Rice histone lysine modification recognition protein and its coding gene and application
A technology for lysine modification and protein recognition, which is applied in the field of histone lysine modification recognition proteins and their coding genes and applications, can solve the problems of differences in the regulation mechanism of growth and development, and achieve high application value and broad application prospects Effect
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Embodiment 1
[0027] Example 1. Rice histone lysine modification recognition protein gene MRG702 the acquisition
[0028] The gene sequence of Arabidopsis MRG1 published in GenBank (GenBank number: NC_003075.7) was compared with the homologous sequence, and the homologous sequence was obtained from the rice genome, and according to the 5' and 3' end sequences of the homologous sequence A pair of primers were designed, and the primer sequences were: 5'-ATGTCCCAAGCTGGATC-3' (SEQ ID No: 3), and 5'-CTATTTGGTCTTTTATCC-3' (SEQ ID No: 4).
[0029] Extract the total RNA of rice yellow seedlings (Promega, SV total RNA isolation system), use the total RNA of rice as a template, and use AMV reverse transcriptase (TaKaRa) to synthesize cDNA (according to the user manual of Plant RT-PCR Kit 2.01 (TaKaRa) conduct). Using cDNA as template, PCR amplifies rice histone lysine modification recognition protein gene MRG702full-length cDNA sequence. The 50ul PCR reaction system contains: template 2ul, high...
Embodiment 2
[0030] Example 2. Rice histone lysine modification recognition protein gene MRG702 protein activity assay
[0031] Using the pUC19-MRG702 obtained in Example 1 as a template, PCR amplification MRG702 From the 1-282 nucleotide sequence at the 5' end (including the active center of the histone lysine modification recognition protein - the chromodomain domain), the 5' and 3' primers are: 5'- GGATCCATGTCCCAAGCTGGATCGGA-3' (SEQ ID No:5), and 5'-CTCGAGTCCAGATTTTACGCTTTTGT-3' (SEQ ID No:6). 200ng of template pUC19-MRG702, 20 pmol of 5' and 3' primers, 5μl of 10Xbuffer, 20μM of dNTP and 1ul of high-fidelity KODplus (TOYOBO) were included in 50μl of PCR amplification system. Amplification was performed under the following conditions: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 1 minute, a total of 30 cycles. The PCR product was Bam HI and xho The restriction site I was cloned into the ...
Embodiment 3
[0033] Example 3. Detection MRG702 Regulatory effect on rice growth and development
[0034] one, MRG702 Construction of antisense expression vector
[0035] Using RNA interference (RNA interference, RNAi) technology to MRG702 The gene is the target gene, and the RNA interference vector is constructed. Using the pUC19-MRG702 obtained in Example 1 as a template, two pairs of different primers were used for PCR amplification MRG702 The nucleotide sequence from the 670th to the 896th position of the 5' end is a complementary DNA duplex as a hairpin structure. The primer pair 1 used is: 5'-aaggatccAAGTATCTCTCTCTAAAGAT-3' (SEQ ID No: 7) and 5'-aaaagcttCTCCTTGGCAGCGGCGGATT-3' (SEQ ID No: 8), and the obtained PCR product is FMRG702i (forward). Primer pair 2 is 5'-aagaattcAAGTATCTCTCTAAAGAT-3' (SEQ ID No: 9) and 5'-aaactagtCTCCTTGGCAGCGGCGGATT-3' (SEQ ID No: 10), and the obtained PCR product is rMRG702i (reversed). In order to facilitate the subsequent vector construction, ...
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