Cloning And Functional Analysis Of Chrysanthemum Morifolium Phospholipase CmPLDα Gene

Phospholipase D (PLD, EC 3.1.4.4) catalyzes phosphodiester bond hydrolysis and nucleotide exchange reaction. It is reported that PLD functions as a phospholipid transfer protein involved in hydrolyzable groups, the hydrolysis products such as phosphatidic acid (PA), diacylglycerol (DAG), free fatty acid (FFA) participate in stress responses or developmental regulation. Chrysanthemum(Chrysanthemum morifolium Ramat.) is one of Chinese top ten flowers and the four traditional flowers cut flowers in the world, drought stress seriously endanger its yield and quality. In recent years, with the development of molecular biology and bioinformatics, a variety of genes and transcription factors involved in stress tolerance regulation are explored. However, how phospholipase and its hydrolysis products participate in tolerance regulation has not been reported. Therefore, this study cloned CmPLDa, analysised its expression profiles and obtained drought-tolerant chrysanthemum via genetic transformation, which lay the foundation for revealing molecular mechanisms involved in chrysanthemum biotic and abiotic stresses. The main conclusions are as follows:1. Cloning and sequence characteristics of CmPLDaUsing degenerate primer cloning, RT-PCR and RACE method, full-length sequences of phospholipase Da gene, designated as CmPLDa, from cut chrysanthemum’Jinba’was cloned. Sequence analysis showed that total length of CmPLDa was 2697bp consisted of 2427bp open reading frame (ORF) encoding 809 amino acids. CmPLDa amino acid sequence present conserved HKD domain and C2 domain. The sequence homology reaches 83%-88%with known phospholipase D amino acid. Phylogenetic analysis found that CmPLDa has the closest relationship with sunflower HaPLDa, and CmPLDa belongs to PLDa family.2. CmPLDα gene expression profile analysis.Expression profiles of CmPLDa in different tissues and in response to abiotic stresses, and exogenous abscisic acid (ABA), aphid, and leaf black spot (Alternaria.alternata) inoculation were investigated using quantitative real-time PCR, and the roles of CmPLDa in stress responses were disclosed. It showed that CmPLDa expressed in root, stem, leaf and flower with higher expression levels in stem, leaf and flower, however it was lowest in the root. Expression of CmPLDa was less affected by cold, while was generally inhibited by mechanical wound. Heat stress significantly decreased the expression of CmPLDa. Pi deficiency and aphid significantly induced expression of CmPLDa. CmPLDa expression was induced in late period of salt and drought treatment, however, was induced quickly by ABA treatment. The expression of CmPLDa increased after leaf black spot (A.alternata) inoculation. It suggested that expression of CmPLDa was constitutive, and the gene was involved in a variety of physiological processes including cold stress, drought stress, Pi deficiency stress, ABA-induced signals, aphid and leaf black spot inoculation.3. CmPLDa genetic transformationOver-expression vectors and antisense expression vector containing the CmPLDa was constructed, the expression vectors were introduced to ’Jinba’ by Agrobacterium-mediated genetic transformation. The putative transformants were identified by PCR and RT-PCR. The Z1, Z2 and F1, F2 lines showing higher and lower expression levels of CmPLDa were selected for futher drought tolerance identification. Drought stress was mimicked using 20% PEG6000. CmPLDa over-expression plants showed a slight shrinking. The survival rate increased significantly which was 1.7-1.8 times of the wild-type. RWC of over-expression plants were nearly 10% higher than the wild-type. Electrical conductivity and MDA content were significantly lower than the wild type, indicating that the cell membrane integrity and stability were better than the wild type. ABA content of the over-expression lines under drought stress were 1.3 times and 1.22 times of wild-type, but ABA content of antisense lines approximately were 0.83,0.81 of wild-type. We supposed that enhanced level of ABA might induce stomatal closure under water stress, thereby reducing the loss of moisture. Drought resistance significantly reduced in antisense lines. Most plants of antisense transgenic lines F1, F2 were wrinkled, and wilting index was 5 and 6. The survival rate was lower than the wild-type after recovery growth. Moisture content of antisense lines were lower than just lines, conductivity and MDA content were significantly higher than the wild-type and antisense lines. In summary, CmPLDa could enhance the drought tolerance via stabilizing the membrane, and might mediate ABA signaled drought tolerance.