Application of arabidopsis thaliana AGP30 gene in reduction of cadmium absorption by plants
A technology for transgenic plants and Arabidopsis, which can be used in applications, plant peptides, plant products, etc., can solve unseen problems, achieve good cadmium stress resistance, reduce cadmium stress absorption, and have a wide range of applications.
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Embodiment 1
[0053] Cloning of embodiment 1 Arabidopsis AGP30 gene
[0054] The CDS sequence of the AGP30 gene was obtained from the Arabidopsis Information Resource Database (TAIR), the length of which is 720bp, and the nucleotide sequence is shown in SEQ ID NO.1.
[0055] (1) CTAB method to extract Arabidopsis genomic DNA
[0056] CTAB extract: CTAB 4g, NaCl 16.364g, add 1M Tris-HCl (pH 8.0) 20mL, 0.5MEDTA 8mL, distilled water to 200mL and then sterilize.
[0057] 1. Grind a small amount of Arabidopsis leaves, add CTAB extract (0.2% mercaptoethanol);
[0058] ②, Divide into several 1.5mL centrifuge tubes, water at 65°C, 30min;
[0059] ③. Centrifuge at 12000rpm for 12min;
[0060] ④. Pour the supernatant into several other 1.5mL centrifuge tubes;
[0061] ⑤Add an equal volume of chloroform, mix well, centrifuge at 12000rpm for 5min, and take the supernatant;
[0062] ⑥ Add 2 times the volume of absolute ethanol, precipitate for 5-10 minutes, and centrifuge at 12000rpm for 12 minutes...
Embodiment 2
[0073] The construction of embodiment 2 recombinant expression vector and the preparation of transgenic plant
[0074] (1) Construction of recombinant expression vector
[0075] i) Obtain the first 2000 bp of the CDS sequence of the myrosinase (PYK10) gene from the Arabidopsis thaliana information resource database (TAIR), and the nucleotide sequence is shown in SEQ ID NO.2.
[0076] ii) Using the Arabidopsis genomic DNA extracted in Example 1 as a template, design primers according to the nucleotide sequence of the first 2000 bp of myrosinase (PYK10) gene, carry out PCR amplification, and carry out 1wt% agarose gelation to the PCR amplification product Gel electrophoresis, and then use Tiangen Biochemical Technology (Beijing) Co., Ltd. DNA recovery kit (article number: DP209) to recover the amplified DNA fragment according to its instructions, and sequence verification, the amplified product is the promoter of the AGP30 gene ;
[0077] Wherein, the primer sequence is as fol...
Embodiment 3
[0094] Example 3 Under cadmium stress, the influence of Arabidopsis AGP30 gene on the growth phenotype of Arabidopsis seedlings
[0095] Take 8 transgenic Arabidopsis seeds prepared in Example 2 and equally divide them into experimental group 1 and experimental group 2; take 4 wild-type Arabidopsis seeds and a batch of transgenic Arabidopsis seeds as a control group.
[0096] The above three groups of Arabidopsis seeds were germinated on 1 / 2 medium for 3 days, and then transferred to the culture medium containing 50 μM CdCl 2 Continue to grow for 7 days on the 1 / 2MS medium to grow into Arabidopsis seedlings, observe its growth situation, the results are as follows figure 1 and figure 2 shown.
[0097] Depend on figure 1 and figure 2 It can be seen that the root length of the transgenic Arabidopsis seeds of the experimental group 1 after growing under cadmium stress reached 3.9cm, and the root length of the transgenic Arabidopsis seeds of the experimental group after grow...
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