Rice root tip-specific expression promoter pro-os04g24469 and its application
A pro-os04g24469, promoter technology, applied in the fields of genetic engineering and molecular biology
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Embodiment 1
[0017] Example 1: Expression Analysis of Os04g24469 Gene in Different Rice Tissues
[0018] 1.1 Rice total RNA sample preparation
[0019] The roots and leaves of rice seedlings grown for 15 days were taken respectively, and stored in liquid nitrogen for quick-freezing; the young ears of rice with a length of about 2 cm when heading began to be taken, and quickly stored in liquid nitrogen. After the rice tissue samples were thoroughly ground with liquid nitrogen, the total RNA of the samples was extracted using a plant RNA extraction kit (QIAGEN). The concentration and quality of RNA samples were measured with NanoDropND-1000 nucleic acid detector.
[0020] 1.2 Synthesis of first-strand cDNA
[0021] Take 2.5 μg of each of the above RNA samples, according to the method provided by Invitrogen The first strand synthesis system first added 1 μl of 10 mM dNTP and 1 μl of Oligo(dT)20, added water to make up the volume to 10 μl, incubated at 65°C for 5 minutes, and immediately p...
Embodiment 2
[0025] Example 2: Cloning of rice root tip-specific expression promoter Pro-Os04g24469
[0026] 2.1 Biomaterials
[0027] 2.1.1 Plant material
[0028] The rice transgenic receptor material is japonica Nipponbare
[0029] 2.1.2 Strains and vectors
[0030] The Agrobacterium strain used was EHA105, and the vector was pCAMBIA1300.
[0031] 2.2 Method
[0032] 2.2.1 Cloning of rice root tip-specific expression promoter Pro-Os04g24469
[0033] Rice Nipponbare leaves were used as materials, and the total DNA of rice was extracted with a plant genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.). According to the genome sequence of rice Os04g24469 published on the rice database website (http: / / rice.plantbiology.msu.edu / index.shtml), the PCR amplification primers for the upstream promoter sequence were designed, and the 5' ends of the two primers were respectively Add restriction site sequences and protection bases. The primer sequences were: pro-F: 5'-TGCTCTAGAT...
Embodiment 3
[0044] Example 3: The rice root tip-specific expression promoter Pro-Os04g24469 regulates the expression of the reporter gene GUS
[0045] GUS histochemical staining and microscopic observation
[0046] The root and leaf materials of the transgenic rice grown for 7 days were taken, immersed in the staining solution containing the GUS display substrate x-gluc, and photographed after being incubated at 37°C for 2-4 hours in the dark. The result is as image 3 As shown, specific staining occurs only at the root tip of the main root and the root tip of the lateral root of rice, while other parts of the root and leaves are not stained, indicating that the Pro-Os04g24469 promoter can regulate the expression of the target gene only at the root tip of rice, and the specificity is very high. powerful. The formula of GUS staining solution is: 100mmol / L phosphate buffer (pH7.0), 5mmol / LEDTA, 0.5mmol / L potassium ferricyanide, 0.5mmol / L potassium ferrocyanide, 0.5%TritonX-100, 0.2mg / LX...
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