Application of Gene osbbx14 in Improving Drought Stress Tolerance in Rice
A technology of drought stress and rice, applied in the field of rice drought stress tolerance, can solve the problems of blind molecular breeding for drought resistance, and achieve the effects of enhancing drought stress tolerance, improving drought stress tolerance, and improving drought stress tolerance
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Embodiment 1
[0024] Embodiment 1: Isolation and cloning are used to construct the DNA fragment of OsBBX14 gene plant expression vector
[0025] 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 ...
Embodiment 2
[0027] Embodiment 2: Construction and genetic transformation of OsBBX14 gene plant expression vector
[0028] 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.
[0029] In order to construct the plant expression vector of OsBBX14 gene, the present application first constructed the plant expression vector of pCDMAR-ubi-Tnos. Firstly, the genomic DNA of maize was extracted by the following method, and the total DNA was extracted from the leaves of maize variety Ludan 981 (a variety reported publicly). The specific steps are as follows: take a small amount of leaves (2-3cm long), put them into a 1.5ml centrifuge tube, freeze them with liquid nitrogen, and grind them quickly. Add 200 μl DNA Extraction buffer (20 mM Tris-HCl pH=7.5; 250 mM NaCl; 25 mM EDTA; 0.5% SDS; 0.2 mg / ml pro...
Embodiment 3
[0090] Example 3: Detection of transcript levels of the OsBBX14 gene in transgenic rice plants and wild-type rice
[0091] Using wild-type rice Nipponbare and 7 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.
[0092]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. Then 100 μl of a mixtu...
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