OsCEBiP (oryza sativa-Chitin elicitor binding protein) gene promoter and application thereof
A promoter and gene technology, applied in the fields of biotechnology and botany, can solve the problems of unstudied gene promoter function and unclear mode of action, and achieve the effect of improving plant disease resistance and insect resistance
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Embodiment 1
[0034] [Example 1] Isolation and identification of promoters
[0035] The inventors selected the range of 1807 bp upstream of the start codon ATG of the rice chitin elicitor binding protein gene OsCEBiP as a candidate promoter region for PCR amplification. According to the position of the cis-acting element, design primers for vector construction, as shown in the table:
[0036]
[0037]
[0038] Genomic DNA of rice material B5 (extracted by CTAB method, ZhangQF et al., 1992, Genetic diversity and differentiation of indica and japonicarice detected by RFLPaysis. Theor. Appl. Genet. 83, 495-499) was used as template for PCR amplification, and the high-fidelity enzyme KOD-Plus-Neo (TOYOBO , Japan) 50μl reaction system amplification. The PCR conditions were: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, annealing and extension at 68°C for 30 s / kb, 40 cycles. The PCR system is as follows:
[0039]
[0040] After the PCR product was recovered, it was ...
Embodiment 2
[0041] [Example 2] Identification of promoter expression activity
[0042] In the embodiment of the present invention, a GUS gene expression vector of the promoter was constructed and transformed into a rice variety, and the tissue-specific expression activity of the promoter was observed by GUS color development. The specific operation is as follows:
[0043] First, the PCR product was double-digested with HindIII / BamHI and EcoRI, and the pCAMBIA1391Z vector was also digested with the same enzymes, purified and recovered by the kit, ligated and sequenced. The correct clone was electroporated into Agrobacterium EHA105. The genetic transformation method mediated by Agrobacterium EHA105 (Hiei et al., 1994, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271-282) was used to introduce the genome vector into rice varieties.
[0044] After the obtained transgene was multiplied, a s...
Embodiment 3
[0045] Example 3: Tissue-specific expression of rice chitin elicitor binding protein gene OsCEBiP
[0046] Taking the receptor material Hejiang 19 (also known as H1493, purchased from the National Rice Germplasm Bank) as the research material, 2 g of the root, stem and leaf tissue were taken from the seedling stage of 18 days of growth and the heading stage of 67 days of growth, respectively, and immediately immersed in Store in liquid nitrogen. Total RNA was extracted with RNAisoPlus (TaKaRa), and then RevertAid TM firststrandcDNAsynthesiskit (Fermentas) reverses the synthesis of cDNA first strand. OsCEBiP in different tissues was detected by quantitative PCR using CFX96TouchTMReal-TimePCRDetectionSystem (Bio-Rad). The 8ul reaction system for PCR is: DNase / RNase-FreeddH 2 O2.9 μl, 2×Supermix 4 μl, primer (5 mM) 0.6 μl, cDNA template 0.5 μl. Reaction conditions: Denaturation at 95°C for 2 minutes; denaturation at 95°C for 5-10s, TM annealing and extension for 10-60s, 40 cy...
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