Sea island cotton receptor albuminoid kinase gene promoter and application thereof
A protein kinase gene and promoter technology, applied in the field of sea island cotton receptor-like protein kinase gene promoter, can solve problems such as slow progress
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Embodiment 1
[0075] The reaction system is:
[0076]
[0077] The reaction procedure is:
[0078] A: 1min at 94°C;
[0079]94℃ 30sec;
[0080] 37℃ 3min
[0081] Ramp to 70°C at 0.2°C / S.
[0082] After PCR reaction A is completed, take out the PCR tube, add 2.5 μl of SP1 and continue PCR;
[0083] B: 94°C 30sec
[0084] 70℃ for 5.5min,
[0085] 94°C 30sec.
[0086] Perform PCR reaction B for 25 cycles, and then perform PCR reaction C for 8 cycles
[0087] C: 94°C 30sec
[0088] 50℃ 30sec
[0089] 70°C for 5min.
[0090] The second round of SEFA-PCR amplification:
[0091] The PCR reaction system is:
[0092]
[0093] The reaction procedure is:
[0094]
[0095] Apply the above-described experimental method, design groove primers SP1, SP2 and SP3 according to the untranslated regulatory sequence at the 5' end of the cloned receptor analogous protein kinase, and design the 3' end sefa-PCR primer as follows:
[0096] SP1: 5'-GCAAACACTATCGCTGCTTTCTC-3';
[0097] SP...
Embodiment 2
[0103] We predicted two transcription start sites, A at -221 and A at -1664, with analysis scores of 0.91 and 0.99, respectively. There is a TATA-Box (ATATA) at the -247 / -251 position, and a CAAT-Box at the -257 / -261 and -289 / -293 positions, both of which are necessary for the basic promoters of higher plants Expression Regulatory Elements. There are two ABREs at -366 / -371, and -1127 / -1132, one W-Box at -752 / -756, -1276 / -1280, -1575 / -1579, and one at -613 / Position -620 has a cis-element in the promoter region of a xylem-associated gene. At the same time, the promoter region also includes some regions that bind to transcription factors such as MYB, MYC and WRKY, four Root-Motifs, and original regulatory elements related to light response.
[0104] Example 3:
Embodiment 3
[0106] FI 5'-CCGGAATTCTTTTAATTAGGGTAGACAG-3';
[0107] F2 5'-CCGGAATTCACAATAAAGTCCCGATTAC-3';
[0108] F3 5'-CCGGAATTCAGGTTGGTCTGGCTCGGGATA-3';
[0109] R1 5'-GAAGATCTACCATGTTTAGTGTTATAGATTGAG-3';
[0110] The constructed vector was verified by PCR amplification, enzyme digestion and sequencing, and the results showed that the constructed vector was correct.
[0111] Example 4:
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