Rice OsABCC9 gene and application thereof in cadmium transport
A gene and rice technology, applied in the field of rice OsABCC9 gene and its application in transporting cadmium
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Embodiment 1
[0028] 1. Plant Material and Growth Conditions
[0029] The materials used in this example are wild-type Nipponbare rice and two OsABCC9 mutant plants.
[0030] Soak the rice seeds in tap water, put them in an incubator at 28°C for 2 days to germinate in the dark, put the seeds on a strainer, and place them in a medium containing 0.5mM CaCl 2 In a 4L plastic container of deionized water, culture was continued for 2 days at 28°C. After the root grows to 2-3cm, it is used for subsequent related experiments.
[0031] 2. Obtaining the complete sequence of the OsABCC9 gene and constructing the phylogenetic tree
[0032] In order to obtain the CDS sequence of OsABCC9, total RNA was extracted from rice roots using the Trizol kit (Lifetechnologies) according to the product instructions. Take 1 μg of total RNA and use the Hiscript II Q RT SuperMix Kit (Vazyme) synthesis kit to synthesize the first cDNA. The obtained cDNA was used as a template to obtain the complete sequence of OsA...
Embodiment 2
[0045] 1. Using the method of PEG-mediated transformation, the plasmid GFP-OsABCC9 or the GFP empty vector obtained in Example 1 were co-transformed with the tonoplast marker plasmid AtTPK1 in rice protoplast cells, and after incubation at room temperature, confocal laser A scanning microscope (TCS SP8; Leica) was used to take pictures, and the results were as follows: Figure 5 shown.
[0046] Depend on Figure 5 As shown in A-D, the GFP empty vector (green fluorescence) localizes in the cytoplasm and nucleus, while the tonoplast marker AtTPK1 (red fluorescence) localizes only on the tonoplast. However, by Figure 5 As shown in E-H, the colocalization results of GFP-OsABCC9 and AtTPK1 revealed that the green fluorescence and red fluorescence can be fused, indicating that OsABCC9 is localized on the tonoplast membrane.
[0047] 2. Phenotype analysis and Cd content analysis of OsABCC9 mutant plants
[0048] In order to study the tolerance of OsABCC9 mutant plants to Cd, 15-...
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