Application of gene osbbx14 in improving rice bacterial blight resistance
A technology for rice bacterial blight and transgenic rice, which can be applied in the fields of application, genetic engineering, plant genetic improvement, etc., and can solve the problems of blind blight resistance molecular breeding.
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Embodiment 1
[0020]Embodiment 1: Isolation and cloning are used to construct the DNA fragment of OsBBX14 gene plant expression vector
[0021] Total RNA was extracted from leaves of rice variety Nipponbare (a publicly reported variety) using TRIZOL reagent (Invitrogen). The specific steps are as follows: put 20 mg leaves into a liquid nitrogen pre-cooled mortar, add liquid nitrogen to quickly grind into powder, put the powder into a 1.5ml centrifuge tube, quickly add 1ml Trizol (Invitrogen) and invert to mix well, stand at room temperature Leave for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Take the colorless aqueous phase into a new 1.5ml centrifuge tube, add 250μl isopropanol, 250μl high salt solution, invert and mix well, and let stand at room t...
Embodiment 2
[0023] Embodiment 2: Construction and genetic transformation of OsBBX14 gene plant expression vector
[0024] In order to better analyze the function of OsBBX14, the applicant increased the expression level of OsBBX14 gene in rice through overexpression technology. The function of the gene was studied according to the agronomic traits of the transgenic plants.
[0025] The construction method of the OsBBX14 gene plant expression vector is as follows: first, the positive clone pMD18-OsBBX14 obtained in Example 1 is double-digested with BamHI and SpeI, and the insert fragment is recovered; and the plant expression vector of pCAMBIA1390-ubi is digested with the same method, and recovered vector fragment. Use the recovered insert and vector fragments for ligation to transform Escherichia coli XL1-Blue. Positive clones were screened by enzyme digestion to obtain a plant expression vector named pCAMBIA1390-ubi-BBX14 (see figure 1 ). pCAMBIA1390-ubi is a commonly used plant genet...
Embodiment 3
[0075] Example 3: Detection of transcript levels of the OsBBX14 gene in transgenic rice plants and wild-type rice
[0076] Using wild-type rice Nipponbare and 8 independent T3 transgenic rice plants as materials, RNA was extracted from rice leaves at the four-leaf stage, and the transcript level of OsBBX14 gene in rice leaves was detected by RT-PCR. The specific method is as follows: use TRIZOL reagent (Invitrogen) to collect 0.03 g of rice leaves from the above materials to extract total RNA, and the specific steps are as described in Example 1.
[0077] Use RNase-free DNase (TaKaRa) to remove DNA in RNA. The specific steps are as follows: Add 50 μg of TotalRNA, 5 μl of 10×DNase I Buffer, 2 μl of DNase I (RNase-free, 5U / μl), and RNase Inhibitor (50U / μl) 0.4 μl, add DEPC water to make up the total volume to 50 μl. The reaction system was mixed and reacted at 37°C for 30min. Then 50 μl of DEPC water was added to the reaction system to make up to 100 μl. Subsequently, 100 μl ...
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