Malus xiaojinensis Cheng et Jiang MxHA5 protein, and coding gene and application thereof
A gene and coding technology is applied to the small golden begonia MxHA5 protein and its coding gene and application field to achieve the effect of increasing iron deficiency tolerance
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Embodiment 1
[0053] Embodiment 1, the acquisition of MxHA5 protein and its coding gene
[0054] 1. Obtaining the full length of the MxHA5 gene
[0055] Total RNA was extracted from leaves of Begonia chinensis and reverse transcribed into cDNA. Using cDNA as a template, using a primer pair composed of F1 and R1, PCR amplification was performed using a high-fidelity HiFi mix to obtain PCR amplification products.
[0056] F1: 5'-ATGGCCGGCGATAAGG-3';
[0057] R1: 5'-AACTGTATAATGTTGTTGAATTGTATCG-3'.
[0058] PCR amplification system (20μl): ddH 2 O 7.0 μl, 2×HiF Mix (0.5 U / μl) 10 μl, F1 (10 mM) 1.0 μl, R1 (10 mM) 1.0 μl and template 1.0 μl.
[0059] PCR amplification program: 94°C pre-denaturation for 5 minutes; then denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 3 min, a total of 33 cycles; finally, extension at 72°C for 10 min.
[0060] Perform 1% agarose gel electrophoresis on the PCR amplification product, see figure 1 A (Swimming lane M is a DNA ...
Embodiment 2
[0099] Example 2, the acquisition of transgenic yeast and the analysis of its resistance to heavy metal toxicity
[0100] 1. Obtaining of transgenic yeast
[0101] 1. Digest the recombinant T vector pEASY-MxHA5 constructed in Example 1 with restriction enzymes KpnI and XbaI, and recover the digested product (about 2965 bp).
[0102] 2. Digest pYES2.0 plasmid (yeast expression vector, Beijing Dingguo Changsheng Biotechnology Co., Ltd.) with restriction enzymes KpnI and XbaI, and recover the vector backbone (about 5800bp).
[0103] 3. Ligate the digested product of step 1 with the vector backbone of step 2 to obtain the recombinant expression vector pYES2.0-MxHA5.
[0104] 4. The recombinant expression vector pYES2.0-MxHA5 was introduced into the yeast strain BJ2168 using the yeast transformation kit (Beijing Pangeno Technology Co., Ltd.), and the transgenic yeast BJ2168 / pYES2.0-MxHA5 was obtained.
[0105] 5. Construction of control plasmid
[0106] (1) Digest pEASY-T1 sampl...
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