Preliminary Study On Construction Of Ramie Atp2RNAi Expression Vector And Its Interference Effects

FiFo-ATP synthase is an important ATP-synthesizing enzyme as the V complex involved in the electron transport chain in mitochondrion. The β subunit of F1is the key site of ATP synthesis and hydrolysis encoded by nuclear genes atp2in higher plants and highly conserved in all species. Usually, deficiency or abnormal distribution of ATP synthase is associated with male sterility, a complicated biological process with unclear mechanisms. We constructed RNAi expression vectors using atp2fragment cloned from ramie maintainer’GS13-X2’and transformed ramie and tobacco mediated by Agrobacterium. The results were as follows:(1) The atp2fragment of ramie maintainer’GS13-X2’ was attained. And atp2RNAi expression vector was constructed in this study, which confirmed through sequence alignment.(2) The constructed expression vector of RNAi was transferred into ramie by Agrobacterium-mediated transformation. As results, all leaf disc died from browning. All transgenic plants did not attain in this study. The genetic transformation system of ramie needs to be optimized.(3) Seven transgenic tobacco plants have been attained by the ramie atp2RNAi expression vector. The atp2relative expression level was confirmed by qRT-PCR which showed lower in transgenic tobacco plants than control.(4) The atp2silencing in general tobacco led to the plant dwarf and the uneven distribution of chlorophyll of leaves, even susceptible to disease. Compared with the control, shorter corolla tube and the stamens couldn’t extend out the tube throat were showed in the transgenic tobacco plants. The female genitals has no obvious abnormalities in all plants.(5) By the pollen germination experiments, all transgenic tobacco pollen germination rates were33.26%lower than the control, the pollen tube length decreased by28.68%and they showed a partial male sterility.(6) Proline content and SOD activity were detected in tobacco leaves and big bud which showned that proline contents of the transgenic tobacco large buds were33.4% lower than the control, while proline contents in leaves compared to the control increased by95.08%, SOD activity in transgenic tobacco leaves and buds were higher than the control tobacco.(7) We harvested some seeds from inbred transgenic tobacco to test the germination rate that was74.23%lower than the control.In this study, we successfully constructed ramie atp2RNAi expression vector and transformed tobacco. Seven transgenic tobacco plants were obtained. The transgenic plants showed significant differences in plant morphology, pollen fertility, seed germination, proline content and SOD activity in comparison with the control plants.