Paeonia ostii PoCAB151 gene, expression vector, and preparation method and application of paeonia ostii PoCAB151 gene
A eukaryotic expression vector, pocab151 technology, applied in the field of plant biology, can solve the problems of Fengdan's weak genetic background and no reports yet
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Embodiment 1
[0034] Example 1 PoCAB151 Cloning of full-length sequence of gene cDNA
[0035] PoCAB151 Acquisition of the cDNA sequence at the 3' end of the gene: the leaves of Fengdan under drought stress were selected as materials, and the total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa) kit. The first strand of cDNA was produced by reverse transcription using 3' full RACECore Set Ver. 2.0 (TaKaRa). The reverse transcription system was: 1 μL RNA, 1 μL 3'-RACE Adapter, 1 μL dNTP Mixture (10 mM each), 2 μL 5× M-MLV Buffer, 0.25 μL RNase Inhibitor, 0.25 μL Reverse Transcriptase M-MLV (RNase H - ), 4.5 μL RNaseFree ddH 2 O; Reverse transcription program: react at 42°C for 60 minutes, and extend at 70°C for 15 minutes. On this basis, 3'-RACE was subjected to two rounds of PCR amplification. The first round of PCR amplification system was: 2 μL cDNA, 8 μL 1× cDNA Dilution Buffer Ⅱ, 2 μL 3'-RACE Outer Primer, 2 μL Gene specific Outer Primer (10 μM) (5'-CGGAACAAAC...
Embodiment 2
[0037] Embodiment 2 Fengdan and other 4 kinds of plants CAB151 Alignment analysis of amino acid sequence homology derived from gene deduction
[0038] Combine Phoenix Dan with 4 other plants CAB151 The amino acid sequences deduced from the genes were expressed in FASTA format, saved as TXT files, and then loaded into the DNAMAN5.2.2 software for homology comparison, and their most homologous amino acid sequences can be observed. The results are shown in figure 2 .
Embodiment 3
[0039] Embodiment 3 Fengdan PoCAB151 Expression of Eukaryotic Expression Vector in Tobacco
[0040] Fengdan PoCAB151 Construction of eukaryotic expression vector: the obtained PoCAB151 The full-length sequence of the gene was sent to Wuhan Sitejin Technology Development Co., Ltd. for whole gene synthesis, and then PCR amplification. The system was: 1 μLdNTP Mixture (25 mMeach), PoCAB151 -Forward Primer (5'-GAGAACACGGGGGACTGGTACCCGGGGATCCATGGCAACTTGTGCAATT-3' (SEQ ID NO. 6)) and PoCAB151 -Reverse Primer (5'-ACAGCTCCTCGCCCTTGCTCACCATGTCGACTTTTCCGGGGACGAAGTT-3'(SEQ ID NO.7)) 2 μL each, 5 μL 10×pfu Buffer, 0.4 μL Pfu high temperature polymerase (5 U / μL), 40 μL RNase Free ddH 2 O. The amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, and 35 cycles; extension at 72°C for 6 min. Cut out the target fragment, perform gel recovery, and connect with the carrier after the de...
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