Plant expression carrier and method for cultivating low-phytic-acid rice
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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
Active Publication Date: 2013-01-09
ZHEJIANG UNIV
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However, studies have found that with the decline of phytic acid content in seeds, other agronomic traits of crops will also change, such as crop yield reduction, seed vigor decline, plant morphology changes, and even affect the resistance of plants to pathogenic bacteria. lethal mutation
At present, there is no report on the cultivation of low phytic acid rice or other crops by silencing other phytic acid metabolism-related genes through RNAi technology
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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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Abstract
The invention discloses a plant expression carrier and a method for cultivating low-phytic-acid rice. The plant expression carrier comprises a hairpin structure unit inoculated into an original carrier and a specific promoter, wherein the specific promoter is a promoter Oleosin 18, and the nucleotide sequence is shown as SEQ ID NO.2. The method comprises the following steps: (1) constructing the plant expression carrier; (2) performing Agrobacterium-mediated transformation on the plant expression carrier to obtain a rice callus tissue; and (3) transferring the rice callus tissue onto a selective culture medium for further culture, transplanting onto a crop-growing field after seedling differentiation, and screening to obtain the low-phytic-acid rice plant. According to the invention, the seed specific promoter Oleosin 18 is used to control gene expression, so that gene silencing only occurs in seed embryos and aleurone layers, thereby effectively preventing the synthesis of phytic acid in other tissue positions of rice from being influenced.
Description
technical field [0001] The invention belongs to the field of genetic engineering, in particular to a plant expression vector and a method for cultivating low phytic acid rice. Background technique [0002] Phytic acid (PA, phytic acid), also known as myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate, abbreviated as InsP6 or IP6, widely exists in cereals, beans, nuts , The seeds of oil crops are the main storage form of plant phosphorus. In plants, phytic acid and its derivatives undertake a variety of physiological functions, such as storing phosphorus and energy during seed development, and providing inorganic phosphorus to seeds during seed germination. In addition, phytic acid and its derivatives are also involved in intracellular signal transduction, energy conversion, ATP metabolism and stress resistance. However, since humans and non-ruminant animals (pigs, chickens, fish, etc.) lack enzymes to digest phytic acid and phytate in their stomachs, the excreta of humans and ...
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