| Potato (Solarium tubemsum L.) is one of the main crops widespread cultivated in arid and semi-arid region. But many factors, such as degeneration of seed tuber, virus infection, increasingly deteriorated nature environment, inconsequent cultivation technique and so on, severely restrict the yield and quality of potato. Brassinosteroids (BRs), a new class of phytohormones, were found in the early1970s. Recently, genetic analysis of Arabidopsis BR-related mutants has provided unequivocal evidence that BRs are required for regulation of cell elongation, stress responses, male fertility, pigment biosynthesis, blooming time, seed germination and numerous other developments in higher plants. CPD (constitutive photomorphogenesis and dwarfism) gene encodes a monooxygenase modulating BR synthesis in plant. Recently a full-length cCPD homologous to the Arabidopsis CPD was cloned from Populous euphratica by our group, named PeCPD. To explore the possibility of improving yield or quality of potato through changing the level of endogenous BRs, the present research was carried out, in which the fusion gene of PeCPD and GFP (PeCPD-GFP) was used to transform potato variety Gan nong shu2. The main results are as follows:1. An efficiently transformation system for getting PeCPD-GFP into potato was established. In this system, the microtubers being0.8~1.0cm in diameter were cut into1-2mm thick slices; After infected in Agrobacterium (OD600nm=0.35) for10min, the slices were cultured on co-cultivation medium with0.2mg/g AS for2days. Later, they were transfered onto shoot induction medium with10mg/L Hyg and0.5mg/L GA3. The getting rate of the Hyg-resistant plants was12.5%. PCR detection often plants of total20Hyg-resistant plants found that five of them, P1, P3, P7, P8and P9, were PCR positive using two pairs of specific primers to GFP and PeCPD genes.2. RT-PCR detection showed that PeCPD expressed in PI, P3, P7and P9transgenic lines but not in wild type (WT).3. Fluorescence microscopic observation revealed that GFP were highly expressed in the root of P3and P9lines. And confocal laser scanning microscopic observation showed that GFP specially accumulated in cytoplasm of meristematic zone and root cap cells, but not in nucleus and cell walls.4. Detecting the tolerance of transgenic test-tube plantlets to NaCl stress found that the survival rate and root fresh weight were significantly improved compared with WT, indicating that their salt-tolerance has been enhanced5. The transgenic test-tube plantlets transplanted in soil of greenhouse showed (a) increased internodes numbers, stem diameter and plant height,(b) flattened petioles,(c) widened and shorted leaf palisade cells,(d) increased density and aperture of leaves stomata,(e) significantly elevated, chlorophyll content, in particularly chlorophyll b, compared with WT.6. The transgenic test-tube plantlets had been induced to produce microtubers. In comparison to the wild type,(a) the microtuber yield per plant and the fresh weight per microtuber obviously increased.(b) microtuber number per plant was reduced.(c) the rate of deformed microtubers increased.(d) starch grains became larger and starch content increased.7. The plants resulted from the transgenic microtubers and grown in soil outside. Exhibited (a) lengthened petiole,(b) increased stomatal density,(c) elevated chlorophyll content, especially for chlorophyll b,(d) enhanced net photosynthetic rate (Pn), transpiration rate (Tr) and stomatic conductance (GS),(e) increased microtuber yield per plant and fresh weight per microtuber, compared with WT.In summary, heterologous expression of PeCPD gene exhibited apparent impact on the phenotype, photosynthesis and biochemistry of plant as well as tuber yield and quality. So it will not only be good for researching the function for PeCPD gene but also will be helpful to molecular breeding of potato. |
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