A rice chloroplast light avoidance movement regulation gene crd1 and its application
A technology for regulating genes and CRD1, applied in the field of molecular biology, which can solve the problems of leaf death, less research, and destruction of chloroplast structure and activity.
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Embodiment 1
[0035] 1. Rice material
[0036] Rice (Oryza sativa) crd1 mutant plants were obtained from the indica rice variety Shuhui 527 (purchased from the Crop Variety Resource Bank of the Chinese Academy of Agricultural Sciences) through EMS mutagenesis treatment.
[0037] After the crd1 mutant plants were treated with strong light, the chloroplast movement to avoid light disappeared. It is significantly different from the wild type (indica rice Shuhui 527). Using laser confocal microscope to observe the autofluorescence of chloroplasts, it is found that the chloroplasts in the mutant are still arranged densely. weakened, such as figure 1 shown.
[0038] 2. Analyze and target groups
[0039] f 2 F 1 Generation, F 1 F 2 group. The individual plants whose chloroplast light-shielding response disappeared were screened out from the F2 population for map-based cloning.
[0040] 3. Localization of CRD1 gene by SSR and STS markers
[0041] Genomic DNA extraction: TBS digestion: Tak...
Embodiment 2
[0053] Functional verification of CRD1:
[0054] Design primers according to the target gene (LOC_Os07g39920):
[0055] pMDC83-DRT1-F: 5'-caccATGAGGAGGAGAGTTGCAC-3';
[0056] pMDC83-DRT1-R: 5'-AGATGACGAGCTGTCCTCAT-3'.
[0057] Use the Q5 high-fidelity enzyme system of NEB Company for PCR amplification. The reaction system is (20 μL): 2 μL of DNA template, 10 μL of 2×Master Mix, 1 μL of upstream and downstream primers, and make up to 20 μL with ultrapure water. The PCR reaction conditions were pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 30s, extension at 72°C for 2min for 30s, 30 cycles; extension at 72°C for 10min.
[0058] After the PCR product was electrophoresed, the target fragment was recovered and inserted into the pETNR / D-TOPO vector. The fragment was 2322bp in length. Using the Gateway recombination system, the sequenced correct fragment was recombined into pMDC83 to construct an overexpression vector, such as image 3 shown...
Embodiment 3
[0060] 1. CRD1 tissue expression analysis
[0061] Total RNA was extracted from the roots, stems, leaves, leaf sheaths, booties, panicles and anthers of Nipponbare plants respectively, and the TOYOBO reverse transcription kit (ReverTra qPCR RT Master Mix with gDNA Remover) was reverse-transcribed into cDNA, and then the CFX96 fluorescent quantitative PCR instrument (Bio-Rad) was used for fluorescent quantitative PCR detection. The qPCR reaction used the SYBR GreenSupermix (Bio-Rad) kit, and all kits involved The methods were operated according to the relevant instructions.
[0062] After the cDNA was obtained by reverse transcription, the expression of CRD1 gene in different tissues and organs was detected by quantitative PCR, and it was found that the expression of CRD1 was higher in leaves and leaf sheaths.
[0063] Subsequently, using pCAMBIA1301 as the backbone vector, according to the promoter sequence, design primers pGUS-F (YP2736) (5'-aagcttAGCCGTGCTCGCGGTTA-3') and ...
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