Promoter of gene me094 related to bacterial blight resistance in Oryza verruca
A technology for resisting bacterial blight and verrucous wild rice, applied in the field of molecular biology, can solve the problem of weakened disease resistance of transgenic rice and achieve the effect of improving resistance
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Embodiment 1
[0048] Example 1 Cloning and analysis of the Me094 promoter of the gene Me094 related to resistance to bacterial blight in Oryza verruca
[0049] (1) Design primers for amplifying the Me094 promoter
[0050] The Me094 gene is a metallothionein gene screened and cloned by the applicant from the SSH library of Oryza verrucosa induced by bacterial blight in the early stage, and its function has been verified, but its upstream promoter sequence is unknown, which limits Study on the regulation of gene expression. According to the full-length cDNA sequence information of the Me094 gene, combined with the principle of TAIL-PCR technology, primers were designed with the biological software Primer5.0 to amplify the promoter of the Me094 gene. The nucleotide sequence of the full-length cDNA of the Me094 gene is shown in the sequence table as SEQ As shown in ID NO: 12, the primers designed to amplify the promoter of the present invention (i.e. the Me094 promoter of the resistance gene M...
Embodiment 2
[0081] Example 2 Construct the high-efficiency expression vector of the Me094 promoter of the present invention, and transform Arabidopsis to study the function of the promoter
[0082] 1. Construction of Me094 promoter expression vector
[0083] The promoter expression vector was constructed on the basis of the obtained Me094 promoter sequence, that is, the 35S promoter on the vector pBI121 was replaced with the Me094 promoter (the vector pBI121 is commercially available). According to the restriction sites on both sides of the 35S promoter on pBI121 and the sequence analysis results of the Me094 promoter, the pBI121 and Me094 promoter cloning vectors were digested with HindⅢ and XbaⅠ respectively, and the restriction system was as follows:
[0084]
[0085] React at 37°C for 3 hours, then electrophoresis on 1% agarose gel, and cut the gel to recover the target band. For the method, refer to the manual of the gel recovery kit (the gel recovery kit was purchased from Shangh...
Embodiment 3
[0109] Example 3: Verification and expression analysis of the expression sequence tag of the gene Me094 related to bacterial blight resistance in Oryza verruca
[0110] (1) Cultivation and processing of materials
[0111] Jinghong warty wild rice (Oryza meyeriana) was planted in a plastic greenhouse until flowering.
[0112] Use NA medium to activate bacterial blight pathogen Y8, culture at 28°C for 2-3 days, wash the bacterial lawn with sterile distilled water, and prepare OD 560 Bacterial solution with a value of 0.6. After 15:00 in the afternoon (this time period is the strongest infection ability of bacterial blight pathogen), the leaves of wild rice verruca in the greenhouse were inoculated by the leaf cutting method, and the control group was simulated inoculated with sterile water. 48h, 72h, 96h, and 120h were sampled in equal amounts, and the same amount of wild rice verruca was also sampled at the same time as the control. Immediately after sampling, the samples wer...
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