Plant expression carrier and method for cultivating low-phytic-acid rice
A plant expression vector, rice technology, applied in the field of genetic engineering, can solve the problems of seed vigor decline, silencing, lethal mutation, etc., and achieve the effect of ensuring the overall quality and avoiding the synthesis of phytic acid
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[0041] 1. Amplification of MIK gene fragments
[0042] (1) Total RNA extraction
[0043] Use the QIAGEN RNeasy Plant Mini kit to extract total RNA from Nipponbare immature embryos 14 days after flowering. The specific steps are as follows:
[0044] ① Grind the sample into powder with liquid nitrogen, transfer it to a self-prepared 1.5mL centrifuge tube, and do the following steps directly or store it in a -70°C refrigerator.
[0045] ② Weigh a sample weighing less than 100 mg into a centrifuge tube, add 450 μL RLT or RLC buffer (add 4.5 μL β-mercaptoethanol before use), shake vigorously, incubate at 56°C for 3 minutes, and vortex to mix the sample.
[0046] ③Transfer the dissolved sample to the purple recovery column, centrifuge at 14000rpm for 2min, transfer the supernatant to a new 1.5mL centrifuge tube (prepared by yourself), do not stir the sediment at the bottom of the tube.
[0047]④ Add 0.5 times the volume of absolute ethanol to the supernatant and mix well.
[0048...
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