Related protein capable of adjusting translation efficiency of chloroplast protein and improving heat resistance of plants, and application thereof
A chloroplast and plant technology, applied in the fields of botany and biology, can solve the problems of undiscovered chloroplast protein translation efficiency, plant heat-resistant thylakoid membrane heat-resistant proteins, etc.
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Embodiment 1
[0087] Identification of rps1 T-DNA mutants
[0088] The inventor purchased the rps1 T-DNA mutant from the Salk website, and in the current planting period, the rps1 T-DNA mutant isolated plants with yellow leaves. According to Salk's prediction, the T-DNA insertion site of rps1 is in the first exon of the RPS1 gene.
[0089] Based on the sequence information, the inventors synthesized the identification primer sequence of RPS1 (CS874869) T-DNA insertion mutant:
[0090] LP(874869-L): 5'-GACTCCAGCTGGTTTAGAGGG-3' (SEQ ID NO.: 4);
[0091] RP(874869-R): 5'-CAAGTAAGCCGATGACTTTGC-3' (SEQ ID NO.: 5);
[0092] LB1: 5'-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3' (SEQ ID NO.: 6); and
[0093] LB2: 5'-GCTTCCTATTATATCTTCCCAAATTACCAATACA-3' (SEQ ID NO.: 7).
[0094] Use the above primers as a primer pair, and use the genomic DNA of the rps1 T-DNA mutant as a template to perform a PCR reaction to identify the T-DNA insertion position in the rps1 mutant (the PCR product is connected to the T...
Embodiment 2
[0106] Downregulation of RPS1 expression reduces the stability of the thylakoid membrane system in a dose-dependent manner
[0107] 1. Thylakoid stacking degree determination method: reference (Khatoon et al., 2009), with appropriate modifications:
[0108] 1). Take 3-4g leaves of Arabidopsis thaliana, add 20ml ice-cold GB buffer solution, and quickly grind it in a pre-cooled mortar until it is homogenized;
[0109] 2). The homogenate is filtered with dust-free paper (2-4 layers);
[0110] 3). The filtrate was centrifuged at 4°C, 2600g, for 3min;
[0111] 4). Discard the supernatant, add 10ml RB buffer, and resuspend;
[0112] 5). Centrifuge at 2600g for 3min at 4°C;
[0113] 6). Repeat steps 4 and 5 once;
[0114] 7). Resuspend the precipitate in Solution C, measure the chlorophyll concentration with the acetone method, and adjust the chlorophyll concentration to 0.25mg / ml with Solution C;
[0115] 8). Incubate at 4°C for 15 minutes in the dark;
[0116] 9). Add digiton...
Embodiment 3
[0126] To construct an estrogen-inducible RPS1-RNAi binary vector, the construction method is as follows:
[0127] The sequence between 25 bp upstream and 457 bp downstream of the initiation codon ATG of RPS1 was amplified by RT-PCR.
[0128] The sense fragment primers are:
[0129] Upstream primer: 5′AAACCCGGGCTCGAGCTGTGTGAGTGAGTGAGACTC-3′
[0130] (SEQ ID NO.: 14)
[0131] Downstream primer: 5′-CGCTCTAGATGACAAACTCTTCCACCATAC-3′
[0132] (SEQ ID NO.: 15)
[0133] Antisense fragment primers are:
[0134] Upstream primer: 5′-AAAGAGCTCCTCGAGCTGTGTGAGTGAGTGAGACTC-3′
[0135] (SEQ ID NO.: 16)
[0136] Downstream primer: 5′-CACGCGGCCGCTGACAAACTCTTCCACCATAC-3′
[0137] (SEQ ID NO.: 17)
[0138] PCR conditions: annealing at 54°C for 15s, extension at 72°C for 0.5min, 30 cycles.
[0139] The amplified sense fragment and antisense fragment were connected to the commercially available pMD19-T vector by TA cloning method, and then transferred into the commercially available host...
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