| Lotus, with rich colors and beautiful patterns, as the only aquatic ornamental plant in China’s top ten famous flowers, is deeply loved by people around the world. The lotus of large size is suitable for deep water, and it is of great significance for ecological restoration of the wetland, while small bowl lotus has very high commercial value.Small plant ‘Lian Xia’ of lotus were selected as experimental material in this study. Three treatments as gibberellin, paclobutrazol and water were conducted to do with "Lian Xia". The height of leaf petiole were measured and paraffin section were made to analyze epidermis cells of leaf petiole under different treatments. The expression of genes that related with gibberellin synthesis were analyzed by quantitative Real-Time PCR in different periods of three treatments. Two genes GA20 ox and GA2 ox which are important for gibberellin synthesis were cloned, and GA2 ox was digested and inserted into expression vector p CAMBIA2300 to transfer into Arabidopsis thaliana. The main results are as follows:1. The size of the different treatments of lotus epidermis cells in different periods are obviously different, gibberellin treatment lead to cell elongation, paclobutrazol shorten the cell. Cell number without significant increase or decrease suggest that raise of the height of lotus is mainly caused by elongation rather than split of cells and the height of lotus may be associated with gibberellin;2. GA20ox1 and GA2ox1 gene were cloned and compared with NCBI databases we found the proteins encoded by the two genes have typical structure of 2-oxoglutarate-dependent-dioxygenases domain, which preliminary confirmed that the cloned genes were gibberellin biosynthetic genes. The full length of ORF of Nn GA2ox1 was 1026 bp, encoding 341 amino acids; while Nn GA20ox1 was 1143 bp, encoding 380 amino acids. Then the over expression vector p CAMBIA2300-GA2ox1 were constructed and transferred into Agrobacterium strain GV3101. Agrobacterium-mediated method was used to transfer p CAMBIA2300-Nn GA2ox1 into the model plant Arabidopsis thaliana, and 15 PCR positive transgenic Arabidopsis were obtained.3. Real-time PCR technology was applied to test the expression of the key genes in gibberellin biosynthesis GA2 ox, GA3 ox, GA20 ox, KAO, KO in lotus leaf and petiole of different developmental stages. The results showed that in the petioles, the expression quantity of five genes in gibberellin treatment reduced at the beginning of the treatment, rose up to a maximum in the growth stage, and paclobutrazol treatment is exactly the opposite; in leaves, the expression of five genes rose sharply in the late growth period. We speculate that the feed forward and feedback mechanism of gibberellin may regulate the lotus petiole from the early stages of growth to the period of productive growth, the height of plant may be associated with the expression quantity of gibberellin synthesis genes; Leaf and petiole growth in lotus plants are greatly different, it has the characteristic of longitudinal elongation in the early, lately rapid lateral growth, ultimately the formation of final shape of the lotus leaf. |
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