Multi-stress-induced promoter derived from alpine ash and application of promoter
A promoter and inducible technology, applied in the field of genetic engineering, can solve problems such as inhibiting plant growth and development, and achieve the effect of saving money and improving the ability to resist various adversities
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Embodiment 1
[0032] promoter discovery
[0033] Genomic DNA of Eucalyptus grandis (variety EG6) was extracted, and the extraction of Eucalyptus genomic DNA referred to conventional methods in the art. Design specific primers (P1: 5'-TCAT TCTAGA GCGTAATTGCTGTC GCACCC-3', such as SEQ ID NO: 2, the underline is the Xba I restriction site) and (P2: 5'-GCTC AAGCTTCGAGAAGGAATCGCTTCTGA-3', such as SEQ ID NO: 3, the underline is the Sac I restriction site) to amplify the genomic DNA, recover and clone the fragments, and perform DNA sequencing to obtain a 720bp DNA fragment, the 720bp DNA fragment The nucleotide sequence is shown in SEQ ID NO:1. The amplified sequence shown in SEQ ID NO: 1 was analyzed for the regulatory region, the start codon ATG of the gene was identified, and the part of the structural gene was removed. Obtain the regulatory sequence at the 5' end of the gene, the regulatory elements of the promoter such as figure 2 As shown, the TATA box is located in the ?150-?155 regi...
Embodiment 2
[0035] 1. Low temperature and drought-induced activity verification of the promoter pG5
[0036] Construction of recombinant expression vector
[0037] 1. Digest pGEM-T: pG5 prepared in Example 1 with restriction endonucleases Xba I and Sac I, and recover the digested product (pG5).
[0038] 2. Digest the binary vector pBI 121 with restriction endonucleases Xba I and Sac I to obtain the pBI 121 vector backbone with the CaMV 35S promoter removed.
[0039] 3. Ligate the digested product of step 1 with the vector backbone of step 2 to obtain a recombinant plasmid, and perform sequencing verification. The sequencing results show that the target plasmid pG5-GUS (replacing pBI with pG5 of the sequence shown in SEQ ID NO: 1) The small fragment between the recognition sites of Xba I and Sac I of 121).
[0040] 2. Transformation of tobacco by transient expression
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