Cloning And Function Identification Of CmNCED3-1 From Chrysanthemum

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of four cut flowers in the world. It is a popular flower due to its rich flower-patterns and easily being cultivated. In recent years, cut chrysanthemum accounts for a large proportion of the flowers consumption, and the cultivation area has been ranked first in the production of cut flowers. Aphids attack chrysanthemum seriously and occur at all stages of cultivation, which seriously affects the ornamental value of chrysanthemum. Aphids infestation has gradually become a bottleneck of chrysanthemum Produce. Therefore, to study the mechnisms involved in the aphid resisitance is important for the improvement of the production and quality of chrysanthemums. ABA, as an important plant hormone, participates in various stresses reponses. In present study, we cloned the key gene in ABA synthesis pathway CmNCED3-1 and generated the CmNCED3-1 overexpression chrysanthemums by Agrobacterium tumefaciens-mediated transformation, and the aphid resisitance of the transgenic plants were assessed as well. In addition, we investigated the effect of ABA on the aphid resistance and its corresponding physiological mechanisms. The main results are summarized as follows:1. Cloning a key gene in ABA synthesis pathwayA key gene in ABA synthesis pathway, named CmNCED3-1, was cloned from cut chrysanthemum’Jinba’, using a strategy based on RT-PCR. Sequence analysis showed that the cloned cDNA fragment consisting of an ORF of 1800bp, coded 600 amino acids. The phylogenetic analysis showed that CmNCED3-1 shared high homology with LsNCED2 from Lactuca saliva.2. The effects of ABA on aphids resistance and corresponding physiological mechanisms0 mg·L-1,15 mg·L-1 and 30 mg·L-1 ABA was applied to the leaf of the chrysanthemum ’Jinba’ followed by aphid inoculation. The activities of the antioxidant enzyme (SOD, POD and CAT), content of the hydrogen peroxide (H2O2), superoxide anion(O2-) producing rate were investigated in ABA treated and aphid inocultated plants. We found that 15mg·L-1 ABA could improve aphids resistance in chrysanthemum, whereas, 30mg·L-1 ABA did not improve the aphid resistance. The results showed that the activity of SOD, POD and CAT presented as a rising, falling and then rising trend, and the peak values were 1.6,2.3 and 1.9 times that of the control. Meanwhile, similar trends existed in the content of H2O2 and O2- producing rate, and they were also significantly changed under 15mg·L-1 ABA compared with the control. It inferred that ABA improved the aphids resistance in chrysanthemum via regulating the ROS levels.3. Generation of CmNCED3-1 overexpressing chrysanthemum plants and its resistance to aphidsCmNCED3-1 was tranformed to the Agrobacterium strain EHA105. Transgenic chrysanthemums resistant to npt Ⅱ were produced by Agrobacterium tumefaciens-mediated transformation. We got two overexpression plants named Z3 and Z4 via qRT_PCR analysis. The level of expression of CmNCED3-1 in Z3 and Z4 were 3.42 and 3.78 times of the control and the level of ABA content in both two lines were 3.2 and 3.6 times of the control Both CmNCED3-1 overexpression plants significantly inhibited the proliferation of aphids. The aphids inhibition rate in Z3 and Z4 was 80.5% and 86.2% respectively.