MicroRNA gene for improving rice tolerance under arsenic stress and application thereof
A tolerance, rice technology, applied in DNA/RNA fragments, applications, genetic engineering and other directions, can solve problems affecting soil ecological structure and function, reduce yield and quality, etc., to alleviate toxicity, improve tolerance, reduce cumulative effect
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Embodiment 1
[0028] Example 1: Cloning of osa-miR812q precursor gene
[0029] The rice variety used for gene cloning and expression was Suxiangjing 3 (Oryza sativa L. subsp. Japonica, cv. Suxiang 3). The cloned osa-miR812q precursor sequence is shown in SEQ ID NO.2 in the sequence table; the mature sequence expressed from the precursor sequence is shown in SEQ ID NO.1.
[0030] Pick plump rice seeds, disinfect with 70% ethanol, rinse with distilled water, transfer to wet filter paper, and accelerate germination with 5% Hoagland nutrient solution for 2 days. After turning white, transfer to a light-temperature incubator (16h light / 8h dark, temperature 25°C, relative humidity 70%), and replace the nutrient solution every 3 days. 18-day-old seedlings were treated with 10 mM NaAsO 2 The stress treatment was carried out, and the untreated group was used as the control, and each group was repeated 3 times. The leaves were sampled after 48 h of treatment, frozen in liquid nitrogen and stored a...
Embodiment 2
[0036] Embodiment 2: Construction of osa-miR812q overexpression vector
[0037] The amplified PCR product was connected to the cloning vector pMD19-T (TakaRa). Add 1 μlpMD19-T vector, 5 μl solution I, and 4 μl purified miR812q precursor DNA to 10 μl system, mix the mixture, and ligate at 16°C for 6 hours. After PCR reaction verification and sequencing identification, the vector pMD19-miR812q is obtained. The carrier pMD19-miR812q was transformed into E. coli DH5α competent cells by heat shock method, cultured overnight, screened by ampicillin (Amp), positive clones were selected, and the correct bacterial solution was selected after sequencing. The plasmid extraction kit was used to extract the completely correct sequence Positive plasmid pMD19-miR812q. Digest with restriction endonucleases Pst I and Kpn I to recover the digested product, and simultaneously use restriction endonucleases Pst I and Kpn I to digest the empty vector pCAMBIA1301 to recover the vector skeleton. 50...
Embodiment 3
[0038] Example 3: Agrobacterium-mediated transformation of rice
[0039] The vector pCAMBIA1301-35S:miR812q was introduced into the Agrobacterium competent cell EHA105, and the operation was as follows:
[0040] 1. Preparation and transformation of competent Agrobacterium: Take a single colony of Agrobacterium, add it to 5 ml of YM liquid culture containing 20 mg / ml rifampicin, and cultivate overnight at 28° C. on a shaker at 250 rpm. Aspirate 2ml of the bacterial liquid and add it to 50ml of YM medium, shake and culture at 28°C until OD600=0.5. Centrifuge at 4000rpm for 10min to collect the bacterial cells, and suspend the bacterial cells in the YM medium for later use. Add 1 μg of miRNA overexpression vector plasmid to 200 μl of Agrobacterium competent cells, pipette and mix with a pipette tip, and place in ice bath, liquid nitrogen, and 37°C water bath for 5 min in turn. Add 800 μl of YM medium, shake and culture at 28°C for 2-4h. Get 200 μ l of bacterial liquid, smear it...
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