A rice osabcc9 gene and its application in transporting cadmium
A rice and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0105] 1. Plant material and growing conditions
[0106] The materials used in this example include wild-type Nipponbare rice and two OsABCC9 mutant plants.
[0107] The rice seeds were soaked in tap water and placed in a 28°C incubator in the dark for 2 days after germination. 2 In a 4L plastic container with deionized water, the culture was continued for 2 days at 28 °C. After the roots grow to 2-3 cm, they are used for subsequent related experiments.
[0108] 2. Obtaining the full sequence of OsABCC9 gene and constructing the phylogenetic tree
[0109] To obtain the CDS sequence of OsABCC9, total RNA was extracted from rice roots using Trizol kit (Lifetechnologies) according to the product's instructions. Take 1 μg of total RNA and use the Hiscript II Q RT SuperMix Kit (Vazyme) synthesis kit to synthesize the first piece of cDNA. The obtained cDNA was used as a template to obtain the complete sequence of OsABCC9 in the following different experiments.
[0110] Phylogen...
Embodiment 2
[0122] 1. Using the method of PEG-mediated transformation, the plasmid GFP-OsABCC9 or GFP empty vector obtained in Example 1 was co-transformed into rice protoplast cells with the tonoplast marker plasmid AtTPK1, and after incubation at room temperature, confocal laser light was used. Scanning microscope (TCS SP8; Leica) to take pictures, the results are as follows Figure 5 shown.
[0123] Depend on Figure 5 As shown in A-D, the GFP empty vector (green fluorescence) is localized in the cytoplasm and nucleus, while the tonoplast marker AtTPK1 (red fluorescence) is only localized on the tonoplast. However, by Figure 5 As shown in E-H in the middle, the co-localization of GFP-OsABCC9 and AtTPK1 showed that green fluorescence and red fluorescence could be fused, indicating that OsABCC9 was localized on the tonoplast membrane.
[0124] 2. Phenotypic analysis and Cd content analysis of OsABCC9 mutant plants
[0125] In order to study the Cd tolerance of OsABCC9 mutant plants,...
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