Application of EdHP1 protein in improving resistance of plants to heavy metal lead
A heavy metal and protein technology, applied in the field of plant genetic engineering
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Embodiment 1
[0054] Embodiment 1, EdHP1 gene in Elymus chinensis to heavy metal Pb 2+ Response analysis of
[0055] 1. Preparation of Plant Material
[0056] The seeds of Elymus chinensis were vernalized at 4°C for three days, then sown in flower pots, and the seedlings after three weeks of growth were taken for Pb 2+ (200mmo1·L -1 Pb(NO 3 ) 2 ) treatment, after treatment for different time (0h, 1h, 5h, 12h and 24h), the treated seedlings were frozen in liquid nitrogen and stored at -70°C for gene expression analysis.
[0057] 2. Northern blot
[0058] 1. Electrophoresis
[0059] ① Prepare MOPS / formaldehyde denaturing gel: Add appropriate amount of agarose (1.2% agarose) to a gel bottle treated with DEPC water, heat to dissolve the agarose, cool to 55°C, add 10×MOPS and formaldehyde, pour glue.
[0060] ② Prepare the RNA sample for loading:
[0061]
[0062] Water bath at 55°C for 15 minutes, place on ice, add 10 μl 5×RNALoadingbuffer, and apply the sample.
[0063] ③ Electrop...
Embodiment 2
[0098] Embodiment 2, the acquisition and identification of EdHP1 transgenic plants
[0099] The EdHP1 gene involved in this example is derived from Elymus dahuricus Turcz., and the sequence of its coding region is sequence 2 in the sequence listing. Sequence 2 is the complete open reading frame, consisting of 2313 nucleotides in total, encoding the protein shown in Sequence 1 in the sequence listing (ie EdHP1 protein). Sequence 1 consists of 770 amino acids in total.
[0100] 1. Construction of recombinant expression vector
[0101] 1. Design of specific primer pairs
[0102] Design specific primers (with BamHI and SacI restriction sites at both ends) as follows:
[0103] Primer-F: 5'-CGGGATCCATGGTGGCGGCGGCGATCCT-3';
[0104] Primer-R: 5'-CGAGCTCCTAAAGGTATTTGAAGAGGA-3'.
[0106] The RNA of Elymus is extracted and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed with Primer-F and Primer-R to obtain PCR ampl...
Embodiment 3
[0151] Embodiment 3, EdHP1 transgenic plant resistance to heavy metal lead
[0152] T obtained in Example 2 2 Homozygous transgenic tobacco VP1-11 and VP1-32, T2 transgenic tobacco, and non-transgenic tobacco W38 were used as experimental materials. Each experimental material was placed in MS 0 When growing to 4 leaves on the culture medium, transplant it to the sand table, grow on the sand table for a week, select the tobacco seedlings with consistent growth, and transplant them into small nutrient flowerpots. Only one seedling is planted in each small pot, and then the Grow in the small pot for 7 days, choose the growth way is good, each experimental material of uniform size is processed, adopts the Pb(CH that contains 16mmol / L 3 COO) 2 The aqueous solution of the lead treatment (every pot pours 200 milliliters, pours once a week). In the test, the plant phenotypes of each experimental material were regularly observed, and after 40 days of lead treatment (by pouring the ...
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