stageof0℃and﹣4℃treatment. With the prolongation of treatment, PLD activities werecontinously induced up to the end of0℃treatment, but inhibited under﹣4℃. These resultsshowed that the PLD activities are related to the different levels of CbPLDα mRNA in somedegree, indicating CbPLDα activity in microsomal membranes was dependent on theCbPLDα gene expression partially. It seems to be a clear correlation between increase inCbPLDδ transcription levels and PLD activities at different days of low temperaturetreatments and the decreases of PLD activities during the low temperature stresses inChorispora bungeana could be due to the down-regulation of CbPLDδ gene.(4) The metabolism regulations of Chorispora bungeana in response to freezing stresswere analyzed. The results showed that Chorispora bungeana have mediated the regulationpathway of membrane stability, osmotic adjustment and regulated in anti-oxidative system. Italso involved in the cycle of active oxygen metabolism and ascorbate-glutathione.(5) We introduced CbPLDα and CbPLDβ into tobacco with the CaMV35S promoter. Thetransgenic tobacco plants were detected by PCR.The sequences results showed that the twogenes have been successfully transferred into tobacco. The resistance of the transgenictobacco plants will be detected in the further step.