A promoter induced by methyl jasmonate and bacterial blight, its preparation method and recombinant expression vector
A technology of bacterial blight and methyl jasmonate, which is applied in the field of plant genetic engineering, can solve the problems that the expression of exogenous resistance genes cannot be finely regulated, and it is not suitable for wide application in the field, so as to avoid adverse effects, low toxicity, and environmental protection. small effect
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Embodiment 1
[0031] The embodiment of the present invention provides a promoter induced by methyl jasmonate and bacterial blight, the promoter contains the sequence shown in SEQ ID NO.1 in the sequence listing, and is named PRJA140 promoter.
[0032] The following briefly introduces the preparation method of the PRJA140 promoter, as follows:
[0033]Extract the genomic DNA of rice, utilize CTAB (Hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide) method (see Murray&Thompson, 1980 for the specific operation method) to extract the genomic DNA of the rice leaf of normal growth, then use nucleic acid protein analyzer Q5000 The quality and concentration of the extracted genomic DNA were detected, and the concentration of the extracted genomic DNA was diluted to 200ng / μl as a template for the PRJA140 promoter. Among them, the PRJA140 promoter is derived from the rice terpene synthase gene LOC_Os02g36140.
[0034] The template is amplified by PCR (Polymerase Chain Reaction, p...
Embodiment 2
[0041] Use EcoRI (purchased from NEB Company) and SalI (purchased from NEB Company) to perform double enzyme digestion on the PCR amplification product obtained in Example 1 and the pCAMBIA1381-GUS vector, and recover the target fragment (PRJA140 promoter) and pCAMBIA1381-GUS respectively Vector, and use T4 DNA ligase (purchased from NEB company) to connect the target fragment and pCAMBIA1381-GUS vector to obtain the pCAMBIA1381-PRJA140-GUS recombinant plasmid, and transfer the pCAMBIA1381-PRJA140-GUS recombinant plasmid into Escherichia coli competent cells by heat shock method Trans-T1 (purchased from Quanshijin Biology), see the instructions of the pEASY-Blunt Cloning Vector kit for specific operation methods, spread the E. On the LB (Luria-Bertani) plate medium of 100mg / L kanamycin, wherein, the LB plate medium of 1L contains 10g tryptone, 5g yeast extract, 10g sodium chloride and 15g agar, the LB plate medium The pH value was 7.4, and the single clone was picked for ampli...
Embodiment 3
[0043] The pCAMBIA1381-PRJA140-GUS recombinant expression vector obtained in Example 2 was transferred into rice by the Agrobacterium-mediated transformation method, and the method reported by Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence a analysis of the boundaries of the T-DNA.Plant J, 1994, 6:271-282) transfer the pCAM BIA1381-PRJA140-GUS recombinant expression vector into the recipient material "Zhonghua 11", the T-DNA used in the transformation process The medium and reagents mainly refer to the authorized patent of Huazhong Agricultural University (Patent No. ZL200710053552.9, the name of the invention: the isolation and application of rice wide-affinity gene S5; patent publication date: June 18, 2008; publication number: CN101200725 ; Patent authorization date: April 21, 2010), the steps of the embodiments of the present invention are different from the above-mentioned documents in that: the method of...
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